CA2659664A1 - Improved radiation therapy methods - Google Patents

Abstract

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 an effective amount of angiotensinogen, angiotensin I(AI), AI analogues, AI
fragments and analogues thereof, angiotensin II (AII), AII
analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists.

Description

IMPROVED RADIATION THERAPY METHODS

This is a divisional application of Canadian Patent Application No. 2,323,237 filed March 8, 1999.

Background of the Invention Radiation therapy is currently one of the most useful methods of treating cancerous tumors. However, radiation therapy damages normal tissue surrounding the tumor (U.S. Patent No. 5,599,712).

This damage can include fibrosis, remodeling of the extracellular matrix, vascular damage, aberrant angiogenesis, pneumonitis, atherogenesis, osteonecrosis, mucositis, imniunosuppression and functional impairment (U.S. Patent No.. 5,616,561).
As a result of these radiation-inctuced 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 treatrrient.

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 (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, neutropluls, and lymphocytes, and the resulting complications ofsuch depletion. For example, in patieMts 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 lo 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 heniatopoietic 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 neQative 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., Aniials N.Y. Acad. Sci. 628:126-139 (1991); Deeg et al., An-z. Hematol. 74:117-122 (1997);
Masse et al., 1998). This treatment is based on the premise that hematopoietic stem ?6909-151D

cells are relatively protected from radiation-related toxicity when quiescent, -particularly when the malignant cells are proliferating (Deeg et al., (1997)).

Bone marrow contains pluripotent stem cells that are capable of reconstituting the entire hematopoietic system. Bone marrow transplantation has been used to treat various- intractable hematopoietic diseases including leukemia and sevefe aplastic aneritia. (U.S: Patent No. 5,186,931).

Typically, a bone iparrow 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 nonnal 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 radiatiori nephropathy, have been viewed as inevitable, progressive and untreatable (Moulder et al., Bone Alarrow 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.

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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 cbemotherapy for cancer would make such treatments safer and possibly permit increases in the intensity of the therapy thereby yielding greater anti-cancer effects.

Summary of the Invention In one aspect, the present invention provides methods and kits for mitigating radiation induced tissue danlage, 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), Al analogues, Al fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists to a patient in need thereof.

In another aspect, the invention provides use of at least one active agent comprising a sequence consisting of at least five contiguous amino acids of groups R1-R8 in the sequence of general formula I

Rl-R2-R3-R4-R5-R6-R7 -RB

wherein R1 is Asp; R 2 is Arg; R3 is selected from the group consisting of Val and Pro; R4 is Tyr; R5 is selected from the group consisting of Ile and Ala; R6 is His; R' is Pro; and R8 is selected from the group consisting of Phe and Ile;
excluding sequences including R4 as an N-terminal Tyr group;
wherein when R3 is Pro, then R5 is Ile; wherein when R5 is Ala, then R3 is Val; wherein when R8 is Ile, then R3, if present, is Val; and RS is Ile; and wherein when R8 is Phe, then one or more of the following is true: the sequence does not consist of 8 contiguous amino acids of R1-R8, R3 is Pro, or RS is Ala; for the preparation of a medicament for the mitigation of damage to one or more of white blood cells, lymphocytes, neutrophils, monocytes, megakaryocytes, and platelets, due to radiation exposure, or for radiation therapy. Preferably, the sequence is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:31 and SEQ ID NO:38.

In another aspect, the invention provides use of at least one active agent comprising a sequence of the following general formula:

Rl-Arg-Rz-R3-R4-His-Pro-RS

wherein Rl is selected from the group consisting of H and Asp; R 2 is selected from the group consisting of Val and Pro;
R3 is Tyr; R4 is selected from the group consisting of Ala and Ile; and R5 is Phe, Ile, or is absent; wherein only one of R' and R8 can be absent; wherein when RZ is Pro, then R4 is Ile; wherein when R4 is Ala, then R2 is Val; wherein when Re is Ile, then R3, if present, is Val; and R5 is Ile; and wherein when R5 is Phe, then one or more of the following is true: the sequence does not consist of 8 contiguous amino acids of R1-R8, R 2 is Pro, or R' is Ala; for the preparation of a medicament for the mitigation of damage to one or more of white blood cells, lymphocytes, neutrophils, monocytes, megakaryocytes, and platelets, due to radiation exposure, or for radiation therapy.

In another aspect, the invention provides use of an active agent comprising a sequence 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, for the preparation of a medicament for the mitigation of damage to one or more of white blood cells, lymphocytes, neutrophils, monocytes, megakaryocytes, and platelets, due to radiation exposure, or for improved methods of radiation therapy.

The invention further provides pharmaceutical compositions and commercial packages based on the inventive medical uses described above and herein.

5a i6909-151D

In another aspect of the present invention, an improved cell culture meditrtn and kits are provided for the production of inegakaryocytes and platelets wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, Al, Al analogues, Al fragments and analogues thereof; AII, AII

analogues, All fragments or analogues thereof or AII AT, type 2 receptor 4gonists.
These aspects and other aspects of the invention become apparent in light of the following detailed description.

io Brief Description of the Drawings Figure 1 is a graph showing the effect of All treatment ttivo days prior to exposure on post-irradiation mouse mortality.

Figure 2 is a graph showing the effect of AII treatment on the day of exposure on post-in adiation mouse mortality.

Figure 3 is a graph showing the effect of All treatment ttii~o days following exposure on post-in-adiation mouse mortality.

Figure 4 is a graph showing the effect of AII 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 AII treatment two days following exposure on white blood cell number after irradiation.

5b Figure 7 is a graph showing the effect of All treatment two days prior to exposure on megakaryocyte number after irradiation.

Figure 8 is a graph showing the effect of All treatment on the day of exposure on megakaryocyte number after irradiation.

Figure 9 is a graph showing the effect of All treatment two days following exposure on megakaryocyte percentage after irradiatio_n_.

Figure 10 is a graph showing the effect of All treatment two days prior to exposure on monocyte number after irradiation.

Figure 11 is a graph showing the effect of All treatment on the day of exposure on monocyte number after itTadiation.

Figure 12 is a graph showirig the cffect of All treatment two days following exposure on monocyte number after irradiation.

Figure 13 is a graph showing the effect of All treatment two days prior to exposure on neutrophil number after irradiation.

Figure 14 is a graph showing the effect of All treatment on the day of exposure on neutrophil number after irradiation.

Figure 15 is a graph showing the effect of All treatment two days following exposure on neutrophil number after irradiation.

Figure 16 is a graph showing the effect of All treatment two days prior to exposure on lymphocyte number after irradiation.

Figure 17 is a graph showing the effect of All treatment on the day of exposure on lymphocyte number after irradiation.

Figure 18 is a graph showing the effect of All treatment two days following exposure on lymphocyte number after irradiation.

Figure 19 is a graph showing is a graph showing the effect of AII 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 iiradiation.

Figure 22 is a graph showing the effect of AII treatment on mouse survival receiving bone mairow transplantafion affter lethal irradiation.

Figure 23 is a graph showing the effect of AIl on white blood cell number in the loblood of mice receiving bone marrow transplantation after lethal irradiation.

Figure 24 is a graph showing the effect of AII on white blood cell number in the blood of mice receiving bone marrow transplantation after lethal irradiation..

Detailed Description of the Preferred Embodiments The present invention fulfills the needs for improved therapeutic methods to mitigate radiation induced tissue damage, to improve the effectiveness of radiation therapy, to support bone marrow transplantation, and to promote 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 titsue type, and also includes hematopoietic stem and progenitor cells, white blood cells and platelets.

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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 signifi;cantly elevated above the normal range of piateiets or megaKaryocyles 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(At), AI
analogues, Al fragments and analogues thereof, angiotensin II (All), All analogues, All fragments or analogues thereof and All AT, type 2 receptor agonists.

Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: lvlolecular Cloning: A
Laboratorv Manual (Sambrook, et al., 1989, Cold Spnng Harbor Laboratory Press), Gene Expression Technology (Methods in Euzymology, Vol. 185, edited by D.
Goeddel, 1991, Academic Press, San Diego, CA), "Guide to Protein Purification"
in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR

Pi-otocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Anintal Cells: A Manual of Basic Technique, 2 d Ed.
(R.I.
Freshney. 1987. Liss, Inc. New York, NY), Gene Trans_ fer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, NJ), and the Ambion 1998 Catalog (Ambion, Austin, TX).

U.S. Patent No. 5,015,629 to DiZerega describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II
(AII) in an amount which is sufficient for said increase. The application of All to .5 wound tissue significantly increases the rate of wound healing, leading tp a more rapid re-epithelialization and tissue repair. The term AII refers to an octapeptide present in humans and other species having the seqtience Asp-Arg-Va1-Tyr-Ile-His-Pro-Phe [SEQ ID NO:I]., The biological formation of angiotensin is initiated by the action of renin on the.plasma substrate angiotensinogen. The substance so formed is a 1o decapeptide called angiotensin I(AI) which is converted to All by the convettittg enzyme angiotensinase which removes the C-terminal His-Leu residues from Al (Asp-Arg-Val-Tyr-IIe-His-Pro-Phe-His-Leu [SEQ ID NO:37]). AII is a known pressor agent and is commercially available. The use of AIl analogues and fragments, AT2 agonists, as well as AIII and AIII analogues and fragments in wound healing has 15 also - been described. (U.S. Patent No. 5,629,292; U.S. Patent No.
5,716,935; WO
96.'39164).

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., 20 - J:liol. Cell. Cardiol. 21:S7 (Supp 111) 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, Ani. J. Pathol. 137:7-i6909-151D

(1990). In addition, All was shown to be angiogenic in rabbit comeal eye and chick chorioallantoic membrane models (Fernandez, et al., J. Lab. Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6 (1991). Therefore, All may accelerate wound repair through increased neovascularization, growth factor release, reepithelialization and/or production of extracellular matrix.

AII has also been implicated in both cell growth and differentiation (Meffert et al., Mot. and Cellul. Endocrin. 122:59 (1996)). Two main classes of All receptors, ATi and AT, have been identified (Meffert, 1996). The growth-promoting effects of All have been attributed to mediation by the ATI receptor, while some evidence suggests that tl-e AT2 receptor tnay bc involved in mediation of the cell differentiation effects of All (Bedecs et al., Biocheni. J. 325:449 (1997)).

The effects of All receptor and All receptor antagonists have been examined in two experimental models of vascular injury and repair which suggest that both All receptor subtypes (AT1 and AT2) play a role in wound liealing (Janiak et al., 1> Hipertension 20:737-45 (1992); Prescott, et al., Ain. J. Pathol. 139:1291-1296 (1991);
Kauffman. et al., Life Sci. 49:223-228 (1991); Viswanathan, et al., Peptides 13:78:3-786 (1992); Kimura, et al., BBRC 187:1083-1090.'(1992).

Many studies have focused upon AII(1-7) (All 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. Pharntacol. 225:57-62 (1992);

Jaiswal, et al., Hypertension 19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J.
Pharniacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol.
Exper.
Ther. 265:664-673 (1991); JaiswaI, et al., Hvpertension 17:1115-1120 (1991);
Portsi, et a., Br. J. Pharrnacol. 111:652-654 (1994).

While a single pilot study has suggested that AII-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 AII, are effective in reducing radiation nephropathy, bone _ L__uu_ u .. a,,,...te ..~J;~t:~,n i_,inrv (Moulder Pt al Tn! /
marI~'ow transplantation liuu'a rieYiya.hy, aia~a cu.u ... ..............
,..., ~.._ _---- -=, _ =

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-(1993); Yoon et al., hit. J. Radiat.- Oncol. Biol. Phvs. 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 ATI receptor by AII

(Moulder et al.. Radiatioiz 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-(1997)).

Furthermore, it has recentlv been demonstrated that angiotensinogen, angiotensin I (Al). Al analogues, Al fragments and analogues thereof, AII, All analogues, AII fragments or analogues thereof or AII AT2 type 2 receptor agonists. are potent stimulators of hematopoietic stem cell proliferation (ILJ.S. Patent No.
6,335,195).
Therefore, it would be expected that the use of these compounds might cause long-term ;6909-151D

hematopoietic damage if used in conjunction with radiation therapy (Masse et al., 1998; Watanabe et al., 1996).

Based on all of the above, it would be unexpected that the use of angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or All AT, type 2 receptqr 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(AI), Al analogues, Al fragments and analogues thereof, angiotensin II (All), All analogties, All fragments or analogues thereof or All AT2 typr.. 2 rGc:cpli-r agonists to stimulate the production and mobilization of inegakaryocytes, or to stimulate the production of platelets.

A peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) 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-NHZ-Phe (Speth and Kim, BBRC 169:997-1006 (1990). This peptide gave binding character istics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharrnacol. 256:93-97 (1994); Bryson, et al., Eur.
J
Pharmacol. 225 `.119-12 7 (1992).

The active Al, Al analogues, Al frarrnents 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'-R8 in the sequence of general formula I

%6909-151D

R '-RZ-R3-W-RS-R6-R'Rg in which R' and R2 together form a group of formula X-R"-RB-, wherein X is H or a one to three peptide group, R" is - suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NHz), Gly, Asp(NH2) and Suc, RB is suitably selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;

R` is selected from the ai-oup consisting of Val, Ala, Leu, norLeu, Ile.
Gly, Pro, Aib, Acpc, Lys, and Tvr;

R4 is selected from the group consisting of Tyr, Tyr(P03)2, Thr, Ser, homoSer, Ala, and azaTyr;

R' is selected from the oroup consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R is His, Arg or 6-NH,-Phe;
R' is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences includina R' as a terminal Tyr group.

Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AII analoQues set forth above subject to the restriction that R6 is p-NHZ-Phe. In addition to peptide agents, various nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.

Particularly preferred combinations for R'' and RB are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys. Particularly preferred embodiments of this class include the following: All, AIII or AIl(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:21;
AII(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID
NO:3];

AH(1-7), 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-lle-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];
A1I(1-5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(1-4), Asp-Arg-Val-Tyr [SEQ ID
NO;10]; and AII(I-3), Asp-Arg-Val [SEQ ID NO:I l]. Other preferred embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO:12] and Arg-Val-Tyr-norLeu--His-Pro-Phe [SEQ ID NO:13]. Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31]. AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tvr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.

A class of particularly preferred compounds in accordance with the present invention consists of those with the following gertoral structure:

R 1-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 Tyr and Tyr(P03)2;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.

'76909-151D

Particularly preferred embodiment of this class are selected from the group consisting of SEQ ID NO:I, SEQ ID NO:4, SEQ ID NO:18, SEQ ID NO:26, SEQ ID
NO:3 1, 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 th-ose of the general formula II

R2-R3-R4-RS-R6-R'-R$
in which R` is selected from the group consisting of H, Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;

R.' is selected froin the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro; Aib, Acpc and R4 is selected from the group consisting of Tyr, Tyr(P03)2a Thr, Ser, homoSer and azaTyr;

R' is selected from the eroup consisting of lle, Ala, Leu, norLeu, Val and Gly;

R6 is His, Are or 6-NH2-Phe;
R' is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.

A particularly preferred subclass of the compounds of general formula II has the formula Rz-R3-Tyr-R'-His-Pro-Phe [SEQ ID NO:16]

wherein R', R3 and R5 are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2].
Other preferred compounds include peptides having the structures Arg-Val-Tyr-Giy-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 l 1o Abbreviation for Amino Acids Me2Gl N,N-dimeth 1 1 c l Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt (betaine) Suc Succinvl Phe(Br) p-bromo-L- henvlalanvl azaTyr aza-a'-homo-L-tvros l Acpc 1-aminoc clo entane carboglic acid Aib 2-aminoisobu fic acid Sar N-methvl 1 cvl (sarcosine) It has been suggested that All and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharnracological Revieivs 26:69 (1974). In general, it is believed that neutral side chains in position R3 , RS and R7 may be involved in maintaining the appropriate distance between active groups in positions R , R
6 and R8 primarily responsible for binding to receptors and/or intrinsic activity.
Hydrophobic ~6909-151D

side chains in positions R3, R5 and R8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.

Appropriate side chains on the amino acid in position R2 may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R2.

For purposes of the present invention, it is believed that R3 may be involved in the formation of linear or nonlinear hydroeen bonds with R5. (in the ganuna turn model) or R6 (in the beta turn model). R' would also participate in the first turn in a beta antiparallel structure (which lias also been proposed as a possible structure). In contrast to other positions in general formula I. it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R3 may suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr. In another preferred embodiment, R3 is Lys.

With respect to R4, conformational analyses have suggested that the side chain in this position (as well as in R 3 and R5) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R4 is preferably selected from Tyr, Thr, Tyr (P03)2, homoSer, Ser and azaTyr. In this position, Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra). In a further preferred embodiment, R4 is Ala.

In position R5, an amino acid with a 0 aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R5, it is preferred that the amino acid in this position be selected from Ile, Ala, Leu, norLeu, Gly and Val.

In the Al, Al analogues, Al fragments and analogues thereof, All, All analogues,; fragments and analogues of fragments of particular interest in accordance with the present invention, R(' is His, Arg or 6-NH2-Phe. The unique properties of the imidazole ring of histidine (e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R6. For examplc, conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R7. Similarly, it is presently considered that R' should be Pro in order to provide the most desirable orientation of R8. In position R8, both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.

Analogues of particular interest include the foJjowing:

Angiotensin II Analogues AIl Amino Acid Sequence Sequence Analogue Identifier Name Analo~ue 1 As -Ar -Val-Tvr-Val-His-Pro-Phe SEQ ID NO: 19 Analogue 2 Asn-Ar -Val-T -Val-His-Pro-Phe SEQ ID NO: 20 Analogue 3 Ala-Pro-Glv-As -Ar -IIe-T r-Val-His-Pro-Phe SEQ ID NO: 21 Analogue 4 Glu-Ar -Val-Tvr-Ile-His-Pro-Phe SEQ ID NO: 22 Analo ue 5 As -L s-Val-Tvr-Ile-His-pro-Phe SEQ ID NO: 23 Analogue 6 As -Ar -Ala-T -Ile-His-Pro-Phe SEQ ID NO: 24 Analogue 7 As -Ar -Val-Thr-Ile-His-Pro-Pbe SEO ID NO: 25 'Analo e 8 As -Ar -VaI-T -Leu-His-Pro-Phe SE ID NO: 26 Analogue 9 As -Ar -Val-T -Ile-Ar -Pro-Phe SE ID NO: 27 Analogue 10 As - -Val-T -Ile-His-Ala-Phe SE ID NO: 28 Analogue 11 Asp-Arg-Val-Tyr-lie-His-Pro-Tyr_ SEQ ID NO: 29 Analogue 12 Pro-Ar -Val-T -Ile-His-Pro-Phe SgQ ID NO: 30 Analo e 13 As -Ar -Pro-T -Ile-His-Pro-Phe SEQ ID NO: 31 Analo- ue 14 As -Ar -Val-T O3 Z-Ile-His-Pro-Phe SE ID NO: 32 Analo ue 15 As -Ar -norLeu-T -IIe-His-Pro-Phe SE ID NO: 33 Analo ue 16 As -Ar -VaI-T -norLeu-His-Pro-Phe SEQ IDNO: 34 Analogue 17 As -Ar -Val-homoSer-T -Ile-His-Pro-Phe SE ID NO: 35 The polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Stnthesis, 2nd ed., Pierce Cheniical Co., Rockford, 111. (1984) and J. Meienhofer, Horn:ona!

Proreins and Peptides, Vol. 2, Academic Prcss, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis:

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616).

Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence,- also suitably protected, is added under conditions 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.

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), Al analogues, Al fragments and analogues thereof, angiotensin II(AII), All analogues, All fragments or analogues thereof or All AT2 type 2 receptor agonists (the "active agents").

In another aspect, the present invention provides improved methods and kits for treating a patient.afflicted with a neoplastic disease state that is being treated with ionizing or nonionizing radiation, the improvement comprising conjunctive therapy whereby an effective radioprotective amount of the active agents is provided.

In another aspcct, the present invention provides improved methods and kits for treating a patient in need of radiation therapy, the improvement comprising the administration of the active agents in conjunction with the radiation therapy.

The invention is appropriate for use with any type of ionizing radiation exposure such as therapeutic or accidental X-ray, gamma ray, or beta particle exposure. Examples of ionizing radiation exposure suitable for treatment with the methods and kits of the present invention include, but are not limited to, clinical radiation therapy, medical diagnostics using radioactive tracers, exposure to naturally occurring ionizing radiation sources such as uranium and radon, wartime exposure, and accidental exposures including occupational exposure at nuclear power facilities, and medical and research institutions. Examples of nonionizing radiation exposure suitable for treatment with the methods and kits of the present invention include, but are not limited to, ultraviolet light, X-rays, microwaves, radio-frequency waves, and electromagnetic radiation.

Virtually any tissue susceptible to radiation-induced tissue damage can gain protection by use of the active agents of the invention. For example, breast tissue is an excellent candidate for receiving the benefit of the subject invention.
Radiation-induced tissue damage can be a fatal side effect of over-exposure to radiation therapy.

Typically, the fibrotic reaction common in normal breast tissue surrounding the canc;erous 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. Haenratol. 94:619-627 (1996)). Repeated or high dose cycles of radiation therapy may be responsible for severe stem cell depletion leading to 17 important long-term hematopoietic sequelea and marrow exhaustion. The methods of the present invention provide for improved mortalitv and blood cell count when used in conjunction with radiation therapy.

Skin exposure is particularlv 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.

-%6909-151D

In another aspect, the present invention provides improved methods and kits for supporting bone marrow transplantation comprising the administration of the active agents to a patient in need thereof. These compounds may be administered in combination with auxiliary agents including, but not limited to interleukin (IL)-3, IL-1, IL-4, Il-5, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colorjy 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 daniage 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, 1~ 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, lipi.d` foams, transdermal patches, topical administrative agents, polyethylene glycol polymers, carboxymethyl cellulose preparations, crystalloid preparations. (e.g., saline, Ringer's lactate solution, phosphate-buffered saline, etc.), viscoelastics, polyethylene glycols. and polypropylene glycols. The means for delivery may either contain the effectiVe amount of angiotensinogen, Al, Al analogues, AI fragments and analogues thereof, All, All analogues, All fragments or analogues thereof or All AT7 type 2 receptor %690y-151D

agonists, or may be separate from the compounds, which are then applied to the means for delivery at ihe 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 ra-Aiation=induced tissue damage and for clinical radiation therapy.

In a further aspect of the present invention, a method of increasing megakaryocyte production and mobilization and platelet production by.exposure to the active agents of the inventions is disclosed. In one embodiment, megakaryocytes are isolated from bone marrow as described in U.S.- Patent No. 5,178,856).
Briefly, marrow is flushed from a subject's femur with Iscove's modification of Dulbeceo'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 37 -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 *Trade-mark 23 ~6909-151D

cytometry as described in U.S. Patent No. 5,155,211)_ Briefly, the appearance of granules and the extensive surface-connected open canalicular membrane system as well as a substantial decrease in the nucleus:cytoplasm volume distribution, indicates that the megakaryocyte population has completed the process of polyploidization but has not yet generated, a major portioh 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 in-adiation and active agent is.
administered subcutaneously (10 pg/kgJday or 100 jig/kg/day) at various times before and after irradiation. The number of white blood cells, megakaryocytes and platelets is preferably detennined by counting with a hemacytometer followed by differential morphologic analysis.

In another einbodiment of this aspect of the- invention; hematopoietic precursor cells are isolated from bone marro% 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 embodiriient, 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 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 colunm 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/mi of the active agents of the invention. The cells are expanded for a period of between 8 and 21 davs 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, granulocy-tz colony-stimulating factor, granulocyte/macrophage colony stimulating factor, macrophage colony-stimulating factor, tumor necrosis factor, epidennal 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 transplatitation, 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.
*Trade-mark 25 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 incteasing 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, intraniuscular, 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, intrastemal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.

The active agents of the invention may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions). The compounds of the invention may be applied in a variety of solutions.
Suitable solutions for use in accordance with the invention are sterile, dissolve sufficient amounts of the peptide, and are not harmful for the proposed application. In i6909-151D

this regard, the compounds of the present invention are very stable but are hydrolyzed by strong acids and bases. The compounds of the present invention are soluble in organic solvents and in aqueous solutions at pH 5-8.

The active agents may be subjected to conventional phatmaceutical operations such as sterilization and/or may contain conventional adj uvants, 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, magnesiuni 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.
Altematively, the compounds of this invention may be dissolved in saline, water, polyethvlene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, com oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers. Other adjuvants and modes of administration are well lcnown in the pharmaceutical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.

Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable_ for administration to the eye, ear, or nose.

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 deterrnined 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, includittg the type of injury, the agc, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound. employed. For example, the active agents are administered to an oncology patient for up to 30 days prior to a course of radiation therapy and for up to 60 days post-radiation exposure. The therapy is administered for I 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 witll;`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.

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 g/kg body weight and about 10.0 mg/kg body weight. This dosage regimen maximizes the therapeutic benefits. of the subject invention while minimizing the amount of agonist or peptide needed. Such an application minimizes costs as well as possible deleterious side effects.

For subcutaneous administration, the active ingredient may comprise from 0.0001 % to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it 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 forntulation are as described for subcutaneous administration.

In a preferred embodiment of all of the aspects of the invention, the active agent is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO:2, SEQ
ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11. SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34; SEQ ID NO:35, SEQ ID NO:36; and SEQ ID NO:37.

In a further preferred embodiment of the above aspects of the invention, administration of the active agent is localized to the area affected by the tissue-damaging radiation.

In another aspect of the present invention, an improved cell culture medium is provided for megakaryocyte and platelet production, wherein the improvement comprises addition to the cell culture medium of an effective amount of between about 0.1 ng and 10 mg/ml of the active agents of the invention. Any cell culture media that can support megakaryocyte and platelet production can be used with the present invention. Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Mediunl, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEMG 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:
lOX) 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 commercialiy 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 cornprise cell culture growth medium. Any cell culture media that can support megakaryocyte and platelet ?6909-151D

production can be used with the present invention. Examples of such cell culture media are described above. The cell culture medium can be supplied in either a concentrated (ie: lOX) 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 serurri 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 anotlier preferred embodimcnt, 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, FungizoneG, 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 invenfion, as defined by the claims appended hereto.

Example I Effect of AII on rat mortality and white blood cell recovery after irradiation Female C57B1/6 mice (Jackson Labs, Bar Harbor, Maine) were irradiated with 600 cGy total body irradiation. Subcutaneous injection with either All (10 gg/kg/day or 100 g/kg/day) or saline (placebo) was initiated two days before (-day 2), on the day of (day 0) or 2 days after (+ day 2) irradiation and continued untii the animals succumbed to the irradiation or were necropsicd. At various times after irradiation, the mice were anaesthetized with Metofane (Pittman-Moore Animal Health, NZ) and bled via the retro-orbital sinus. Red blood cells were lysed with 0.3% acetic acid and. the number of wliite 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 1), but that AII administration on the day of irradiation (Figure 2) or two days after irradiation (Figure 3) substantially increased survivaL
Ftuthermore, AII administration at all time periods tested increased the number of circulating white blood cells (Figures 4-6). Further experiments demonstrated that AII
administration increased the number of megakaryocytes (Figures 7-9), monocytes (Figures 10-12), neutrophils (Figures * 13-15), and lymphocytes (Figures 16-18). 'Ihese. _ data demonstrate : that in vivo administration of All can improve hematopoietic recovery after irradiation.

Example 2. Effect of AII and AII 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 aRcr irradiation.
The data are shown in Figures 19 and 20. and show that the peptides increase the production of both of these blood eleinents.

*Trade-mark 32 %6ti09-151D

Table 3: Designation for Analogues/Fragments Name Abbreviation Sequence SEQ ID NO:
GSD 28 IleB-AII DRVYIHPI SEQ ID NO: 38 GSD 24B Pro3-AII DRPYIHPF SEQ ID NO:31 GSD 22A Ala4-AIII RVYAHPF SEQ ID NO:18 AII(1-7) DRVYIHP SEQ ID NO:4 All DRVYIHPF SEQ ID NO. I

Example 3. Effect of AII on survival of mice receiving bone marrow transplantation after lethal ir-radiation 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 g/ml angiotensin 11 (0.1 ml, 100 g/kg) subcutaneously for fourteen days. At the end of this penod, 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 ~,vere then injected intravenously into recipient mice (female C57B1/6, 6-8 weeks old) that had been lethally irradiated (900 cGy total body irradiation) at two. concentrations: 1 x 1,06 or I x 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 I x 10' Saline All I x 106 Saline All 1 x 105 All Saline 1 x 106 All Saline I x 105 All All 1 x 106 All All 1x105 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 t in Figures 21-24, and demonstrate that All treatment increased both survival and white blood cell number in mice receiving bone marrow transplantation after: irradiation. The.greatest benefit was conferred by treatment of both tlie donor bone marrow cells and the recipient nuce with AII.

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 damasz and for clinical radiation therapy, as well as bone marrow transplantation by increasing the survival rate of patients and accelerating the reconstitution of the patient's hematopoietic system. Similarly, by providing a method for megakaryocyte and platelet production, the present invention will greatly augment clinical cancer treatments and bone marrow transplantation and other conditions that lead to decreased megakaryocyte production and mobilization and platelet production.

The method of the present invention also increases the potential utility of megakaryocytes as vehicles for gene therapy in hematopoietic disorders, bv 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.

Claims (35)

1. A use of an effective amount of at least one active agent for bone marrow transplantation, the active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A-R B-, wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, MezGly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc;

R B is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group.

2. A use of an effective amount of at least one active agent in the manufacture of a medicament for bone marrow transplantation, the active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A-R B-, wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly, Asp (NH2) and Suc;

R B is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group.

3. The use of claim 1 or 2 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 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.

4. The use of claim 1 or 2 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.

5. A bone marrow transplantation kit, comprising:

(a) an amount effective to support bone marrow transplantation of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A-R B-, wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu(NH2), Gly, Asp(NH2) and Suc;

R B is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;

R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group, and (b) instructions for using the effective amount of active agent for supporting bone marrow transplantation.

6. The kit of claim 5 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 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.

7. The kit of claim 5 wherein the active agents are 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.

8. The kit of any one of claims 5 to 7 further comprising a means for delivery of the active agent.

9. A use of an effective amount of at least one active agent for increasing megakaryocyte production and mobilization and platelet production in a mammal, the active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A R B-, wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2), Gly, Asp (NH2) and Suc;

RB is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group.

10. A use of an effective amount of at least one active agent in the manufacture of a medicament for increasing megakaryocyte production and mobilization and platelet production in a mammal, the active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A R B- , wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2) , Gly, Asp(NH2) and Suc;

RB is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group.

11. The use of claim 9 or 10, 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 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.

12. The use of claim 9 or 10 wherein the active agents are 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.

13. An improved cell culture medium for megakaryocyte and platelet production, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate megakaryocyte and platelet production of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A-R B-, wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2) , Gly, Asp(NH2) and Suc;

R B is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group.

14. The improved cell culture medium of claim 13 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 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.

15. The improved cell culture medium of claim 13 wherein the active agents are 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.

16. A kit for megakaryocyte and platelet production, comprising:

(a) an amount effective to increase megakaryocyte and platelet production, of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R1-R8 in the sequence of general formula I

in which R1 and R2 together form a group of formula X-R A-R B-, wherein X is H or a one to three peptide group R A is selected from Asp, Glu, Asn, Acpc, Ala, Me2Gly, Pro, Bet, Glu (NH2) , Gly, Asp(NH2) and Suc;

R B is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc, Lys, and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(PO3)2, Thr, Ser, homoSer, Ala, and azaTyr;

R5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R6 is His, Arg or 6-NH2-Phe;
R7 is Pro or Ala; and R8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R4 as a terminal Tyr group; and (b) instructions for using the amount effective of active agent as a cell culture medium supplement.

17. The kit of claim 16, further comprising cell growth medium.

18. The kit of claim 16 further comprising a sterile container.

19. The kit of claim 16 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 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.

20. The kit of claim 16 wherein the active agents are 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.

21. A use of at least one active agent for bone marrow transplantation, the 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 (PO3) 2 ;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.

22. A use of at least one active agent in the manufacture of a medicament for bone marrow transplantation, the 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 (P03) 2;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.

23. The use of claim 21 or 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. A bone marrow transplantation kit, comprising:
(a) an amount effective to support bone marrow transplantation of at least one 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 (PO3) 2 ;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent, and (b) instructions for using the effective amount of active agent for supporting bone marrow transplantation.

25. The kit of claim 24 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.

26. The kit of claim 24 or 25 further comprising a means for delivery of the active agent.

27. A use of at least one active agent for increasing megakaryocyte production and mobilization and platelet production in a mammal, the 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 (PO3) 2;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.

28. A use of at least one active agent in the manufacture of a medicament for increasing megakaryocyte production and mobilization and platelet production in a mammal, the 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 (PO3) 2 ;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.

29. The use of claim 27 or 28, 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.

30. An improved cell culture medium for megakaryocyte and platelet production, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate megakaryocyte and platelet production of at least one 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;

R 2 is selected from the group consisting of Val and Pro;

R3 is selected from the group consisting of Tyr and Tyr ( PO3 ) 2 ;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent.

31. The improved cell culture medium of claim 30 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.

32. A kit for megakaryocyte and platelet production, comprising:

(a) an amount effective to increase megakaryocyte and platelet production, of at least one active agent comprising a sequence of the following general formula:

R1-Arg-R2-R3-R4-His-Pro-R5 wherein R' 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(PO3)2;

R4 is selected from the group consisting of Ala, Ile, Leu, and norLeu; and R5 is Phe, Ile, or is absent; and (b) instructions for using the amount effective of active agent as a cell culture medium supplement.

33. The kit of claim 32, further comprising cell growth medium.

34. The kit of claim 32 or 33, further comprising a sterile container.

35. The kit of any one of claims 32 to 34 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.