Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation Number:7,118,748 from the United States Patent and Trademark Office (PTO) owispatent

Title: Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation

Abstract: The present invention provides methods, improved cell culture medium and kits for promoting hematopoietic and mesenchymal stem and lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (Al), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof or AII AT.sub.2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.

Patent Number: 7,118,748 Issued on 10/10/2006 to Rodgers,   et al.

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Inventors:	Rodgers; Kathleen E. (Long Beach, CA), diZerega; Gere S. (Pasadena, CA)
Assignee:	University of Southern California (Los Angeles, CA)
Appl. No.:	09/564,045
Filed:	May 3, 2000

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	09012400	Jan., 1998	6335195
	60066593	Nov., 1997
	60065612	Nov., 1997
	60063910	Oct., 1997
	60063684	Oct., 1997
	60046859	May., 1997
	60036507	Jan., 1997

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Current U.S. Class:	424/185.1 ; 435/325; 435/372; 435/375; 530/329; 530/330
Current International Class:	A61K 39/00 (20060101); A61K 38/04 (20060101); C12N 5/00 (20060101); C12N 5/08 (20060101)
Field of Search:	435/2,325,372,283.1,306.1,70.5,375 424/185.1 530/329,330

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References Cited [Referenced By]

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U.S. Patent Documents

	6335195		January 2002		Rodgers et al.

Other References

Smilek et al. PNAS (199) 88:9633-9637. cited by examiner . Mrug et al., "Angiotensin II facilitates EPO-mediated Proliferation of Early Erythroid Progenitors" Blood 88:64a (1996)Abstract 2574. cited by other.

Primary Examiner: Chan; Christina
Assistant Examiner: Belyavskyi; Michail A.
Attorney, Agent or Firm: McDonnell Boehnen Hulbert & Berghoff LLP

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Parent Case Text

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CROSS REFERENCE

This application claims priority to U.S. application Ser. No. 09/012,400 filed Jan. 23, 1998, now U.S. Pat. No. 6,335,195, which claims priority to U.S. Provisional Application Nos. 60/036,507, filed Jan. 28, 1997, 60/046,859, filed May 8, 1997; 60/063,684 filed Oct. 28, 1997; 60/063,910 filed Oct. 31, 1997; 60/065,612 filed Nov. 18, 1997; and 60/066,593 filed Nov. 26, 1997.

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Claims

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We claim:

1. A method of accelerating the proliferation of hematopoietic pluripotent progenitor cells or hematopoietic lineage-specific cells comprising contacting the hematopoietic pluripotent progenitor cells or lineage-specific cells with an amount effective to accelerate proliferation of hematopoietic pluripotent progenitor cells or lineage-specific cells of a formulation comprising one or more amino acid sequence consisting of at least five contiguous amino acids of SEQ ID NO:4.

2. The method of claim 1 wherein the formulation comprises a sequence consisting of six contiguous amino acids of SEQ ID NO:4.

3. The method of claim 1 wherein the formulation comprises a sequence consisting of the amino acid sequence of SEQ ID NO:4.

4. The method of claim 1, 2, or 3 wherein the contacting occurs in vivo.

5. The method of claim 4 wherein the dosage of active agent is between 0.1 ng/kg and 1.0 mg/kg body weight.

6. The method of claim 1 wherein the method is used for hematopoietic pluripotent progenitor cell transplantation therapy.

7. The method of claim 1 wherein the method is used to augment chemotherapy.

8. The method of claim 1 wherein the method is used to augment radiation therapy.

9. The method of claim 2 wherein the method is used for hematopoietic pluripotent progenitor cell transplantation therapy.

10. The method of claim 2 wherein the method is used to augment chemotherapy.

11. The method of claim 2 wherein the method is used to augment radiation therapy.

12. The method of claim 3 wherein the method is used for hematopoietic pluripotent progenitor cell transplantation therapy.

13. The method of claim 3 wherein the method is used to augment chemotherapy.

14. The method of claim 3 wherein the method is used to augment radiation therapy.

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Description

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FIELD OF THE INVENTION

The present invention relates to methods, pharmaceutical compositions, and articles of manufacture for treating a patient undergoing chemotherapy, particularly for increasing hematopoietic cell survival and stem cell mobilization, and reducing the incidence and/or severity of chemotherapy-related side effects.

BACKGROUND OF THE INVENTION

People diagnosed as having cancer are frequently treated with single or multiple cytotoxic chemotherapeutic agents (cytotoxic agents) to kill cancer cells at the primary tumor site or at distant sites to where cancer has metastasized. (U.S. Pat. No. 5,605,931 incorporated by reference herein in its entirety.) Chemotherapy treatment is given either in a single or in several large doses or, more commonly, it is given in small doses 1 to 4 times a day over variable times of weeks to months. There are many cytotoxic agents used to treat cancer, and their mechanisms of action are generally poorly understood.

Irrespective of the mechanism, useful chemotherapeutic agents are known to injure and kill cells of both tumors and normal tissues. The successful use of chemotherapeutic agents to treat cancer depends upon the differential killing effect of the agent on cancer cells compared to its side effects on critical normal tissues. Among these effects are the killing of hematopoietic blood forming cells, and the killing and suppression of the white blood cells, which can lead to infection. Acute and chronic bone marrow toxicities are also major limiting factors in the treatment of cancer. They are both related to a decrease in the number of hemopoietic cells (e.g., pluripotent stem cells and other progenitor cells) caused by both a lethal effect of cytotoxic agents or radiation on these cells, and via differentiation of stem cells provoked by a feed-back mechanism induced by the depletion of more mature marrow compartments. (U.S. Pat. No. 5,595,973 incorporated by reference herein in its entirety.) Stimulators and inhibitors of bone marrow kinetics play a prominent role in the induction of damage and recovery patterns (Tubiana, M., et al., Radiotherapy and Oncology 29:1, 1993).

Prevention of, or protection from, the side effects of chemotherapy would be a great benefit to cancer patients. The many previous efforts to reduce these side effects have been largely unsuccessful. For life-threatening side effects, efforts have concentrated on altering the dose and schedules of the chemotherapeutic agent to reduce the side effects. Other options are becoming available, such as the use of granulocyte, colony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), epidermal growth factor (EGF), interleukin 11, erythropoietin, thrombopoietin, megakaryocyte development and growth factor, pixykines, stem cell factor, FLT-ligand, as well as interleukins 1, 3, 6, and 7, to increase the number of normal cells in various tissues before the start of chemotherapy (See Jimenez and Yunis, Cancer Research 52:413415; 1992). The mechanisms of protection by these factors, while not fully understood, are most likely associated with an increase in the number of normal critical target cells before treatment with cytotoxic agents, and not with increased survival of cells following chemotherapy.

Acute myelosuppression as a consequence of cytotoxic chemotherapy is well recognized as a dose-limiting factor in cancer treatment. (U.S. Pat. No. 5,595,973) Although other normal tissues may be adversely affected, bone marrow is particularly sensitive to the proliferation-specific treatment such as chemotherapy or radiotherapy. For some cancer patients, hematopoietic toxicity frequently limits the opportunity for chemotherapy dose escalation. Repeated or high dose cycles of chemotherapy may be responsible for severe stem cell depletion leading to serious long-term hematopoietic sequelea and marrow exhaustion.

Despite advances in the field of chemotherapy, prior art methods have proven to be of limited utility in minimizing chemotherapy-induced depletion of hematopoietic stem cells and their progeny. Thus, there is a need for improved therapeutic methods and pharmaceutical compositions for increasing hematopoietic cell survival following chemotherapeutic treatments, as well as for decreasing the adverse effects of chemotherapy on the bone marrow.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods, pharmaceutical compositions, and articles of manufacture for treating a patient undergoing chemotherapy, for increasing hematopoietic cell survival following chemotherapy, for reducing or preventing other side effects of chemotherapy, such as anemia, and for mobilizing hematopoietic progenitor cells from bone marrow into peripheral blood, comprising administering an amount effective for such purposes of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AIII), AIII analogues, AII fragments or analogues thereof, AII AT.sub.2 type 2 receptor agonists, or ACE inhibitors.

These aspects and other aspects of the invention become apparent in light of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of AII treatment on white blood cell number in the blood 7 days after 5FU treatment.

FIG. 2 is a graph showing the effect of AII treatment on white blood cell number in the spleen 7 days after 5FU treatment.

FIG. 3 is a graph showing the effect of AII treatment on white blood cell number in the thymus 7 days after 5FU treatment.

FIG. 4 is a graph showing the effect of AII treatment on white blood cell number in the bone marrow 7 days after 5FU treatment.

FIG. 5 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following blood harvest 7 days after 5FU treatment.

FIG. 6 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following spleen harvest 7 days after 5FU treatment.

FIG. 7 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following bone marrow harvest 7 days after 5FU treatment.

FIG. 8 is a graph showing the effect of AII treatment on CFU-GM cell number in the blood on day 7 after 5FU treatment.

FIG. 9 is a graph showing the effect of AII treatment on white blood cell number in the spleen on day 14 after 5FU treatment.

FIG. 10 is a graph showing the effect of AII treatment on white blood cell number in the thymus on day 14 after 5FU treatment.

FIG. 11 is a graph showing the effect of AII treatment on white blood cell number in the bone marrow on day 14 after 5FU treatment.

FIG. 12 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following spleen harvest 14 days after 5FU treatment.

FIG. 13 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following blood harvest 14 days after 5FU treatment.

FIG. 14 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following blood harvest 14 days after 5FU treatment.

FIG. 15 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following spleen harvest 14 days after 5FU treatment.

FIG. 16 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following bone marrow harvest 7 days after 5FU treatment.

FIG. 17 is a graph showing the effect of AII treatment on CFU-GM cell number on day 7 after culture initiation following femur harvest 7 days after 5FU treatment.

FIG. 18 is a graph showing the effect of AII treatment on CFU-GM cell number in the bone marrow on day 7 after 5FU treatment.

FIG. 19 is a graph showing the effect of AII treatment on CFU-GM cell number in the spleen on day 7 after 5FU treatment.

FIG. 20 is a graph showing the effect of AII treatment on white blood cell number in the blood on day 14 after 5FU treatment.

FIG. 21 is a graph of a different experiment showing the effect of AII treatment on white blood cell number in the blood on days 4, 7, and 10 after 5FU treatment.

FIG. 22 is a graph showing the effect of AII(1 7) treatment on white blood cell number in the blood on day 14 after 5FU treatment.

FIG. 24 is a graph showing the effect of 2GD treatment on white blood cell number in the blood on day 14 after 5FU treatment.

FIG. 25 is a graph showing the effect of 5GD treatment on white blood cell number in the blood on day 14 after 5FU treatment.

FIG. 26 is a graph showing the effect of 9GD treatment on white blood cell number in the blood on day 14 after 5FU treatment.

FIG. 27 is a graph showing the effect of 10 .mu.g AII and AII analogues and fragments on GM-CFU numbers in the bone marrow on day 10 after 5FU treatment.

FIG. 28 is a graph showing the effect of 100 .mu.g AII and AII analogues and fragments on GM-CFU numbers in the bone marrow on day 10 after 5FU treatment.

FIG. 29 is a graph showing the effect of 10 .mu.g AII and AII analogues and fragments on GM-CFU numbers in the blood on day 10 after 5FU treatment.

FIG. 30 is a graph showing the effect of 100 .mu.g AII and AII analogues and fragments on GM-CFU numbers in the blood on day 10 after 5FU treatment.

FIGS. 31a and b Female C57B1/6 mice, 6 8 weeks old, were treated with 200 mg/kg cytoxan by intravenous injection. Two days after (panel a) and two days before (panel b) cytoxan injection, subcutaneous administration of AII(1 7) was initiated. Various times after cytoxan injection, the animals were necropsied and peripheral blood harvested. The number of white blood cells was counted by hematocytometer after red blood cell lysis. An asterisk indicates a result significantly different from saline control (p.ltoreq.0.05). These data are mean and standard error of 4 animals per group (dose and time point).

FIGS. 32a and b Female C57B1/6 mice, 6 8 weeks old, were treated with 200 mg/kg cytoxan by intravenous injection. Two days after and two days before cytoxan injection, subcutaneous administration of AII(1 7) was initiated. Twenty eight after cytoxan injection, the animals were necropsied and bone marrow (panel a) or peripheral blood (panel b) harvested. The number of GM-CFU formed from cells isolated from bone marrow or peripheral blood after red blood cell lysis by culturing in semi solid medium containing recombinant colony stimulating factors was counted. An asterisk indicates a result significantly different from saline control (p.ltoreq.0.05). These data are mean and standard error of 3 animals per group (dose and time point).

FIG. 33 Effect of AII analogues and enalapril on the increase in white blood cells in the peripheral blood after intravenous administration of 5FU.

FIG. 34 Effect of AII analogues and enalapril on the increase in white blood cells in the peripheral blood after intravenous administration of 5FU.

FIG. 35 Effect of AII analogues and enalapril on the increase in white blood cells in the peripheral blood after intravenous administration of 5FU.

FIG. 36 Effect of AII analogues and enalapril on platelet increase after intravenous administration of 5FU.

FIG. 37 Effect of AII analogues and enalapril on platelet increase after intravenous administration of 5FU.

FIG. 38 Effect of AII analogues and enalapril on platelet increase after intravenous administration of 5FU.

FIG. 39 Effect of AII analogues and enalapril on hemoglobin increase after intravenous administration of 5FU.

FIG. 40 Effect of AII analogues and enalapril on hemoglobin increase after intravenous administration of 5FU.

FIG. 41 Effect of AII analogues and enalapril on hemoglobin increase after intravenous administration of 5FU.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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, Calif.), "Guide to Protein Purification" in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109 128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

As defined herein the phrase "hematopoietic cells" refers to undifferentiated hematopoietic stem cells, to committed hematopoietic progenitor cells, and to differentiated hematopoietic cells including, but not limited to megakaryocytes, platelets, red blood cells, monocytes, neutrophils macrophages, and lymphocytes.

As defined herein, "chemotherapy side effects" encompass any deleterious effects suffered as a result of chemotherapy, including but not limited to hematopoietic toxicity, decreased mobilization of hematopoietic progenitor cells from bone marrow into the peripheral blood; anemia, myelosuppression, pancytopenia, thrombocytopenia, neutropenia, lymphopenia, leukopenia, stomatitis, alopecia, headache, and muscle pain.

Unless otherwise indicated, the term "angiotensin converting enzyme inhibitors" or "ACE inhibitors" includes any compound that inhibits the conversion of the decapeptide angiotensin I to angiotensin II, and include but are not limited to alacepril, alatriopril, altiopril calcium, ancovenin, benazepril, benazepril hydrochloride, benazeprilat, benzazepril, benzoylcaptopril, captopril, captopril-cysteine, captopril-glutathione, ceranapril, ceranopril, ceronapril, cilazapril, cilazaprilat, converstatin, delapril, delapril-diacid, enalapril, enalaprilat, enalkiren, enapril, epicaptopril, foroxymithine, fosfenopril, fosenopril, fosenopril sodium, fosinopril, fosinopril sodium, fosinoprilat, fosinoprilic acid, glycopril, hemorphin-4, idapril, imidapril, indolapril, indolaprilat, libenzapril, lisinopril, lyciumin A, lyciumin B, mixanpril, moexipril, moexiprilat, moveltipril, muracein A, muracein B, muracein C, pentopril, perindopril, perindoprilat, pivalopril, pivopril, quinapril, quinapril hydrochloride, quinaprilat, ramipril, ramiprilat, spirapril, spirapril hydrochloride, spiraprilat, spiropril, spiropril hydrochloride, temocapril, temocapril hydrochloride, teprotide, trandolapril, trandolaprilat, utibapril, zabicipril, zabiciprilat, zofenopril and zofenoprilat. (See for example. Jackson, et al., Renin and Angiotensin in Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th ed., eds. Hardman, et al. (McGraw Hill, 1996); and U.S. Pat. No. 5,977,159.)

U.S. Pat. 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 that is sufficient for said increase. The application of AII to wound tissue significantly increases the rate of wound healing, leading to amore 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 (Clouston et al., Genomics 2:240 248 (1988); Kageyama et al, Biochemistry 23:3603 3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196 2200 (1983); each reference hereby incorporated in its entirety). The substance so formed is a decapeptide called angiotensin I (AI) which is converted to AII by the angiotensin converting enzyme (ACE) which removes the C-terminal His-Leu residues from AI [SEQ ID NO: 37]. AIII is a known pressor agent and is commercially available.

Studies have shown that AII 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. Pat. No. 5,015,629; Dzau et. al., J. Mol Cell. Cardiol. 21:S7 (Supp III) 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 19--(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 (1990). In addition, AII 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).

We have previously demonstrated that angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments or analogues thereof, and AII AT.sub.2 type 2 receptor agonists are effective in accelerating wound healing and the proliferation of certain cell types, such as hematopoietic stem and lineage specific cells. See, for example, co-pending U.S. patent application Ser. No. 09/012,400, filed Jan. 23, 1998; Ser. No. 09/198,806 filed Nov. 24, 1998; Ser. No. 09/264,563, filed Mar. 8, 2000; 09/287,674, filed Apr. 7, 1999; Ser. No. 09/255,136 filed Feb. 19, 1999; Ser. No. 09/245,680, filed Feb. 8, 1999; Ser. No. 09/250,703 filed Feb. 15, 1999; Ser. No. 09/246,525 filed Feb. 8, 1999; Ser. No. 09/266,293 Mar. 11, 1999; Ser. No. 09/332,582 filed Jun. 14, 1999; Ser. No. 09/373,962 filed Aug. 13, 1999; and Ser. No. 09/352,191 filed Jul. 12, 1999; as well as U.S. patent Ser. Nos. 5,015,629; 5,629,292; 5,716,935; 5,834,432; and 5,955,430; 6,096,709; 6,110,895.

Angiotensin II and its sarcosine analogue have also been used in combination with cytotoxic drugs to induce hypertension in humans and experimental animals undergoing intra-arterial and intraperitoneal chemotherapy. (Taniguchi et al., J. Nuclear Medicine 37:1522 1523 (1996); Morita et al., Am. J. Clin. Oncol. 15:188 193 (1992); Ohigashi et al., Hepato-Gastroenterology 43:338 345 (1996); Cancer Chemother. Pharmacol. 39:113 121 (1996); Kuroiwa et al., Cancer Chemother. Pharmacol. 35:357 363 (1995); Li et al., Br. J. Cancer 67:975 980 (1993); Dworkin et al., Br, J. Cancer 76:1205 1210 (1997); Sato et al., World J. Surg. 19:836 842 (1995); Mutoh et al., Urol. Int. 48:175 180 (1992). In each of these cases, the use of angiotensin II was intended to selectively increase blood flow to the tumor vasculature relative to normal vasculature, thereby increasing the delivery of cytotoxic agent to the tumor. None of these studies demonstrated or suggested that the use of angiotensin II or its sarcosine analogue would be effective in increasing hematopoietic cell survival, hematopoietic stem cell mobilization into peripheral blood following chemotherapy, or the reduction in chemotherapy side effects.

Based on all of the above, it would be unexpected that the use of angiotensinogen, angiotersin I (AI), AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof, AII AT.sub.2 type 2 receptor agonists, or ACE inhibitors would be effective in increasing hematopoietic cell survival following chemotherapy, for reducing or preventing other side effects of chemotherapy, such as anemia, and for mobilizing hematopoietic progenitor cells from bone marrow into peripheral blood.

A peptide agonist selective for the AT2 receptor (AII has 100 times higher affinity for AT2 than AT1) has been identified. This peptide is p-aminophenylalanine 6-AII ["(p-NH.sub.2-Phe)6-AII)"], Asp-Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH.sub.2-Phe (Speth and Kim, BBRC 169:997 1006 (1990). This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93 97 (1994); Bryson, et al., Eur. J. Pharmacol. 225:119 127 (1992).

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 AII receptor subtypes (AT1 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 AII to evaluate their activity AII(1 7) elicits some, but not the full range of effects elicited by AII. 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. Phammacol. 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 a., Br. J. Pharmacol. 111:652 654 (1994).

Other data suggests that the AII fragment AII(1 7) acts through a receptor(s) that is distinct from the AT1 and AT2 receptors which modulate AII activity. (Ferrario et al., J. Am. Soc. Nephrol. 9:1716 1722 (1998); Iyer et al., Hypertension 31:699 705 (1998); Freeman et al., Hypertension 28:104 (1996); Ambuhl et al., Brain Res. Bull. 35:289 (199.4)). Thus, AII(1 7) activity on a particular cell type cannot be predicted based solely on the effect of AII on the same cell type. In fact, there is some evidence that AII(1 7) often opposes the actions of AII. (See, for example, Ferrario et al., Hypertension 30:535 541 (1997))

As hereinafter defined, a preferred class of AT2 agonists for use in accordance with the present invention comprises angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, AII, AII analogues, AII fragments or analogues thereof or AII AT.sub.2 type 2 receptor agonists having p-NH-Phe in a position corresponding to a position 6 of AII. 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, as well as compounds fused to the active agent to provide some further desired property.

The active AII analogues, fragments of AII and analogues thereof of particular interest in accordance with the present invention comprise a sequence of at least three contiguous amino acids of groups R.sup.1 R.sup.8 in the sequence of general formula I R.sub.1--R.sup.2--R.sup.3--R--R.sup.5--R.sup.6--R.sup.7--R.sup.8

wherein R.sup.1 is selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me.sup.2Gly, Pro, Bet, Glu(NH.sub.2), Gly, Asp(NH.sub.2) and Suc,

R.sup.2 is selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R.sup.3 is selected from the group consisting of Val, Ala, Leu, Lys, norLeu; Ile, Gly, Pro, Aib, Acpc and Tyr;

R.sup.4 is selected from the group consisting of Tyr, Tyr(PO.sub.3).sub.2, Thr, Ser, Ala, homoSer and azaTyr;

R.sup.5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R.sup.6 is selected from the group consisting of His, Arg or 6-NH.sub.2-Phe;

R.sup.7 is selected from the group consisting of Pro or Ala; and

R.sup.8 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr,

excluding sequences including R.sup.4 as a terminal Tyr group.

Particularly preferred combinations for R.sup.1 and R.sup.2 are Asp-Arg, Asp-Lys, Glu-Arg and Glu-Lys.

In alternate embodiments, the active agents comprise a sequence of at least four, five, six, seven, or eight contiguous amino acids of groups R.sup.1 R.sup.8 in the sequence of general formula I. In a further alternative, the active agents consist essentially of a sequence of at least four, five, six, seven, or eight contiguous amino acids of groups R.sup.1 R.sup.8 in the sequence of general formula I.

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.sup.6 is p-NH.sub.2-Phe. In a further preferred embodiment of all of the aspects of the invention the active agent comprises a sequence selected from the group consisting of angiotensinogen, 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, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42.

Particularly preferred embodiments of this class comprise the following sequences: AII (SEQ ID NO:1), AII or AII(2 8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AII(3 8), also known as des1-AII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(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-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(1 5), Asp-Arg-Val-Tyr-Ile [SEQ ID NO:9]; AII(1 4), Asp-Arg-Val-Tyr [SEQ ID NO:10]; and AII(1 3), Asp-Arg-Val [SEQ ID NO:11]. 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), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.

In a particularly preferred embodiment, the active agents of the present invention comprise an amino acid sequence of the following general formula: Asp-Arg-R1-R2-Ile-His-Pro-R3, wherein

R1 is selected from the group consisting of Val, Pro, Lys, Norleu, and Leu;

R2 is selected from the group consisting of Ala, Tyr, and Tyr(PO.sub.3).sub.2; and

R3 is Phe or is absent.

In a most particularly preferred embodiment, the active agent comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.

Another class of compounds of particular interest in accordance with the present invention are those comprising a sequence of the general formula II R.sup.2--R.sup.3--R.sup.4--R.sup.5--R.sup.6--R.sup.7--R.sup.8

in which R.sup.2 is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;

R.sup.3 is selected from the group consisting of Val, Ala, Leu, norLeu, Lys, Ile, Gly, Pro, Aib, Acpc and Tyr;

R.sup.4 is selected from the group consisting of Tyr, Tyr(PO.sub.3).sub.2, Thr, Ser, Ala homoSer and azaTyr;

R.sup.5 is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;

R.sup.6 is selected from the group consisting of His, Arg or 6-NH.sub.2-Phe;

R.sup.7 is selected from the group consisting of Pro or Ala; and

R.sup.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 R.sup.2--R.sup.3--Tyr-R.sup.5-His-Pro-Phe [SEQ ID NO:16]

wherein R.sup.2, R.sup.3 and R.sup.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 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-US-00001 TABLE 1 Abbreviation for Amino Acids Me.sup.2Gly N,N-dimethylglycyl Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide inner salt (betaine) Suc Succinyl Phe(Br) p-bromo-L-phenylalanyl azaTyr aza-.alpha.'-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.sup.3, R.sup.5 and R.sup.7 may be involved in maintaining the appropriate distance between active groups in positions R.sup.4, R.sup.6 and R.sup.8 primarily responsible for binding to receptors and/or intrinsic activity. Hydrophobic side chains in positions R.sup.3, R.sup.5 and R.sup.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.sup.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.sup.2.

For purposes of the present invention, it is believed that R.sup.3 may be involved in the formation of linear or nonlinear hydrogen bonds with R.sup.5 (in the gamma turn model) or R.sup.6 (in the beta turn model). R.sup.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.sup.3 may suitably be selected from Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr. Lys has also been found to be effective at position R.sup.3.

With respect to R.sup.4, conformational analyses have suggested that the side chain in this position (as well as R.sup.3 and R.sup.5) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors. Thus, R.sup.4 is preferably selected from Tyr, Thr, Tyr (PO.sub.3).sub.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). Ala has also been found to be effective at position R.sup.4.

In position R.sup.5, an amino acid with a .beta. aliphatic or alicyclic chain is particularly desirable. Therefore, while Gly is suitable in position R.sup.5, it is preferred that the amino acid in this position be selected from Ile, Ala, Leu, norLeu, Gly and Val.

In the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AII analogues, fragments and analogues of fragments of particular interest in accordance with the present invention, R.sup.6 is His, Arg or 6-NH.sub.2-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 R.sup.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.sup.7. Similarly, it is presently considered that R.sup.7 should be Pro in order to provide the most desirable orientation of R.sup.8. In position R.sup.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-US-00002 TABLE 2 Angiotensin II Analogues AII Analogue Sequence Name Amino Acid Sequence Identifier Analogue 1 Asp-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 19 Analogue 2 Asn-Arg-Val-Tyr-Val-His-Pro-Phe SEQ ID NO: 20 Analogue 3 Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His- SEQ ID NO: 21 Pro-Phe Analogue 4 Glu-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 22 Analogue 5 Asp-Lys-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 23 Analogue 6 Asp-Arg-Ala-Tyr-Ile-His-Pro-Phe SEQ ID NO: 24 Analogue 7 Asp-Arg-Val-Thr-Ile-His-Pro-Phe SEQ ID NO: 25 Analogue 8 Asp-Arg-Val-Tyr-Leu-His-Pro-Phe SEQ ID NO: 26 Analogue 9 Asp-Arg-Val-Tyr-Ile-Arg-Pro-Phe SEQ ID NO: 27 Analogue 10 Asp-Arg-Val-Tyr-Ile-His-Ala-Phe SEQ ID NO: 28 Analogue 11 Asp-Arg-Val-Tyr-Ile-His-Pro-Tyr SEQ ID NO: 29 Analogue 12 Pro-Arg-Val-Tyr-Ile-His-Pro-Phe SEQ ID NO: 30 Analogue 13 Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe SEQ ID NO: 31 Analogue 14 Asp-Arg-Val-Tyr(PO.sub.3).sub.2-Ile-His- SEQ ID NO: 32 Pro-Phe 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- SEQ ID NO: 35 Pro-Phe

The polypeptides of the instant invention may be produced by any standard method, including but not limited to recombinant DNA technology and conventional synthetic methods including, but not limited to, 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 (U.S. Pat. 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.

Preferably, peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art. Alternatively, the active agents can be prepared by standard recombinant DNA techniques.

In one aspect, the present invention provides methods and kits for increasing hematopoietic cell survival following chemotherapy, and treating and preventing the adverse effects of chemotherapy, comprising the administration of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AI), AII analogues, AII fragments or analogues thereof or AII AT.sub.2 type 2 receptor agonists (hereinafter referred to as "active agents") to a patient undergoing chemotherapy.

In another aspect, the present invention provides methods and kits for mobilizing hematopoietic progenitor cells from bone marrow into peripheral blood comprising the administration of the active agents of the invention to a patient in need of such treatment. This aspect of the invention can also be used to treat a patient in need of chemotherapy.

The methods of the invention are appropriate for use with chemotherapy using any cytotoxic agent, including, but not limited to, cyclophosphamide, taxol, 5-fluorouracil, adriamycin, cisplatinum, methotrexate, cytosine arabinoside, mitomycin C, prednisone, vindesine, carbaplatinum, and vincristine. The cytotoxic agent can also be an antiviral compound which is capable of destroying proliferating cells. For a general discussion of cytotoxic agents used in chemotherapy, see Sathe, M. et al., Cancer Chemotherapeutic Agents: Handbook of Clinical Data (1978), hereby incorporated by reference.

The methods of the invention are also particularly suitable for those patients in need of repeated or high doses of chemotherapy. For some cancer patients, hematopoietic toxicity frequently limits the opportunity for chemotherapy dose escalation. Repeated or high dose cycles of chemotherapy may be responsible for severe stem cell depletion leading to severe 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 chemotherapy.

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, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneally.

The active agents 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 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 (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.

The dosage regimen of active agents for the methods of the invention 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. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg body weight of the active agents per body weight are useful for all methods of use disclosed herein.

In all of these embodiments, the compounds of the invention can be administered prior to, simultaneously with, or subsequent to chemotherapeutic exposure.

In a preferred embodiment, the active agent is administered subcutaneously. A suitable subcutaneous dose of the active agent is preferably between about 0.1 ng/kg and about 10 mg/kg administered twice daily for a time sufficient to increase white blood cell survival after chemotherapy treatment or to mobilize hematopoietic progenitor cells from bone marrow into peripheral blood. 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 2.5 .mu.g/kg body weight and about 100 .mu.g/kg body weight. This dosage regimen maximizes the therapeutic benefits of the subject invention while minimizing the amount of agonist needed. Such an application minimizes costs as well as possible deleterious side effects. For example, the active agents are administered to an oncology patient for up to 30 days prior to a course of chemotherapy and for up to 60 days post-chemotherapy. The therapy is administered for to 6 times per day at dosages as described above. In a further preferred embodiment, the active agent is administered once per day.

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 a preferred embodiment, subcutaneous administration of between about 1 to 1000 .mu.g/kg/day of the active agents is initiated at between one week before to one week after administration of a chemotherapeutic agent. In a most preferred embodiment, administration of the active agents begins either at the time chemotherapy is initiated, or 0 10 days after initiation.

In another preferred embodiment of the invention, a subject undergoes repeated cycles of treatment according to the method of this invention. Preferably, a subsequent treatment cycle commences only after administration of the compounds of the invention has terminated, and the subject's blood cell counts (e.g., white blood cell count, as well as platelet and megakaryocyte count) have returned to a therapeutically acceptable level (as determined by the attending veterinarian or physician), permitting the repeated chemotherapy.

In a further aspect, the present invention provides kits for increasing hematopoietic cell survival following chemotherapy, reducing the incidence and/or severity of anemia and other side effects of chemotherapy, and/or mobilizing hematopoietic progenitor cells from bone marrow into peripheral blood wherein the kits comprise an effective amount of the active agents for increasing hematopoietic cell survival following chemotherapy, reducing chemotherapy-induced side effects, or for mobilizing hematopoietic progenitor cells from bone marrow into peripheral blood, 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 (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 the active agents, or may be separate from the compounds, which are then applied to the means for delivery at the time of use.

In a further embodiment, the present invention provides an article of manufacture, comprising the pharmaceutical composition of the invention preloaded into a syringe or other delivery system, for home use by patients undergoing chemotherapy.

In another aspect, the present invention provides a pharmaceutical composition, comprising an amount effective of the active agents to increase hematopoietic cell survival and/or to reduce the side effects of chemotherapy in a chemotherapy patient, and a pharmaceutically acceptable carrier. In a preferred embodiment, the active agent is AII(1 7), and the effective dosage is between about 2.5 .mu.g/kg/day and 100 .mu.g/kg/day.

In a further embodiment, the pharmaceutical composition further comprises an amount of cytokine effective for increasing the production of hematopoietic cells. According to this aspect of the invention, cytokines appropriate for use include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), epidermal growth factor (EGF), interleukin 11, erythropoietin, thrombopoietin, megakaryocyte development and growth factor, pixykines, stem cell factor, FLT-ligand as well as interleukins 1, 3, 6, and 7. In a most preferred embodiment, the cytokine is granulocyte colony stimulating factor.

The methods, kits, and pharmaceutical compositions of the present invention, significantly enhance the utility of presently available treatments for clinical chemotherapeutic treatments, by providing improved methods for increasing hematopoietic blood cell survival following chemotherapy, reducing the side effects of chemotherapy, and mobilizing hematopoietic progenitor cells from bone marrow into peripheral blood, and also by their cytokine sparing effect, in that significantly reduced amounts of cytokines are needed when administered with the active agents to a patient undergoing chemotherapy.

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 AII on White Blood Cell Mobilization and Recovery after 5 Fluorouracil Treatment

This study was designed to test the effect of AII on the recovery of white blood cells in lymphoid organs and on the level of granulocyte macrophage precursors (CFU-GM) in the blood (ie: mobilization), spleen (mobilization), and bone marrow (recovery) after intravenous administration of 5-fluorouracil (5FU).

Subcutaneous administration of AII (either 10 or 100 .mu.g/kg/day) was initiated either two days before (-d2), the day of (d0), or two days after (d2) intravenous administration of 5FU. On either day 7 or 14 after 5FU administration, the animals were necropsied and the spleen, thymus, peripheral blood, and bone marrow were harvested. The number of white blood cells in each of the lymphoid organs, or the number of CFU-GM present in all the organs except the thymus, were then assessed. The number of white blood cells per lymphoid organ was assessed after (1) dissociation of the tissues into a single cell suspension (thymus and spleen), (2) flushing of bone marrow from the femur, or (3) lysis of red blood cells by a hypotonic ammonium chloride solution (blood). An aliquot of the cell suspension was diluted with 0.04% trypan blue and the number of cells was determined by microscopic analysis using a hematocytometer. After counting, the number of cells were adjusted to allow a 1:10 dilution of cells into semi-solid medium containing fetal bovine serum, bovine serum albumin methyl cellulose, stem cell factor, interleukin 3, interleukin 6, L-glutamine, 2 mercaptoethanol, human transferrin and bovine insulin. On day 7 after culture initiation, the number of CFU-GM per well (and then per organ) was determined by microscopic analysis (FIGS. 1 20). These data demonstrate that AII treatment after chemotherapy leads to significantly enhanced white blood cell mobilization and/or recovery in all of the tissues tested.

EXAMPLE 2

Effect of AII Analogues and Fragments on White Blood Cell Mobilization and Recovery after 5Fluorouracil Treatment

The method was conducted as described above in Example 1, except that mice were injected subcutaneously with 150 mg/kg body weight of 5FU, and AII peptide analogues and fragments were tested. Administration of the peptides (see Table 3) was begun 2 days after and continued until 10 days after 5FU administration, at which time-the mice were euthenized for evaluation of bone marrow and blood GM-CFU progenitors. On days 4 and 7 after 5FU administration, blood was taken under anesthesia from the retro-orbital sinus. On day 10, blood was taken by cardiac puncture.

The data for these experiments is shown in FIGS. 21 30, and demonstrate that all peptides tested accelerated the recovery of white blood cells after chemotherapy (FIGS. 21 26), increased the number of GM-CFU progenitors in the bone marrow (FIGS. 27 28), and increased the mobilization of GM-CFU progenitors from the bone marrow into the peripheral blood (FIGS. 29 30), relative to controls. The peptides were effective at both concentrations tested (10 .mu.g/kg/day and 100 .mu.g/kg/day), and the efficacy generally increased with increasing length of treatment.

TABLE-US-00003 TABLE 3 Designation for Analogues/Fragments Name Abbreviation Sequence SEQ ID NO: 1GD Ala4-AII(1 7) DRVAIHP SEQ ID NO:38 2GD Pro3-AII(1 7) DRPYIHP SEQ ID NO:39 5GD Lys3-AII(1 7) DRKYIHP SEQ ID NO:40 9GD NorLeu-AII(1 7) DR(nor)YIHP SEQ ID NO:41 AII(1 7) DRVYIHP-- SEQ ID NO:4 AII DRVYIHPF SEQ ID NO:1

EXAMPLE 3

Effect of AII(1 7) on Hematopoietic Recovery after Cytoxan

In this study, female C57B1/6 mice were injected with 200 mg/kg cyclophosphamide ("cytoxan") intravenously. Administration of AII(1 7) by subcutaneous injection was begun 2 days before or 2 days after administration of the antineoplastic and continued daily until necropsy for evaluation of mature formed blood elements in the circulation and the number of GM-CFU in the bone marrow and peripheral blood. On days 5, 9, 14, 21 and 28 after cytoxan administration, blood was taken under metofane anesthesia from the retro-orbital sinus to assess white blood cell (WBC) and platelet number.

The data showed that AII(1 7) accelerated the recovery of WBC after intravenous administration of cyclophosphamide when treatment with peptides was initiated two days after the chemotherapeutic (FIG. 31a). The increase in WBC number was observed within 9 days after cyclophosphamide treatment. However, if AII(1 7) treatment was initiated two days prior to the administration of cyclophosphamide, there was a decrease in WBC number compared with control starting on day 14 after exposure to the chemotherapeutic (FIG. 31b).

Administration of cyclophosphamide also significantly reduced the number of myeloid progenitors (GM-CFU) in the bone marrow. Treatment with AII(1 7) (100 .mu.g/kg/day) 2 days prior to treatment with cytoxan slightly reduced the number of GM-CFU in the bone marrow of treated animals (FIG. 32a). However, initiation of AII(1 7) administration 2 days after chemotherapy increased the number of myeloid progenitor cells in the bone marrow (FIG. 32a). Further, administration of AII(1 7), initiated after cyclophosphamide treatment, increased the number of myeloid progenitors in the peripheral blood (FIG. 32b).

EXAMPLE 4

Effect of Angiotensin Fragments and Ace Inhibitor on Recovery after Chemotherapy

The next study was designed to test the effect of angiotensin peptides and the angiotensin converting enzyme inhibitor, enalapril on recovery of white blood cells, platelets and hemoglobin after chemotherapy, as well as on granulocyte-macrophage colony forming units (GM-CFU) in the blood and bone marrow.

Female C67B1/6 mice (4 per group), 6 8 weeks old, were injected with 150 mg/kg 5FU intravenously. Administration of the peptides (AII (100 .mu.g/kg/day), AII(1 7) (10 or 100 .mu.g/kg/day), AII(1 5) (10 or 100 .mu.g/kg/day), and AII(1 6) (100 .mu.g/kg/day)), by subcutaneous injection, or enalapril (30 mg/kg/day) by oral gavage, was begun 2 days after administration of the antineoplastic and continued daily until 28 days after 5FU administration, at which time the mice were euthanized for evaluation of white blood cell numbers, platelets, hemoglobin levels and the number of GM-CFU in the bone marrow and peripheral blood. On days 7, 10, 14 and 21 after 5FU administration blood was taken under anesthesia from the retro-orbital sinus to assess white blood cell number, platelet number, and hemoglobin levels.

Retro-Orbital Bleeding of Mice

The mice were bled from the retro-orbital sinus at days 7, 10, 14 and 21. The mice were anesthetized with Metofane (an inhaled anesthesia). Approximately 150 200 .mu.l of blood were obtained from the retro-orbital sinus with a heparinzed capillary tube. The blood was then placed in a 1.7 ml microfuge tube containing 10 mM EDTA and held on ice until further processing.

Hemoglobin Assay

One hundred .mu.l of blood was pipetted into a centrifuge tube, to which 900 .mu.l of distilled water was added. The blood and water were mixed by inversion and allowed incubate at 4.degree. C. for 20 minutes. The tube was then centrifuged at 12000 rpm to precipitate the cellular debri for 20 to 30 minutes at room temperature. Twenty .mu.l of the supernatant from this centrifugation was then added into triplicate wells of a 96 well plate containing 180 .mu.l of distilled water. The optical density was then read using a microplate reader at 570 nM.

WBC and Platelet Evaluation

Twenty .mu.l of blood was mixed with 200 .mu.l of red blood cell (RBC) lysing solution (0.83% NH.sub.4Cl, 10 mm EDTA, 0.5% NaHCO.sub.3). The mixture was then incubated for 10 minutes at 4.degree. C. After this incubation, the supernatant was removed and the pellet was resuspended in 100 .mu.l of PBS. To this, 100 .mu.l of 0.04% trypan blue was added. This mixture was vortexed and the number of WBC (baseline was approximately 10.sup.7 cells/ml) was evaluated by hematocytometer under light microscopy and the number of platelets (baseline was approximately 2.5.times.10.sup.8 platelets/ml) was evaluated by hematocytometer under phase contrast microscopy.

Evaluation of GM-CFU Progenitors in the Blood and Bone Marrow

The blood was harvested by cardiac puncture on day 10 to assess mobilization of myeloid progenitors into the peripheral blood. The femurs and tibia were also collected and the bone marrow was harvested by flushing with PBS containing 2% fetal calf serum. After collection of the blood and bone marrow, the red blood cells were lysed with a hypotonic solution (described above), mixed with 0.04% trypan blue, and the number of nucleated cells assessed by hematocytometer under light microscopy. Aliquots of cells were then resuspended at 1.times.10.sup.5 cells/ml (bone marrow) or 1.times.10.sup.6 cells/ml (blood). One hundred .mu.l of each suspension was added to 900 .mu.l of semisolid medium containing 0.9% methyl cellulose in Iscove's MDM, 15% fetal calf serum, 1% bovine serum albumin, 10 .mu.g/ml bovine pancreatic insulin, 200 .mu.g/ml human transferrin, 10.sup.-4 M 2-mercaptoethanol, 2 mM glutamine, 10 ng/ml recombinant murine interleukin 3, 10 ng/ml recombinant human