METHODS OF TREATING BLOOD CELL DEPLETION

This application claims priority to U.S. Provisional Application Serial No. 60/952,691, filed July 30, 2007, U.S. Provisional Application Serial No. 60/965,580, filed August 20, 2007, U.S. Provisional Application Serial No. 60/966,948, filed August 29, 2007, U.S. Provisional Application Serial No. 61/039,860, filed March 27, 2008, and U.S. Provisional Application Serial No. 61/039,866, filed March 27, 2008, and claims priority under 35 U.S. C. sections 120, 363 and/or 365 to co-pending International Application

PCT/US filed on even date herewith (also known as Attorney Docket No.

IRB-004PC) each of which is incorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION

[0001] The methods provided herein relate to the field of hematology. In particular, the methods relate to the replenishment of blood cells in a mammal after depletion of blood cells from various causes such as exposure to therapeutic radiation and therapeutic drugs.

2. BACKGROUND OF THE INVENTION

[0002] Blood cells, including red blood cells, white blood cells, macrophages, platelets and the like are vital for a number of functions including proper oxygenation of tissues, fighting infections, and proper blood clotting. The depletion of blood cells in mammals can be caused by a number of events including illness, exogenous substances (e.g., drugs or pharmaceuticals), or radiation exposure.

[0003] Vast improvements have been made in the last century in drug treatments of illnesses. However, many drugs have adverse effect that can be life threatening. For example, drugs for treating cancer often cause severe myelosuppression. In some instances, the adverse effects of a drug are severe enough that the drug therapy must be discontinued. Methods of decreasing or ameliorating the adverse effects of medications are needed.

[0004] Human exposure to ionizing radiation, which causes cell damage by disruption of DNA, occurs in a variety of settings including, treatment or amelioration of cancer. However, even non-lethal exposures to radiation, for example, in a cancer treatment

setting, can cause myelosuppression. Methods replenishing blood cells in human receiving therapeutic radiation are also needed.

3. SUMMARY OF THE INVENTION

[0005] The methods and compositions provided herein can be used for reducing or ameliorating an adverse effect of a drug or medication in an animal, comprising administering to an animal substance P or a substance P analog in an amount effective to decrease or ameliorate one or more adverse effects of a drug or medication in the animal. In a preferred embodiment, the adverse effect is one or more blood dyscrasias. [0006] In certain embodiments, the methods and compositions provided herein can be used to prevent, treat, or ameliorate disorders using substance P or substance P analogs in animals, including humans, associated with radiation exposure, including therapeutic radiation. The methods and compositions provided herein are based, in part, on the Applicants' surprising discovery that certain substance P analogs can increase blood cell levels in an animal following exposure of the animal to sub-lethal doses of radiation. [0007] In certain embodiments, the methods and compositions provided herein can be used to stimulate blood cell regeneration in a mammal exposed to radiation by administering a therapeutic amount of substance P or a substance P analog. In one embodiment, a therapeutic amount of substance P or a substance P analog can be administered to stimulate or promote the development and mobilization of granulocytes in a human. [0008] In a preferred embodiment, the methods and compositions can be used to regenerate granulocytes after therapeutic radiation by administering a therapeutic amount of a substance P analog.

[0009] In certain embodiments, the methods and compositions provided herein can be used to reduce poly(ADP-ribose) polymerase (PARP) activity in an animal comprising administering to an animal substance P or a substance P analog in an amount effective to reduce poly(ADP-ribose) polymerase (PARP) activity in the animal. In certain embodiments, the methods and compositions provided herein can be used to reduce poly(ADP-ribose) polymerase (PARP) expression in an animal.

[0010] In another embodiment, the methods and compositions provided herein can be used to treat radiation illness by administering a therapeutically effective amount of a substance

P analog to an animal in need thereof. In certain embodiments, such administration reduces poly(ADP-ribose) polymerase (PARP) activity in the animal. In one embodiment, the animal is a mammal. In a preferred embodiment, the animal is human.

[0011] In one embodiment, reduction of PARP activity can be assessed by levels of nicotinamide adenine dinucleotide (NAD), poly(ADP-ribosyl)ation (PAR), loss of cell membrane integrity or morphological indicators of apoptosis or necrosis.

[0012] In certain embodiments, substance P analog comprises substance P (SEQ ID

NO.:1).

[0013] In certain embodiments, the substance P analog is of Formula (I):

Z ₁ -Xaa ¹ -Xaa ² -Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ^{1 I} -Z ₂ (I) wherein:

Xaa ¹ is Arg, Lys, 6-N methyllysine or (6-N, 6-N) dimethyllysine;

Xaa ² is Pro or Ala;

Xaa ³ is Lys, Arg, 6-N-methyllysine or (6-N, 6-N) dimethyllysine;

Xaa ⁴ is Pro or Ala;

Xaa ⁵ is GIn or Asn;

Xaa ⁶ is GIn or Asn;

Xaa ⁷ is Tyr, Phe or Phe substituted with chlorine at position 2,3 or 4;

Xaa ⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa ⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa ¹⁰ is Leu, VaI, He, Norleucine, Met, Met sulfoxide, Met sulfone, N- methylleucine, or N-methylvaline;

Xaa ¹ ' is Met, Met sulfoxide, Met sulfone or Norleucine; Zi is R ₂ N- or RC(O)NR-; Z ₂ is -C(O)NR ₂ or -C(O)OR or a salt thereof; each R is independently R is H, (Ci -C ₆ ) alkyl, (Ci -C ₆ ) alkenyl, (Ci -C ₆ ) alkynyl, (C5 -C ₂₀ ) aryl, (C ₆ -C ₂₆ ) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa ¹ through Xaa ^{1 x} independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

In certain embodiments, the substance P analog is of Formula (I):

Z , -Xaa ¹ -Xaa ² -Xaa ³ -Xaa ⁴ -Xaa ^s -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa' °-Xaa' '-Z ₂ (I) wherein:

Xaa ¹ is Arg;

Xaa ² is Pro;

Xaa ³ is Lys;

Xaa ⁴ is Pro;

Xaa ⁵ is GIn;

Xaa ⁶ is GIn;

Xaa ⁷ is Tyr, Phe or Phe substituted with chlorine at position 4;

Xaa ⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4;

Xaa ⁹ is GIy, Pro, or N-methylglycine;

Xaa ¹⁰ is Leu; and

Xaa ¹¹ is Met, Met sulfoxide, Met sulfone or Norleucine; Zi is H ₂ N- or HC(O)NH-; and Z ₂ is -C(O)NH ₂ or -C(O)OH or a salt thereof.

[0014] In certain embodiments, the substance P analog is selected from the group consisting of

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO.: 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO.: 9); and

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[0015] In an even more preferred embodiment, the substance P analog can be Z,-RPKPQQFFMeGIyLM(O ₂ )-Z ₂ ; wherein Z, is NH ₂ and Z ₂ is C(O)NH ₂ .

[0016] In one embodiment, the animal is a mammal. In a preferred embodiment, the animal is a human.

4. DESCRIPTION OF THE FIGURES

[0017] Fig. 1 provides a graph of the colony count of BFU-E vs. concentration for

HOMSPERA® (Sar ⁹ -SP) and substance P (SP).

[0018] Fig. 2 provides a graph of the colony count of CFU-E vs. concentration for

HOMSPERA® (Sar ⁹ -SP) and substance P (SP).

Fig. 3 provides a graph of the colony count of CFU-GM vs. concentration for

HOMSPERA® (Sar ⁹ -SP) and substance P (SP).

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 DEFINITIONS

[0019] The terms "adverse effect," "adverse reaction," "adverse drug event," and "adverse drug reaction" refer to any unexpected or dangerous reaction to a drug or an unwanted effect caused by the administration of a drug or medication. The onset of an adverse reaction can be sudden or develop over time.

[0020] The term "alkyl" refers to a saturated branched, straight chain, or cyclic hydrocarbon radical. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like. In preferred embodiments, the alkyl groups are (Ci -C ₆ ) alkyl.

[0021] The term "alkenyl" refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond. The radical may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert- butenyl, pentenyl, hexenyl and the like. In preferred embodiments, the alkenyl group is (C, -C ₆ ) alkenyl.

[0022] The term "alkynyl" refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon triple bond. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl, and the like. In preferred embodiments, the alkynyl group is (Ci -C ₆ ) alkynyl.

[0023] The term "aryl" refers to an unsaturated cyclic hydrocarbon radical having a conjugated π electron system. Typical aryl groups include, but are not limited to, penta- 2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, chrysenyl, coronenyl, fluoranthenyl, indacenyl, idenyl, ovalenyl, perylenyl, phenalenyl, phenanthrenyl, picenyl, pleiadenyl, pyrenyl, pyranthrenyl, rubicenyl, and the like. In preferred embodiments, the aryl group is (C ₅ -C ₂₀ ) aryl, with (C ₅ -C ₁₀ ) being particularly preferred. [0024] The term "alkaryl" refers to a straight-chain alkyl, alkenyl or alkynyl group wherein one of the hydrogen atoms bonded to a terminal carbon is replaced with an aryl moiety. Typical alkaryl groups include, but are not limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl, naphthenobenzyl and the like. In preferred embodiments, the alkaryl group is (C ₆ -C2 ₆ ) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (Ci -C ₆ ) and the aryl moiety is (C ₅ -C2 ₀ ). In particularly preferred embodiments, the alkaryl group is (C ₆ -C ₁₃ ) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (Ci -C ₃ ) and the aryl moiety is (C ₅ -C ₁₀ ).

[0025] The term "alkheteroaryl" refers to a straight-chain alkyl, alkenyl or alkynyl group where one of the hydrogen atoms bonded to a terminal carbon atom is replaced with a heteroaryl moiety. In preferred embodiments, the alkheteroaryl group is 6-26 membered alkheteroaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkheteroaryl is (Ci -C ₆ ) and the heteroaryl is a 5-20-membered heteroaryl. In particularly preferred embodiments the alkheteroaryl is 6-13 membered alkheteroaryl, i.e., the alkyl, alkenyl or alkynyl moiety is a 5-10 membered heteroaryl.

[0026] The term "drug" or "medication" refers to any article, substance, compound or composition intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or any article, substance, compound, or composition intended to affect the structure or any function of an animal, or any article, substance, compound, or composition that can modify a chemical process or processes in the body. Drugs or medication can be either prescription or nonprescription pharmaceutical composition, a biologically-derived composition or product such as a vaccine, serum, or blood derived product, tissues or organs, or radiopharmaceuticals. The term "drug" also refers to an illegal or illicit substance or compound, including, for example, marijuana, methamphetamine, heroin, and the like.

[0027] The term "drug induced blood dyscrasia" refers to an abnormality in the blood or bone marrow cellular components caused, induced, or precipitated by or believed to be caused, induced or precipitated by a drug or medication.

[0028] The term "heteroaryl" refers to an aryl moiety wherein one or more carbon atoms is replaced with another atom, such as N, P, O, S, As, Se, Si, or Te. Typical heteroaryl groups include, but are not limited to, acridarsine, acridine, arsanthridine, arsindole, arsindoline, carbazole, β-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isoarsindole, isoarsinoline, isobenzofuran, isochromene, isoindole, isophosphoindole, isophosphinoline, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phosphoindole, phosphinoline, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, selenophene, tellurophene, thiophene, and anthenes. In preferred embodiments, the heteroaryl group is a 5-20 membered heteroaryl, with 5-10 membered aryl being particularly preferred. [0029] The term "side effect" refers to any problem that occurs when treatment goes beyond the desired effect or any undesired effect that occurs in addition to the desired effect. For example, excessive bleeding caused by the anticoagulant heparin is a side effect of heparin. Fatigue, nausea, vomiting, decreased blood cells counts, hair loss and mouth sores are instances of side effects that occur during cancer treatment in addition to the desired therapeutic effect. As used herein a "side effect" is an "adverse effect." [0030] The term "substituted alkyl, alkenyl, alkynyl, aryl alkaryl, heteroaryl or alkheteroaryl" refers to an alkyl, alkenyl, alkynyl, aryl, alkaryl, heteroaryl or alkheteroaryl group in which one or more hydrogen atoms is replaced with another substituent. Preferred substituents include -OR, -SR, -NRR, -NO ₂ , -CN, halogen, -C(O)R, -C(O)OR and -C(O)NR, where each R is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, alkaryl, heteroaryl, or alkheteroaryl.

[0031] The term "therapeutic radiation" refers to the use of ionizing radiation for curative or palliative treatment of cancer.

[0032] The term "treatment" refers to treating, ameliorating, or reducing the severity or duration of signs or symptoms of sickness, injury, disease or illness in an animal. 5.2 METHODS OF TREATMENT

[0033] Substance P has been shown to induce the production of hematopoietic growth factors. Rameshwar, et al., 1993, Blood, 81(2): 391-398. However, Applicant's have surprisingly discovered that substance P analogs can induce proliferation and differentiation of hematopoietic cells to a greater extent than native substance P. Furthermore, the hematopoietic effects of certain substance P analogs can be achieved at concentrations much lower than native substance P.

5.2.1 Methods of Treating Radiation Exposure

[0034] In certain embodiments, substance P analogs provided herein can be used to treat therapeutic radiation illness in mammals, including humans, for which increasing blood cells is beneficial. Treatment in the methods provided herein can be administration of a substance P analog to a mammal after the mammal has been exposed to therapeutic radiation but the mammal does not yet show signs or symptoms of radiation sickness. In one embodiment, treatment of the mammal in need thereof can be after the mammal has been exposed to therapeutic radiation but the mammal shows signs or symptoms of radiation sickness.

[0035] In a preferred embodiment, the methods provide for treating anemia, leukocytopenia, or thrombocytopenia in a mammal in need thereof by administering a substance P analog. In another preferred embodiment, the methods provide for treating anemia, leukocytopenia, or thrombocytopenia in a mammal in need thereof by administering [SaT ⁹ MeI(O) ₂ ¹¹ J- substance P (Homspera®, SEQ ID NO :10). [0036] In one embodiment, the methods provide for prevention, treatment or amelioration of myelosuppression induced or caused by therapeutic radiation. Therapeutic radiation can be any use of ionizing radiation to treat, prevent or ameliorate a condition in an animal. Therapeutic radiation is most commonly used in the field of radiation oncology to treat and possibly eradicate cancer from an animal or as a palliative by reducing tumor load in an animal.

[0037] The radiation therapy can be any supplied by any means known in the art. In one embodiment the therapeutic radiation can be external beam radiation. In another embodiment, the radiation is brachytherapy. In another embodiment, the radiation is an unsealed source radiation.

[0038] Radiation doses used in radiation therapy can vary depending on a number of factors including, for example, the type and stage of cancer, whether the patient will receive or has received chemotherapy, whether the patient will undergo or has undergone surgery. Total doses for solid tumors tend to be about 60 to 80 Gy down to about 20 Gy for lymphomas.

[0039] Radiation doses are fractionated, e.g., administered over a period of time to allow the non-cancerous cells to recover. Fractionation regimes vary widely depending on the type of cancer, anatomical region to be irradiated, overall health of the individual, equipment availability and the like. There is little standardization. For example, radiation therapy schedules for treatment of metastatic spinal cord compression can be 1 x 8 Gy in 1 day, 5 x 4 Gy in 1 week, 10 x 3 Gy in 2 weeks, 15 x 2.5 Gy in 3 weeks or 20 x 2 Gy in 4 weeks. Rades, et al, 2005, J. Clin. Oncol. 23(15): 3366-75. See also, Lukka, et al, 2005, J. Clin. Oncol. 23(25): 6132-38, Fujii, et al, 2008, Breast Cancer 15(1): 86-92, Butler, et. al, 1999, Int. J. Radiat. Oncol. Biol. Phys. 45(1): 21-32. Supportive Care Guidelines Group. Radiotherapy fractionation for the palliation of uncomplicated painful bone metastases. Toronto (ON): Cancer Care Ontario (CCO); 2003 Mar. 22 p. (Practice guideline; no. 13-2).

[0040] In certain embodiments, substance P analogs provided herein can promote or stimulate the production of blood cells following exposure to non-lethal, therapeutic radiation. In one embodiment, the blood cells can be erythrocytes, leukocytes, granulocytes, neutrophils, monocytes or platelets.

[0041] In certain embodiments, determining counts of blood cell types, classes, subclasses and subtypes can be done according to any method known in the art. Many such methods are automated and are provided in a standard complete blood count. Parameters of the blood which can be determined and used for monitoring include red blood cell count, white blood cell count, platelet count, hemoglobin concentration, hematocrit, MCV, red blood cell size, count of any blood cell type or subtype, etc. The determining of counts or other blood parameters can be performed before and/or after treatment and can be performed multiple times for continued monitoring.

[0042] Red blood cells express hemoglobin which binds to oxygen and transports it around the body. Proper oxygenation of tissues is dependent on the amount and function of red

blood cells in general, and hemoglobin in particular. For humans, the normal range of red blood cell counts is 4.7 to 6.1 x 10 ⁶ cells/microliter for males and 4.2 to 5.4 x 10 ⁶ cells/microliter for females. Platelets are required for proper clotting of blood. For humans, normal values for platelets are 15,000 to 400,000/mm ³ .

[0043] Leukocytes or white blood cells are required for defense against infectious agents, such as bacteria, fungi, parasites, and viruses. Leukocytes include granulocytes (i.e., neutrophils, eosinophils and basophils), band cells, T lymphocytes, B lymphocytes, and monocytes.

[0044] Hematocrit is the percentage of red blood cells in whole blood. It is a measure of the number and size of erythrocytes. Normal values of hematocrit for human males is 40.7 to 50.3 % and for human females 36.1 to 44.3 %. Normal values of hemoglobin are 13.8 to 17.2 gm/dL for male humans and 12.1 to 15.1 gm/dL for female humans. [0045] Homspera® was effective at stimulating differentiation of several hematopoietic progenitor cells under both optimal and sub-optimal growth factor conditions. Under sub- optimal conditions HPP-SP, GM-CFC, CFC-GEMM and B-CFC were noticeably stimulated to differentiate. Under optimal conditions, HPP-SP, GM-CFC, T-CFC, Mk- CFC, CFC-GEMM and B-CFC progenitor cells were stimulated to differentiate. [0046] Accordingly, it is contemplated that the methods can be used to increase the numbers of any blood cell class or subclass. These include erythrocytes, leukocytes, and platelets. It further includes neutrophils, band cells, T lymphocytes, B lymphocytes, monocytes, eosinophils, and basophils. More particularly, these include T helper cells, CD45 positive cells, antigen presenting cells, CD4 positive cells, CD8 positive cells, and T cells expressing Toll Like Receptor (TLR) types.

[0047] In certain embodiments, the methods can be used to increase differentiation of cell populations selected from HPP-SP, GM-CFC, CFC-GEMM and B-CFC progenitor cells. In certain embodiments, the methods can be used to increase differentiation of cell populations selected from HPP-SP, GM-CFC, T-CFC, Mk-CFC, CFC-GEMM and B-CFC progenitor cells. In certain embodiments, the methods can be used to increase differentiation of cell populations selected from BFU-E, CFU-E, CFU-GM and CFU-Mk cell progenitor cells.

[0048] In certain embodiments, the methods can be used to increase proliferation of cell populations selected from HPP-SP, GM-CFC, T-CFC, Mk-CFC, CFC-GEMM and B-CFC progenitor cells. In certain embodiments, the methods can be used to increase proliferation of cell populations selected from B-CFC and T-CFC cells. In certain embodiments, the methods can be used to increase proliferation of cell populations selected from BFU-E, CFU-E, CFU-GM and CFU-Mk cells.

[0049] The HPP-SP population comprises stem and progenitor cells that can express CD90+/CD133+/CD34+ markers. The BFU-E population comprises erythroid cells that can express CD38+/Glycophorin-A+ markers. The GM-CFC population comprises granulocyte-macrophage colony forming cells that can express CD38+/CD14+/CD15+ markers. The Mk-CFC population comprises megakaryocyte colony forming cells that can express CD41+/CD61+ markers. The T-CFC population comprises T-lymphocyte colony forming cells that can express CD3+/CD4+/CD8+ cell markers. The B-CFC population comprises B-lymphocyte colony forming cells that can express CD 19+ markers. The CFC-GEMM population comprises granulocyte, erythroid, macrophage and megakaryocyte cells. Cells of the CFC-GEMM population can express cell markers for their particular lineage as well as CD34+ and CD133+ (e.g., granulocytes can express CD38+/CD14+/CD15+ as well as CD34+ and CD133+ markers, erythroid cells can express CD38+/Glycophorin-A+ markers as well as CD34+ and CD 133+ markers). [0050] In one embodiment, the methods provide for prevention, treatment, or amelioration of myelosuppression associated with radiation therapy by the administration of an effective amount of a substance P analog to a human in need thereof wherein the substance P analog is of Formula (I):

Zi-Xaa ¹ -Xaa ² -Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Z2 (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa ¹ is Arg, Lys, 6-N methyllysine or (6-N, 6-N) dimethyllysine;

Xaa ² is Pro or Ala;

Xaa ³ is Lys, Arg, 6-N-methyllysine or (6-N, 6-N) dimethyllysine;

Xaa ⁴ is Pro or Ala;

Xaa ⁵ is GIn or Asn;

Xaa ⁶ is GIn or Asn;

Xaa ⁷ is Tyr, Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa ⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4;

Xaa ⁹ is GIy, Pro, Ala or N-methylglycine;

Xaa ¹⁰ is Leu, VaI, He, Norleucine, Met, Met sulfoxide, Met sulfone, N- methylleucine, or N-methylvaline;

Xaa ¹ ' is Met, Met sulfoxide, Met sulfone, or Norleucine;

Z, is R ₂ N- or RC(O)NR-;

Z ₂ is -C(O)NR ₂ or -C(O)OR or a salt thereof; each R is independently R is H, (Ci -C ₆ ) alkyl, (Ci -C ₆ ) alkenyl, (Ci -C ₆ ) alkynyl, (C ₅ -C ₂ o) aryl. (Ce -C ₂ 6) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa ¹ through Xaa ¹ ' independently designates an amide linkage, a substitute amide linkage or an isostere of an amide. In one embodiment, the substance P analogs can be of Formula (I) with the proviso that the substance P analog is not substance P (SEQ ID NO: 1).

[0051] In one embodiment, the substance P analog can be of Formula (I) as described herein wherein Xaa ¹ is Arg; Xaa ² is Pro; Xaa ³ is Lys; Xaa ⁴ is Pro; Xaa ⁵ is GIn; Xaa ⁶ is GIn; Xaa ⁷ is Tyr, Phe or Phe substituted with chlorine at position 4; Xaa ⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa ⁹ is GIy, Pro or N-methylglycine; Xaa ¹⁰ is Leu; and Xaa ¹ ' is Met, Met sulfoxide, Met sulfone or norleucine. [0052] In a preferred embodiment, the substance P analog can be of Formula (I) as described herein wherein the "— " between residues Xaa ¹ through Xaa ¹¹ designates - C(O)NH-; Zi is H ₂ N-; and Z ₂ is -C(O)NH ₂ . [0053] In another preferred embodiment, the substance P analog can be:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); or

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[0054] In another preferred embodiment, the substance P analog can be Zi-RPKPQQFFMeGlyLM(O ₂ )-Z ₂ ; wherein Z ₁ is NH ₂ and Z ₂ is C(O)NH ₂ . In one embodiment, the substance P analog is not substance P (SEQ ID NO.:1). [0055] In certain embodiments, the substance P analog can be used alone or in combination with other drugs used for the treatment of radiation sickness or the signs or symptoms of radiation sickness. Radiogardase (also called Prussian blue), pentetate calcium trisodium (Ca-DTPA) and pentetate zinc trisodium (Zn-DTPA) have been approved by the Food and Drug Administration (FDA) for treatment of radiation contamination from an industrial accident or a dirty bomb. In one embodiment, the methods provide for administration of a substance P analog with Radiogardase, pentetate calcium trisodium (Ca-DTPA) or pentetate zinc trisodium (Zn-DTPA). [0056] In certain embodiments, radiogardase, also known as Prussian blue, can be used to treat mammals exposed to radiation containing harmful amounts of cesium- 137 or thallium. Ca-DTPA and Zn-DTPA can be used for contamination with radioactive forms of plutonium, americium and curium. All three drugs work to eliminate radioactive substances from the body.

[0057] In certain embodiments, the substance P analog can be used alone or in combination with other drugs used to treat or mitigate the symptoms of radiation illness. Drugs used to treat the symptoms of radiation illness can be antiemetics, antidiarrheals, bandages (Silvasorb®, Jansen) or creams (Silvadene®, King) to treat skin burns, fluids (normal saline) and electrolyte replenishment fluids {e.g., Pedialyte®, Abbott), antiinflammatories (non-steroidal anti-inflammatory, steroids), treatments for the minimizing damage due to ulceration of the oral mucosa, esophagus, stomach, intestines such as histamine 2 antagonists (cimetidine, ranitidine and the like) or proton pump inhibitors (Protonix®, Wyeth, Prevacid®, Tap and the like). In one embodiment, other drugs that stimulate progenitor cells of the bone marrow can be used in combination. In one embodiment, the substance P analog can be used in combination with granulocyte- colony stimulating factors such as filgrastim (Neupogen®, Amgen, , Neulasta®, Amgen),

potassium iodide, ethylenediaminetetraacetic acid (EDTA), penicillamine, intraveous fluids, blood transfusions, or erythropoiesis stimulating agents such as erythropoietin ( Epogen®, Amgen), darbepoetin (Aranesp®, Amgen). See generally, Merck Manual, 18 ^th edition, Merck Research Laboratories, Whitehouse Station, NJ 2006. [00S8] In one embodiment, provided herein are methods and compositions for reducing poly(ADP-ribose) polymerase (PARP) activity in an animal. In one embodiment, the poly(ADP-ribose) polymerase (PARP) activity is caused by radiation exposure. In one embodiment, provided herein are methods and compositions for reducing poly(ADP- ribose) polymerase (PARP) activity expression in an animal. In one embodiment, provided herein are methods and compositions for treating radiation illness by reducing poly(ADP-ribose) polymerase (PARP) activity in an animal.

[0059] One of the earliest nuclear events that follows DNA strand breakage caused by agents such as ionizing irradiation, carcinogens, or alkylating agents during DNA repair is the poly(ADP-ribosyl)ation of various proteins that are localized near DNA strand breaks. Poly(ADP-ribose) polymerase (PARP) catalyzes the poly (ADP-ribosyl)ation of various nuclear proteins; however, only when PARP is bound to single -or double stranded DNA ends. PARP cycles on and off the DNA ends during DNA repair in vitro. PoIy(ADP- ribosyl)ation occurs on nuclear DNA binding proteins, such as histone, topoisomerases I and II SV40 large T antigen, DNA polymerase a, PCNA, and -15 protein components of the DNA synthesome, as well as the automodification of PARP itself. It is hypothesized that this modification of nucleosomal proteins changes the nucleosomal structure of the DNA surrounding strand breaks and accordingly promotes access of various replicative and repair enzymes to these sites. PARP has been shown to undergo proteolytic cleavage into 89-and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced and spontaneous apoptosis. Caspase-3, a member of the family of aspartate -specific cysteine proteases plays a role in the execution of the apoptotic program, and is responsible for the cleavage of PARP during cell death. There appears to be a transient positive requirement for PARP and poly(ADP-ribosyl)ation very early in apoptosis as demonstrated using well-characterized cell lines stably transfected with inducible PARP-antisense constructs as well as with immortalized fibroblasts derived from PARP knock out mice. Poly(ADP-ribosyl)ation has also been

shown to play a unique role in cellular differentiation, since cellular depletion of PARP by PARP antisense induction markedly inhibited differentiation of 3T3-L1 preadipocytes into adipocytes by preventing a transient increase in PARP activity, that appears essential for entering the differentiation process, and precisely correlates with an essential round of DNA replication, required for onset of terminal differentiation. Additionally, it has been established that the tumor suppressor p53, which is required for apoptosis in many cell systems, was shown to be poly(ADP-ribosyl)ated in vitro and in vivo demonstrating that the modification of p53 by poly(ADP-ribosyl)ation occurs in vivo, and represents one of the early acceptors of poly(ADP-ribosyl)ation during apoptosis in human osteosarcoma cells. It has been hypothesized that when various proteins are in a highly negative poly(ADP-ribosyl)ated state they become "DNA-phobic" and cannot bind to sites in DNA whether on breaks or promoters; however, when poly(ADP-ribose) glycohydrolase cleaves the polymer from these DNA binding proteins they are then able to cycle back to sites in DNA, such as the p53 consensus promoter sequence, which induces pro-apoptosis genes such as BAX, p21 , and FAS. The human PARP gene chromosome was mapped to Iq31 - q42 and PARP-like sequences to 14ql3-q32 and 13q-34. PARP (-/-) mice, with a disrupted PARP gene, neither express immunodetectable PARP nor exhibit significant poly(ADP- ribosyl)ation. Boulares, et al. 2002, J. Biol. Chem. 277(1): 372-378, Simbulan-Rosenthal, et al, 2001, Neoplasia, 3: 175088, Boulares, et ah, 2001 J. Biol. Chem. 276(41): 38185- 92, Stoica, et al., 2001, Tox. Appl. Phar. 171 : 94-106, Rosenthal, et al., 2001, J. Invest. Dermatology 117: 1566-1573, Simbulan-Rosenthal, et al., 2000, PNAS USA 97: 11274- 1 1279, Simbulan-Rosenthal, et al, 1999, PNAS 96: 13191-13196, Simbulan-Rosenthal, et al. 1999, Cancer Res. 59: 2190-2194, Boulares, et al, 1999, J. Biol. Chem. 274: 22932- 2294, Simbulan-Rosenthal, et al, 1999, Oncogene 18: 5015-5023. The PARP enzyme polymerizes ADP-ribose from nicotinamide adenine dinucleotide (NAD). PARP assists in the repair of DNA damage by mediating base excision repair. Activation of PARP has been shown to induce apoptosis and cellular death in response to injuries such as radiation exposure and ischemia-reperfusion, activation of PARP has been shown to induce cell death via apoptosis. Zhang et al. 2005, J. Cereb. Blood Flow Metab. 25(7): 868-77, Williams, et al, 2006, Endocrinology 147(5), 2496-2505.

[0060] In one embodiment, provided herein are methods and compositions for treating radiation illness in an animal by reducing poly(ADP-ribose) polymerase (PARP) activity in an animal comprising administering to the animal an effective amount of a substance P analog as described herein . In preferred embodiments, the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[0061] In one embodiment, provided herein are methods and compositions for reducing poly(ADP-ribose) polymerase (PARP) expression in an animal comprising: administering to the animal an effective amount of a substance P analog. In certain embodiments, the substance P analog is selected from the group consisting of:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); and

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[0062] In one embodiment, the PARP is Poly(ADP-ribose) polymerase- 1 (PARP-I).

[0063] In one embodiment, the PARP gene is cytogenetic band Iq41-q42, location 222,855,240 bp to 222,902,526 bp. See, Human Genome Organisation (HUGO), HUGO Gene Nomenclature Committee.

[0064] In one embodiment PARP activity can be determined by caspase 3 activity or the presence of 89-and 24-kDa fragments. In one embodiment PARP expression can be determined by gene expression techniques known in the art. See, Simbulan-Rosenthal, et al, 2000, PNAS, 97(21): 11274-11279, Clayton and Holtz 1996, MoL Biochem. Parasitology, 11 (1): 1-6, Bieche, et al, 1996, Clin. Cancer Res. 2(7): 1163-1167. [0065] In one embodiment, treatment of radiation illness by reducing PARP expression can be reduction or amelioration of the signs or symptoms of radiation illness. [0066] In one embodiment, provided herein are methods and compositions for reducing PARP expression by administering a substance P analog and one or more drugs to decrease the symptoms of radiation illness. In one embodiment, the drugs can be antiinflammatories (e.g. ibuprofen, naproxen, aspirin, prednisone and the like), decongestants (e.g. pseudoephedrine), antihistamines (e.g. loratadine, diphenhydramine), antipyretics (e.g. acetaminophen), cough suppressants (e.g. dextromethorphan), and the like. Such drugs are known to those skilled in the art. United States Pharmacopeia and Drug Facts and Comparisons, updated monthly, Wolters Kluwer.

[0067] In one embodiment, the substance P analog can be provided to an animal after a radiation incident that places an animal at risk of radiation illness. For example, the substance P analog can be provided to an animal after a round of therapeutic radiation or a fractionated radiation treatment but prior to the animal's having signs or symptoms of radiation illness.

[0068] In one embodiment, the methods and compositions can be provided to an animal after exposure to ionizing radiation. In one embodiment the methods can be providing a substance P analog to an animal one hour after the animal has been exposed to ionizing radiation. In one embodiment, the methods can be providing a substance P analog to an animal two hours after the animal has been exposed exposed to ionizing radiation. In one embodiment, the methods can be applied 6 hours, 12 hours, 24 hours, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 30 days, 45 days, 90 days, 180 days, or 365 days after exposure to ionizing radiation.

[0069] In one embodiment, the methods provide for treating an animal afflicted with radiation illness whereby the methods and compositions are provided or applied to the animal within 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours or more after the animal develops signs or symptoms of radiation illness. [0070] In one embodiment, the animal is a mammal. The methods can be used for any mammal exposed ionizing radiation, including domestic or commercial animals, including cows and horses, or pets, including cats and dogs. In a more preferred embodiment the mammal is human.

5.5.2 Methods of Treating Adverse Effects of Drugs

[0071] In certain embodiments, provided herein are methods and compositions for reducing, treating or ameliorating an adverse effect of a drug in an animal. In certain embodiments, provided herein are methods and compositions for preventing an adverse effect of a drug or medication in an animal, comprising administering to an animal one or more substance P analogs in an amount effective to decrease or ameliorate one or more adverse effects of a drug in the animal.

[0072] In certain embodiments, provided herein are methods and compositions for preventing an adverse effect of a drug or medication in an animal, comprising administering to an animal substance P or a substance P analog in an amount effective to decrease or ameliorate one or more adverse effects of a drug or medication in the animal. In one embodiment, the animal is a mammal. In a preferred embodiment, the animal is a human.

[0073] The methods and compositions described herein can be used to treat, prevent or ameliorate hematologic disorders. In one embodiment, the hematological disorder can be neutropenia, thrombocytopenia, leucopenias, granulocytopenia, agranulocytosis, or anemias including drug induced hemolytic anemia, drug induced aplastic anemia, or drug induced macrocytic anemia. In one embodiment the animal is a mammal. In a preferred embodiment the mammal is human.

[0074] In one embodiment, the substance P analog can be of Formula (I) as described herein. The severity of drug or medication adverse effects run the spectrum from the mild annoying and generally self-limiting to life-threatening.

[0075] In one embodiment, the adverse effects of a drug or medication can be hematological. Hematological effects or blood dyscrasias occur when the bone marrow or peripheral blood cells are adversely affected by drugs or medications. Lubran, 1989, Annals Clin. Lab. Science 19(2): 114-121, Meulenhoff, 1983, Pharmacy World & Science, 6(1) 39-47. In one embodiment, the hematological effect can be neutropenia, thrombocytopenia, leucopenias, agranulocytosis, or anemias including hemolytic anemia, aplastic anemia or macrocytic anemia.

[0076] For example clozapine (Clozaril® ) is an anti-psychotic medication that can cause agranulocytosis in about 1-2% of patients taking clozapine. The prescribing and dispensing of clozapine must adhere to the guidelines of the Clozaril® National Registry. These guidelines include regular, documented complete blood cell counts with differentials in patients taking clozapine.

[0077] In one embodiment, the blood dyscrasias can be induced by any drug, medication, herb or botanical. The blood dyscrasias can be due to an unanticipated reaction in an animal, for example an allergic reaction, or can be an anticipated adverse effect based on pharmacologic principles as is the case with chemotherapeutic drugs. Blood dyscrasias caused by or believed to be caused by any mechanism are within the scope of the methods and compositions as described herein.

[0078] In one embodiment, the drug can be an anti-infective (antibiotic, antifungal, antimalarial, antiprotozoal and the like), anticonvulsant, antihistamine, appetite suppressant, tricyclic antidepressant, decongestant, antifungal, antipsychotic, or benzodiazepine. In a preferred embodiment, the drug can be a chemotherapeutic drug, for example, a drug used to combat cancer.

[0079] In one embodiment, the drug can be aminophylline, an aminoglycoside antibiotic (e.g., gentamicin, tobramycin, amikacin), clozapine, carbamazepine, lithium, phenytoin, theophylline, warfarin, heparin, cyclosporin, digoxin, procainamide, quinidine, valproic acid, pyrimethamine, chloramphenicol, levamisole, sulphamethoxazole, trimethoprim, sulphapyridine, sulfasalazine, glutethimide, hydroxychloroquine, isoniazid, meprobamate, methazolamide, perphenazine, amitriptyline, phenacemide, pimozide, rifampin, thioxanthenes, trimethobenzamide, quetiapine, ziprasidone, terbinafine, ticlopidine, lamotrigine, or phenylbutazone.

[0080] In one embodiment, the substance P analogs can be used to ameliorate the side effects of an herbal, botanical or dietary supplement, for example, echinacea, St. John's Wort, vinpocetine, evening primrose oil, or α-lipoic acid.

[0081] Chemotherapeutic drugs are known to damage rapidly dividing cells including cells produced by the bone marrow. The decrease in blood cell counts, induced by chemotherapeutic drugs is generally expressed as the nadir. Nadir periods for chemotherapeutic drug classes {e.g., antimetabolites, alkylating agents) as well as individual drugs are known in the art and chemotherapeutic drug regimens and post- treatment care are often designed to take nadir time periods into consideration. In one embodiment, the methods and compositions can be used to prevent, ameliorate, or minimize the severity or duration of a nadir after chemotherapy. [0082] In one embodiment, the drug can be one used to treat cancer (chemotherapeutic drug). In one embodiment the chemotherapeutic drug can be 5-azacitidine, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, actinomycin-D (dactinomycin), alemtuzumab, altretamine, aminoglutethimide, anagrelide, arsenic trioxide, asparaginase, bevacizumab, bexarotene, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunomycin, daunorubicin, decitabine, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, gefitinib, gemcitabine, gemtuzumab (gemtuzumab ozogamicin), goserelin, hydroxyurea, ibritumomab, idarubicin, ifosfamide, irinotecan, imatinib, lapatinib, lenalidomide, leuprolide, lomustine, mechlorethamine, mercaptopurine, methotrexate, melphalan, mitoxantrone, mitomycin, nelarabine, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, sorafenib, streptozocin, sunitinib, tretinoin, tositumomab (with or without I ¹³¹ ), temozolomide, temsirolimus, teniposide, thalidomide, thioguanine, thiotepa, topotecan, toremifene, vinblastine, vincristine, vinorelbine, or vorinostat.

[0083] In one embodiment, the substance P analogs can be used to potentiate or enhance a biological response modifier. In one embodiment the biological response modifier can be interferon (interferon- alpha or interferon alpha-2b), interleukin, Bacillus Calmette-Guerin (BCG), denileukin, G-CSF (filgrastim), GM-CSF (sargramostim), erythropoietin

(Procrit™, Epogen™, Aranesp™), interleukins (interleukin-11 (oprevelkin) and interleukin-2 (aldesleukin)), denileukin, or difitox.

[0084] Drug induced blood dyscrasias can be prevented, treated or ameliorated by the administration of an effective amount of a substance P analog according to Formula (I) as described herein.

[0085] In one embodiment, provided herein are methods and compositions for preventing an animal from adverse effects of a drug prior to the animals' exposure to the drug. [0086] In one embodiment, the methods provide for reducing or ameliorating adverse effects of a drug whereby the methods and compositions are provided or administered to the animal within 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours or more after the animal develops adverse effects.

[0087] In certain embodiments, the methods provided herein can be applied to any mammal that produces red blood cells. In a preferred embodiment, the manmmal is a human. In one embodiment the mammal can be a commercial animal such as cows (bovine), sheep (ovine) or primates. In one embodiment, the mammal can be a domestic animal such as a canine, feline or equine.

5.3 SUBSTANCE P ANALOGS

[0088] In certain embodiments, the substance P analog is of Formula (I):

Z'-Xaa'-Xaa^Xaa^Xaa^Xaa'-Xaa^Xaa'-Xaa'-Xaa'-Xaa^-Xaa ¹¹ ^ ² © wherein:

Xaa ¹ is Arg, Lys, 6-N methyllysine or (6-N, 6-N) dimethyllysine;

Xaa ² is Pro or Ala;

Xaa ³ is Lys, Arg, 6-N-methyllysine or (6-N, 6-N) dimethyllysine;

Xaa ⁴ is Pro or Ala;

Xaa ⁵ is GIn or Asn;

Xaa ⁶ is GIn or Asn;

Xaa ⁷ is Tyr, Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa is Tyr, Phe, or Phe substituted with chlorine at position 2,3 or 4;

Xaa ⁹ is GIy, Pro, Ala, or N-methylglycine;

Xaa ¹⁰ is Leu, VaI, He, Norleucine, Met, Met sulfoxide, Met sulfone, N- methylleucine, or N-methylvaline;

Xaa ¹ ' is Met, Met sulfoxide, Met sulfone, or Norleaucine _; Zi is R ₂ N- or RC(O)NR-; Z ₂ is -C(O)NR ₂ or -C(O)OR or a salt thereof; each R is independently R is — H, (Ci -C ₆ ) alkyl, (Ci -C ₆ ) alkenyl, (Ci -C ₆ ) alkynyl, (C ₅ -C ₂ o) aryl, (C ₆ -C ₂₆ ) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa ¹ through Xaa ¹¹ independently designates an amide linkage, a substitute amide linkage or an isostere of an amide. In one embodiment, the substance P analogs can be of Formula (I) with the proviso that the substance P analog is not substance P (SEQ ID NO: 1). [0089] In certain embodiments, the substance P analog is of Formula (I):

Z ¹ -Xaa ¹ -Xaa ² -Xaa ³ -Xaa ⁴ -Xaa ⁵ -Xaa ⁶ -Xaa ⁷ -Xaa ⁸ -Xaa ⁹ -Xaa ¹⁰ -Xaa ¹¹ -Z ² (I) wherein:

Xaa ¹ is Arg; Xaa ² is Pro; Xaa ³ is Lys; Xaa ⁴ is Pro; Xaa ⁵ is GIn; Xaa ⁶ is GIn;

Xaa ⁷ is Tyr, Phe or Phe substituted with chlorine at position 4; Xaa ⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa ⁹ is GIy, Pro, or N-methylglycine; Xaa ¹⁰ is Leu; and

Xaa ¹ ' is Met, Met sulfoxide, Met sulfone or Norleucine; Zi is R ₂ N- or RC(O)NR-; Z ₂ is -C(O)NR ₂ or -C(O)OR or a salt thereof; each R is independently R is H, (Ci -C ₆ ) alkyl, (Ci -C ₆ ) alkenyl, (Ci -C ₆ ) alkynyl, (C ₅ -C ₂₀ ) aryl, (C ₆ -C ₂₆ ) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and

each "— " between residues Xaa ¹ through Xaa ¹ ' independently designates an amide linkage, a substitute amide linkage or an isostere of an amide. In one embodiment, the substance P analogs can be of Formula (I) with the proviso that the substance P analog is not substance P (SEQ ID NO: 1). In certain embodiments, the substance P analog can be in a salt, e.g., associated with a cation or anion, form. Any pharmacuetically acceptable salt known to one skilled in the art can be used in the salt forms of the substance P analogs. [0090] As will be understood by those of skill in the art, substance P (SEQ ID NO.:1) refers to peptide: Arg Pro Ly s Pro GIn GIn Phe Phe GIy Leu Met, or the single letter representation RPKPQQFFGLM-NH2 (SEQ ID NO.:1). As such, a substance P analog as used herein refers to a substance P derivative that comprises one or more amino acids substitutions relative to SEQ ID NO.:1 and can either compete with substance P for binding to its receptor (NK-I) or agonize the NK-I (neurokinin) receptor according to an assay conventional to the art, e.g., as described in Shue, et ai.Bioorgan Med Chem Letters 2006, 16(4): 1065-1069. The amino acid substitutions can be conservative or nonconservative substitutions. Further, the amino acid substitutions can include substitutions of non-standard amino acids (e.g., amino acids other than the 20 amino acids normally encoded by the genetic code). For example, the substance P analogs can comprise Met-OH, a methionine amino acid where the amide group of the terminal Met- NH ₂ has been replaced with a carboxyl group. In another example, this carboxyl group can further be methylated. In yet another example, the substance P analog can comprise norleucine. In yet another example, the substance P analog can comprise sarcosine. In yet another example, the substance P analog can comprise N-methylglycine. In yet another example, the substance P analog can comprise phenylalanine that is substituted with between 1 and 4 chlorines, more preferably 1 chlorine.

[0091] In one embodiment the methionine residue side chain sulfur (S) can be oxidated. In one embodiment the methionine is methionine sulfoxide (-NH-CH(CO)-CH ₂ -CH ₂ - S(O)CH ₃ ). In one embodiment the methionine is methionine sulfone or methionine S, S, dioxide, (-NH-CH(CO)-CH ₂ -CH ₂ -S(O ₂ )CH ₃ ), also referred to herein as Met(O) ₂ . [0092] It will be apparent to one skilled in the art that the amino (designated herein as Zi) or carboxy terminus (designated herein as Z ₂ ) of the substance P analogs can be modified.

Provided herein are "blocked" forms of the substance P analogs, i.e., forms of the substance P analogs in which the N- and/or C-terminus is blocked with a moiety capable of reacting with the N-terminal -NH ₂ or C-terminal -C(O)OH. In some embodiments the N- and/or C-terminal charges of the substance P analogs can be an N-acylated peptide amide, ester, hydrazide, alcohol and substitutions thereof. In a preferred embodiment, either the N- and/or C-terminus (preferably both termini) of the substance P analogs are blocked. Typical N-terminal blocking groups include RC(O)-, where R is — H, (Ci -C ₆ ) alkyl, (Cj - C ₆ ) alkenyl, (Ci -C ₆ ) alkynyl, (C5 -C20) aryl, (C ₆ -C26) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl. Preferred N-terminal blocking groups include acetyl, formyl and dansyl. Typical C-terminal blocking groups include -C(O)NRR and -C(O)OR, where each R is independently defined as above. Preferred C-terminal blocking groups include those wherein each R is independently methyl. In another preferred embodiment the C-terminal group is amidated.

[0093] In certain embodiments, substituted amides generally include, but are not limited to, groups of the formula -C(O)NR--, wherein R is (Ci -C ₆ ) alkyl, substituted (Ci -C ₆ ) alkyl, (Ci -C ₆ ) alkenyl, substituted (C ₁ -C ₆ ) alkenyl, (C ₁ -C ₆ ) alkynyl, substituted (Ci -C ₆ ) alkynyl, (C ₅ -C ₂ o) aryl, substituted (C ₅ -C2 ₀ ) aryl, (C ₆ -C ₂₆ ) alkaryl, substituted (C ₆ -C ₂₆ ) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl and substituted 6-26 membered alkheteroaryl.

[0094] In certain embodiments, amide isosteres generally include, but are not limited to, -CH ₂ NH-, -CH ₂ S-, -CH ₂ CH ₂ - -CH=CH- (cis and trans), -C(O)CH ₂ -, -CH(OH)CH ₂ — and -CH ₂ SO-. Compounds having such non-amide linkages and methods for preparing such compounds are well-known in the art (see, e.g., Spatola, March 1983, Vega Data Vol. 1, Issue 3; Spatola, 1983, "Peptide Backbone Modifications" In: Chemistry and Biochemistry of Amino Acids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267 (general review); Morley, 1980, Trends Pharm. Sci. 1 :463-468; Hudson et al, 1979, Int. J. Prot. Res. 14:177-185 (-CH ₂ NH-, -CH ₂ CH ₂ -); Spatola et al, 1986, Life Sci. 38:1243-1249 (-CH ₂ -S); Hann, 1982, J. Chem. Soc. Perkin Trans. I. 1 :307-314 (-CH=CH-, cis and trans); Almquist et al., 1980, J. Med. Chem. 23:1392-1398 (-COCH ₂ -); Jennings- White et al., Tetrahedron. Lett. 23:2533 (-COCH ₂ -); European Patent Application EP 45665 (1982) CA 97:39405 (-CH(OH)CH ₂

-); Holladay et al, 1983, Tetrahedron Lett. 24:4401-4404 (-C(OH)CH ₂ -); and Hruby, 1982, Life Sci. 31 : 189- 199 (-CH ₂ -S-).

[0095] In certain embodiments, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides. Suitable amide mimetic moieties are described, for example, in Olson et al., 1993, J. Med. Chem. 36:3039-3049. [0096] In one embodiment, the substance P analog is [Nle"]-substance P, e.g., the substance P analog wherein the 11 amino acid position is norleucine, i.e., the peptide: Arg Pro Lys Pro GIn GIn Phe Phe GIy Leu NIe (RPKPQQFFGLNIe) (SEQ ID NO.:2). In one embodiment, the substance P analog is [Pro ⁹ ]-substance P, which refers to the substance P analog wherein the 9 ^th amino acid position is proline and has the sequence: Arg Pro Lys Pro GIn GIn Phe Phe Pro Leu Met (RPKPQQFFPLM) (SEQ ID NO.:3). In one embodiment, the substance P analog is [Sar ⁹ ]-substance P, which refers to the substance P analog wherein the 9 ^th amino acid position is Sarcosine or N-Methylglycine and has the sequence: Arg Pro Lys Pro GIn GIn Phe Phe MeGIy Leu Met (RPKPQQFFMeGIyLM) (SEQ ID NO.:4). In one embodiment, the substance P analog is [Tyr ⁸ ]-substance P refers to the substance P analog wherein the 8 ^th amino acid position is tyrosine and has the sequence: Arg Pro Lys Pro GIn GIn Phe Tyr GIy Leu Met (RPKPQQFTGLM) (SEQ ID NO 5). [p-Cl-Phe ' ]-substance P refers to the substance P analog wherein the Phenylalanine residue at positions 7 and 8 are chlorinated at the 4 position and has the sequence: Arg Pro Lys Pro GIn GIn Phe(4-Cl) Phe(4-Cl) GIy Leu Met-NH ₂ (RPKPQQF(4- CL)F(4-CL)GLM) (SEQ ID NO.:6). In one embodiment, the 11 ^th amino acid residue is Methionine sulfoxide, RPKPQQFFGLM(O) (SEQ ID NO:7). In one embodiment, the 9 ^th amino acid residue is Sarcosine and the 11 ^th residue is Methionine sulfoxide, RPKPQQFFMeGIyLM(O) (SEQ ID NO:8). In one embodiment, the 11 ^th amino acid residue is Methionine sulfone, RPKPQQFFGLM(O) ₂ (SEQ ID NO:9). [Sar ⁹ , Met (O ₂ ) ¹¹ ]- substance P refers to the substance P analog wherein the 9 ^th amino acid position is Sarcosine or N-Methylglycine and the 11 ^th amino acid position is Md(O ₂ ) and has the sequence: Arg Pro Lys Pro GIn GIn Phe Phe MeGIy Leu Met-O ₂ (RPKPQQFFMeGIyLM- O ₂ ) (SEQ ID NO.: 10). [0097] In a preferred embodiment the substance P analog can be:

RPKPQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNIe (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); or

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[0098] In another preferred embodiment the substance P analog can be:

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); or

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[0099] A substance P bioactive analog, preferably [Sar ⁹ , Met (O ₂ ) ¹ ^-substance P (Homspera®) in one embodiment, can be administered. Other compounds which function in the same way can be identified by their ability to compete with substance P for binding to its receptor (NK-I) and for their ability to agonize the NK-I (neurokinin). As discussed above, substance P analogs, are those which act as competitive inhibitors of substance P by binding to the substance P receptor (NK-I receptor) or which agonize the NK-I receptor. Substance P analogs other than those specifically described above as are known in the art and/or commercially available (e.g., from Sigma) can be used in the methods and compositions described herein. In addition, substance P fragments and derivatized substance P fragments that retain the ability to compete with substance P for binding to the NK-I receptor or can agonize the NK-I receptor are considered to be within the scope of

the present invention. Substitution, deletion, or insertion of one to eight amino acid residues, and preferably from one to three amino acid residues, are also specifically contemplated. In addition, functional groups may be modified on the substance P analogs while retaining the same amino acid backbone. The substitutions, deletions, and/or modifications can be of consecutive or nonconsecutive amino acids. Further, the substitutions, deletions, and/or modifications can be at either or both of the C-terminal, N- terminal, or both, or neither.

[00100] In certain embodiments, the substance P analog is not substance P (SEQ ID

NO.-l)..

5.4 COMPOSITIONS AND KITS

[00101] In one embodiment, provided herein are compositions for administration of a substance P analog to prevent, treat, or ameliorate myelosuppression induced by therapeutic radiation or an adverse effect of one or more drugs. In one embodiment, provided herein is a composition comprising an effective amount of a substance P analog according to Formula (I) as described herein.

[00102] In one embodiment, the composition is a pharmaceutical composition. In a preferred embodiment, the composition can be a pharmaceutical composition of [Sar ⁹ , Met(O ₂ ) ^π ]-substance P (SEQ ID NO.: 10). In one embodiment, an effective amount of a substance P analog is an amount sufficient to reduce or diminish PARP activity. In one embodiment, an effective amount of a substance P analog is an amount sufficient to reduce or diminish PARP expression. In one embodiment, the pharmaceutical composition comprising a substance P analog can be used to treat, prevent, reduce the severity, ameliorate a symptom, or otherwise provide clinical benefit to an animal exposed to radiation or a drug as described herein.

[00103] Pharmaceutical compositions of the substance P analogs comprise a therapeutically effective amount of a compound described herein, formulated together with one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" or "carrier" refers to a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch;

cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, or as an oral or nasal spray, or a liquid aerosol or dry powder formulation for inhalation. [00104] Injectable parenteral preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of iηjectables.

[00105] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[00106] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution that, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).

[00107] Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[00108] In preferred embodiments, the parenteral composition can be administered intravenously, intramuscularly, subcutaneously or intradermally.

[00109] Intravenous, aerosol inhalation, topical, intratracheal, intrabronchial, intranasal, subcutaneous, sublingual, and oral administrations can be used. Suitable concentration ranges of substance P or its bioactive analog in an aerosol administered is between about 0.1 μM and about 5000 mM, Exemplary concentrations which can be used include about 1 mM, about 10 mM, about 50 mM, about 75 mM, about 100 mM, about 300 mM and about 1000 mM. For intramuscular injections, a volume of about 0.1 to 1.0 ml/kg of body weight can be used.

[00110] One skilled in the art can routinely determine dosages of substance P analogs for use in the methods and compositions described herein according to conventional parameters such as, for example, mass, distribution and clearance rates, etc. Doses will generally be from about 0.5 ng/kg to about 500 mg/kg.

[00111] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound can be mixed

with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium-phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form can also comprise buffering agents.

[00112] Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[00113] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[00114] Solid compositions of a similar type can also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[00115] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release

controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound can be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms can also comprise buffering agents. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[00116] Absorption through the gastrointestinal tract can be accomplished with peptides particularly if formulated in an appropriate composition. For example, peptides, such as the substance P analogs can be made in a pro-drug composition to provide oral absorption. See, Borchardt 1999, J. Controlled Release 62(1-2): 231-238, Catnach et ai, 1994, Gut 35(4): 441-444. In another embodiment, the oral routes of administration (including but not limited to ingestion, buccal and sublingual routes) can be used. In preferred embodiments, appropriate formulations (e.g., enteric coatings) are used to avoid or minimize degradation of the active ingredient, e.g., in the harsh environments of the oral mucosa, stomach and/or small intestine. Oral pharmaceutical compositions of the substance P analog peptide can be conjugated with Nobex™. (Protein Delivery Inc.) polymers as described in U.S. Pat. Nos. 5,359,030; 5,438,040; 5,681,811; 6,191,105; 6,309,633 and 6,380,405, incorporated herein by reference.

[00117] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol,

tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[00118] Compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[00119] Dosage forms for topical or transdermal administration of a compound include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component can be admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of these embodiments.

[00120] The ointments, pastes, creams and gels can contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[00121] Compounds and compositions can also be formulated for use as topical powders and sprays that can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

[00122] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[00123] Pharmaceutical compositions can also be formulated for delivery as a liquid aerosol or inhalable dry powder. Liquid aerosol formulations can be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles where bacteria reside in patients with bronchial infections, such as chronic bronchitis and pneumonia. Pathogenic bacteria are commonly present throughout airways down to bronchi, bronchioli and lung parenchema, particularly in terminal and respiratory bronchioles. During exacerbation of infection, bacteria can also be present in alveoli. Liquid aerosol and inhalable dry powder formulations are preferably delivered throughout the endobronchial tree to the terminal bronchioles and eventually to the parenchymal tissue.

[00124] Aerosolized formulations can be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of aerosol particles having with a mass medium average diameter predominantly between 1 to 5 μm. Further, the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosolizable volume able to deliver effective dose of the compounds to the site of the delivery. Additionally, the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.

[00125] Aerosolization devices suitable for administration of aerosol formulations, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation into aerosol particle size predominantly in the size range from 1-5 μm. Predominantly means that at least 70% but preferably more than 90% of all generated aerosol particles are 1 to 5 μm range. A jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. A variety of suitable devices are available, including, for example, AeroNeb and AeroDose vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, Calif.), Sidestream7 nebulizers (Medic-Aid

Ltd., West Sussex, England), Pan LC7 and Pan LC Star7 jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Va.), and Aerosonic (DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden, Germany) and UltraAire7 (Omron Healthcare, Inc., Vernon Hills, 111.) ultrasonic nebulizers.

[00126] The substance P analogs can be advantageously administered as a liquid dosage form at a concentration between about 0.1 μM and IM. More preferably from abut 0.1 mM to about 100 mM. In an even more preferred embodiment, the substance P analogs can be administered based on the subject's weight. In one embodiment, the substance P analog is administered at a dose of about 0.01 mg/kg to about 10 mg/kg. In a more preferred embodiment, the compositions are administered at a dose of about 0.05 mg/kg to about 5 mg/kg. Other exemplary dosage forms include about ImL of about 10OmM substance P analog solution, about ImL of about ImM substance P analog solution or about ImL of about 10 μM substance P analog solution administered parenterally or by inhalation.

[00127] Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions for use in the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art. It will be appreciated that the preferred route of administration and thus the type of pharmaceutical composition can vary with the condition, age and compliance of the recipient.

[00128] The methods and compositions can be administered in a frequency and duration for prevention or amelioration of decreased blood cells due to drugs or therapeutic radiation. In one embodiment, the compositions can be administered one time (e.g. single dose). In one embodiment, the compositions can be administered multiple times, for example concomitantly with a medicament or following a medication regimen, for example. In one embodiment, the composition can be given hours, days, weeks or even months after a medicinal or therapeutic regimen (i.e. chemotherapy or radiation therapy).

In one embodiment, the compositions can be administered intermittently, for example, every 3 days, every 7 days, every 14 days, every 30 days, every 60 days, every 90 days, every 180 days, every 360 days and the like.

[00129] In one embodiment, the compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[00130] In one embodiment, stable substance P analog formulations that have a long shelf life can be made by lyophilizing the substance P analog protein—either to prepare bulk for reformulation, or to prepare individual aliquots or dosage units which can be reconstituted by rehydration with sterile water or an appropriate sterile buffered solution prior to administration to a subject.

[00131] In certain embodiments, suitable compositions of the substance P analogs for administration are any which are pharmaceutically acceptable and in which the substance P analogs retain biological activity. Generally, such compositions are substance P analogs dissolved in sterile normal saline, buffered normal saline or sterile water. Other compositions for changing stability, absorption and half-life characteristics can be used, including liposomal compositions, carrier molecules that physically or chemically retard the release of the substance P analogs into physiological spaces and slow-release (depot) compositions.

[00132] In certain embodiments, provided herein are compositions that can be administered in a frequency and duration for prevention or amelioration of myelosuppression of therapeutic radiation. In one embodiment, the compositions can be administered one time (e.g., single dose). In one embodiment, the compositions can be administered multiple times, for example concomitantly with a radiation treatment or following radiation treatment. In one embodiment, the composition can be given hours, days, weeks or even months after radiation treatments. In one embodiment, the compositions can be administered intermittently, for example, every 3 days, every 7 days,

every 14 days, every 30 days, every 60 days, every 90 days, every 180 days, every 360 days and the like.

[00133] In certain embodiments, provided herein are compositions that can be administered in a frequency and duration for reducing PARP activity, reducing PARP expression, or treating radiation illness in an animal. In one embodiment, the compositions can be administered to the animal one time (e.g., single dose). In one embodiment, the compositions can be administered once, twice, three times, four times daily or more. In one embodiment, the compositions can be administered on a single day. In one embodiment, the compositions can be administered continuously, for example, for 3 days, 7 days, 10 days, 14 days, 21 days, 30 days, 90 days and the like. In one embodiment, the compositions can be administered intermittently, for example, every 7 days, every 10 days, every 21 days, every 30 days, 90 days, every 180 days, and the like. [00134] In certain embodiments, the compositions provided herein can be administered alone or in combination with other preventative or therapeutic agents. In one embodiment, the composition can comprise an effective amount of a substance P analog. In certain embodiments, the substance P analog can be: RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO.: 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO. : 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); or

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[00135] In certain embodiments, the substance P analog can be used in combination with one or more biological response modifiers to ameliorate myelosuppression or adverse effects. In a preferred embodiment, the biological response modifier can increase hematopoiesis. In one embodiment, the biological response modifier and the substance P analogs can be administered contemporaneously, for example, on the same day. In one

embodiment, the biological response modifier and the substance P analog can be administered at intervals, based on the radiation schedule of a human. For example, the biological response modifier can be given on Day 1 and the substance P analog, with or without a concomitant biological response modifier, can be administered on Day -3, Day - 1, Day 2, Day 3, Day 7, Day 10 or Day 14 of treatment. Such combinations can be administered either before or after radiation treatments. Such combinations can also be administered either before or after the myelosuppression is manifested. [00136] The substance P analogs can be advantageously administered as a liquid dosage form at a concentration between about 0.1 μM and IM. More preferably from abut 0.1 mM to about 100 mM, In an even more preferred embodiment, the substance P analogs can be administered based on the subject's weight. In one embodiment, the substance P analog is administered at a dose of about 0.01 mg/kg to about 10 mg/kg. In a more preferred embodiment, the compositions are administered at a dose of about 0.05 mg/kg to about 5 mg/kg. Other exemplary dosage forms include about ImL of about 10OmM substance P analog solution, about ImL of about ImM substance P analog solution or about ImL of about 10 μM substance P analog solution administered parenterally or by inhalation.

[00137] In one embodiment, provided herein is a kit for administering a substance P analog and a preventative and/or therapeutic agent. For example, such a kit can comprise both a treatment agent for radiation illness and at least one substance P analog. In certain embodiments, the substance P analog can be:

RPKPQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO.: 9); or

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[00138] The treatment agent for radiation illness and the substance P analog can be in separate, or divided or undivided containers. The two agents can be in liquid, dried, lyophilized, or frozen form, as is convenient for the end user and good for shelf life. The treatments can be administered at one time or sequentially, over a period of, for example, one day, one week, one month, six months or twelve months. [00139] In certain embodiments, provided herein are kits for providing the substance P analog alone or in combination with other drugs used for the treatment or amelioration of radiation sickness or the signs or symptoms of radiation sickness. In one embodiment, the drugs used for the treatment or amelioration of radiation sickness can be Radiogardase (also called Prussian blue), pentetate calcium trisodium (Ca-DTPA) and pentetate zinc trisodium (Zn-DTPA), antiemetics, antidiarrheals, bandages (Silvasorb®), creams (Silvadene®), (normal saline), electrolyte replenishment fluids (e.g. Pedialyte®), antiinflammatories (non-steroidal anti-inflammatory, steroids), histamine 2 antagonists (cimetidine, ranitidine and the like), proton pump inhibitors (Protonix®, Prevacid® and the like), granulocyte-colony stimulating factors such as filgrastim (Neupogen®, Neulasta®), potassium iodide, ethylenediaminetetraacetic acid (EDTA), penicillamine, blood transfusions, or erythropoiesis stimulating agents such as erythropoietin ( Epogen®), darbepoetin (Aranesp®).

[00140] In one embodiment, provided herein are a kit for administering a substance

P analog and a drug. For example, such a kit can comprise both an anti-cancer drug and at least one substance P analog and optionally, one or more biological response modifier. In certain embodiments, the substance P analog can be of Formula I as described herein. The drug and substance P analog can be in separate, or divided or undivided containers. The two agents can be in liquid, dried, lyophilized, or frozen form, as is convenient for the end user and good for shelf life. The treatments can be administered at one time or sequentially, over a period of, for example, one day, one week, one month, six months or twelve months.

[00141] In certain embodiments, the substance P analog can be:

RPKPQQFFGLM (SEQ ID NO. : 1 );

RPKPQQFFGLNIe (SEQ ID NO. : 2);

RPKPQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGIyLM (SEQ ID NO. : 4);

RPKPQQFTGLM (SEQ ID NO. : 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO. : 7);

RPKPQQFFMeGIyLM(O) (SEQ ID NO. : 8);

RPKPQQFFGLM(O ₂ ) (SEQ ID NO. : 9); or

RPKPQQFFMeGIyLM(O ₂ ) (SEQ ID NO. : 10).

[00142] Various embodiments of the invention have been described. The descriptions and examples are intended to be illustrative of the invention and not limiting.

Indeed, it will be apparent to those of skill in the art that modifications may be made to the various embodiments of the invention described without departing from the spirit of the invention or scope of the appended claims set forth below.

[00143] All references cited herein are incorporated herein by reference in their entireties for all purposes.

6. EXAMPLES

6.1 Example 1 : Preliminary analysis of effects of a substance P analog following lethal radiation of mice

[00235] One purpose of the study is to determine the effects of Homspera ^® (formerly Radilex™; [Sar 9,Met (02) 1 1] Substance P) after exposure to a lethal dose of 60 Co radiation exposure. Another purpose of the study is to determine if intra-muscular injections of RadilexTM can be as effective as aerosol inhalation of RadilexTM at 8Gy 60 Co radiation level.

[00236] Blood draws (2 animals per group rotating every 5 days) for erythrocytes, leukocytes and platelets were taken to assess anemia, neutropenia and thrombocytopenia. Counts of erythrocytes, leukocytes and platelets were taken to assess anemia, neutropenia and thrombocytopenia and act as indicators of bone marrow damage and/or immune system destruction.

[00237] The mice received Homspera ^® treatment within 2 hours of radiation exposure. The Homspera ^® dosage was administered in a volume of 0.01 ml in a daily single treatment via IM injection into the hind limb muscle a single time, for the duration of the study (30 days), or until death. [00238] The following regimens were tested on groups of 9-11 mice:

10 mM Homspera ^® single intramuscular treatment

1 mM Homspera ^® single intramuscular treatment

1 mM Homspera ^® daily intramuscular treatment

75 uM Homspera ^® single intramuscular treatment

75 uM Homspera ^® daily intramuscular treatment

10 uM Homspera ^® daily via nebulizer

50 uM Homspera ^® daily via nebulizer

75 uM Homspera ^® daily via nebulizer

100 uM Homspera ^® daily via nebulizer

200 uM Homspera ^® single administration via nebulizer at 2 hours post radiation exposure

1 mM Homspera ^® single administration via nebulizer at 2 hours post radiation exposure

10 mM Homspera ^® single administration via nebulizer at 2 hours post radiation exposure

[00239] Radioprotective effects of Homspera ^® were confirmed as the effects of lethal exposure were reversed. In particular, neutropenia and anemia were reversed in surviving animals. The normal white cell count for C57BL/6J mice is 6.2 + 2.7 K/microliter for males, 5.9 + 1.1 for females. Normal hematocrit is 45.4 + 1.7 % for males, 46.2 + 1.2 % for females. The mice that did not survive our highest radiation dose, 8 Gy*, had white cell counts 1/10 of normal or less, and hematocrits of less than half normal. Mice that did survive had, on average, white cell counts at 1/10 of normal or higher, and hematocrits at half normal or higher. It is clear, although not statistically significant because of small sample size per group, that more mice administered Homspera ^® survived the 8 Gy dose than would be expected to have survived, suggesting efficacy of Homspera ^® treatment. In view of these promising results, furhter experiments described in the examples that follow were performed.

6.2 Example 2. Half-life of plasma Homspera® relative to native substance P

[00240] The objective was to determine the half-life of Homspera ^® (RPKPQQFFMeGlyLM(O ₂ )-NH ₂ (SEQ. ID. NO: 10)) in plasma from three animal species. [00241] Frozen plasma from mice, human and non-human primates (designated hereinafter as primates for simplicity) was obtained from Biochemed (Winchester, VA) (human: Lot BC061107-07, primate: Lot CYNBREC-27070, mouse: Lot S-74242). EDTA was added as an anticoagulant during isolation of the plasma for all samples. [00242] The plasma was thawed and 990 μL was added to a 1.5 mL microcentrifuge vial. To have a final concentration of Homspera ^® in the plasma, two different stock solutions at either 1 mg/mL or 10 mg/mL were prepared using phosphate buffered saline (PBS), pH 7.4 at a IX concentration. Ten μL of a 1 mg/mL solution were added to 990 μL of plasma for a final Homspera ^® concentration of 7 μM and samples were vortexed to mix. Ten μL of a 10 mg/mL solution were added to 990 μL of plasma for a final Homspera ^® concentration of 70 μM and samples were vortexed to mix. In a 96-deep-well plate, 50 μL of either the 7 μM or the 70 μM plasma samples were mixed with 50 μL of a IX PBS, pH 7.4 solution.

[00243] For the preliminary half-life assessment, the 96- well plate was placed in a 37 ⁰ C oven for 0, 3, 10, 60, 120, and 180 min. Each sample per time point was terminated with the addition of 400 μL of 450 ng/mL glyburide in 90% acetonitrile (ACN) and 0.1% formic acid in water. Each sample was tested in duplicate. The plate was centrifuged at 4,000 rpm for 10 minutes and the supernatant was transferred to another 96- well plate. The supernatants were stored at -80 ⁰ C while other time points were being collected. Samples were evaporated using a Turbovap ^® and then reconstituted with 50 μL of 10 μg/mL of Pro ⁹ -substance P (SEQ. ID. NO:3) in 40% acetonitrile and 0.1% formic acid in water. Pro ⁹ -substance P functioned as an internal standard. Once the preliminary assessment was completed (data not shown), the time points were refined for the definitive portion. The time points assessed for the definitive study were 0, 10, 20, 30, 60, and 120 minutes. Samples were analyzed and quantitated by liquid chromatography/mass spectrometry (LC/MS).

[00244] The MS instrument was manufactured by Applied Biosystems (model: API 4000) and the LC portion of the instrument included a high pressure liquid chromatography (HPLC) pump made by Shimadzu (part number: LC-10ADvp) and an Agilent Poroshell HPLC column (part number: 300 SB-C 18 (2.1 X 75 mm). The system solvent consisted of two different mobile phases, designated as A or B, to resolve Homspera ^® . Mobile phase A was 95% H ₂ O, 5% ACN and 0.1% formic acid, and mobile phase B was 90% ACN, 10% H ₂ O, and 0.1% formic acid. The program used for separation on the HPLC column was a linear gradient from 0 to 1.5 minutes going from 0% B to 50% B, and from 50% B to 90% B at 1.5 minutes to 1.6 minutes, a hold at 90% B from 1.6 minutes to 2 minutes followed by a gradient from 90% B to 0% B until 2.1 minutes and a hold at 0% B from 2.1 to 2.5 minutes. The LC/MS was programmed to operate using a flow rate of 0.6 mL/min and 5 μL of sample were injected onto the HPLC column for each run. Additionally, the instrument was equipped with a turbo ion spray source, which was set to operate in the positive ion interface and multiple reaction monitoring acquisition modes. The source temperature was 500 ⁰ C and the total run time was 2.5 minutes. The parent/daughter ion pair of Homspera® was 698.8/348.4 and was 695.2/21 1.3 for the internal standard, Pro ⁹ -substance P.

[00245] At the lower concentration, 7μM, the mean half-life in mice was 33 minutes (st. dev. 3). The mean half-life for humans and primates at 7 μM was very similar with a mean half-life in primates of 37 minutes (st. dev. 4). At higher concentrations, 70 μM, the mean mouse half-life was 38 minutes, (st. dev. 1). The mean half-life for primates at 70 μM was 59 minutes (st. dev. 6) and 66 minutes for humans (st. dev. 0). Without being bound to any theory, it is proposed that the higher concentration of Homspera ^® saturated the ex vivo system perhaps by binding to other proteins in the plasma that would stabilize or protect Homspera ^® from enzymatic degradation. Accordingly, it is concluded the half-life of Homspera ^® is between 30-60 minutes for the three species examined.

Table 1. Ex Vivo Homspera® Half-Life in Plasma in Three Animal Species.

[00246] With regard to substance P, Berger et ah, reported that native substance P is rapidly degraded in rat brain fractions and in human plasma ex vivo. Berger et ah, 1979, Biochem. Pharmacol. 28: 3173-3180. At concentrations below 10 ^"7 M, the native peptide had a half-life of 9.3 minutes when incubated in a 1 mg/mL rat brain homogenate fraction. The half-life of the native peptide in plasma, ex vivo, was 24 minutes. [00247] Blumberg and Teichberg determined native substance P has a half-life of about 5 minutes (± 2 min) when 0.2 μM was incubated in 1 mg/mL rat brain homogenate. Blumberg and Teichberg, 1979, Biochem. Biophys. Res. Comm. 90(1): 347-354.

6.3 Example 3: The exemplary substance P analog Homspera® stimulates cellular proliferation and differentiation following radiation treatment

[00248] This study was done to determine the effect of treating irradiated mice with an exemplary substance P analog.

6.3.1. Materials and Methods

[00249] Homspera ^® was provided by ImmuneRegen via CSBio, Inc. (Menlo Park, CA, catalog number CS2663) as a lyophilized powder of the trifluoroacetate salt. The sample was stored at -20° C until solubilized. Homspera ^® was dissolved in dilute sterile saline and dilute acetic acid to obtain a solution of 300 μM concentration.

[00250] Seventy-two (72) Balb/c mice of age 5-6 weeks and normal physiological state (Taconic) were separated into 4 groups: Non-irradiated control (or Non-treatment control) (n=12), Irradiated control (vehicle controls) (n=20), Irradiated / Treated pre-exposure (n=20), and Irradiated / Treated post-exposure (n=20). Animals were housed individually in ventilated microisolator cages (4-5 mice per cage), fed ad libitum Lab Diet pellets, and acclimated for 5-7 days prior to treatment. On Day 1, animals were placed into the X-ray irradiator (RadSource 2000) for 4 minutes. Non-irradiated controls received no radiation exposure while the irradiated controls were exposed to radiation at the level of 1

Gy/minute. Animals were either treated with vehicle control or 300 μM Homspera in the same vehicle solution. The Non-irradiated control group and Irradiated control group were administered 25 μL of sterile saline intranasally daily for 7 days following radiation exposure. Animals treated with Homspera ^® pre-radiation exposure were administered 25 μL of 300 μM solution intranasally 1 day prior to radiation exposure and daily thereafter for 7 days. Animals treated with Homspera ^® post-radiation exposure were administered 25 μL of 300 μM solution intranasally daily for 7 days following radiation exposure as described in Table 2.

Table 2. Study Design

[00251] Following radiation exposure, gross observations were made at least once daily. Animal body weights were recorded at Days 1, 2, 3, 4, 5, 9, and 12 for all irradiated animals. Three mice from each group, including controls, were sacrificed at each timepoint listed in Table 2 or when each mouse became moribund. The remaining mice, 8 each from the Irradiated control, Irradiated / Treated pre-exposure, and Irradiated / Treated post-exposure groups were observed for survival until Day 30 or when moribund.

Table 3. Blood collection timepoints

* Three mice sacrificed per timepoint

[00252] Deaths and unanticipated adverse reactions were reported to the institutional veterinarian as soon as noted. The mice were sacrificed by regulated CO ₂ upon the animal being moribund. Mice were considered moribund if one or more of the following criteria were met: 1) loss of body weight of 20% or greater in a 1 week period; 2) prolonged, excessive diarrhea leading to excessive weight loss (>20%); 3) persistent wheezing and respiratory distress; 4) extreme lethargy; 5) dehydration indicated by loose skin; 6) fever indicated by shivering or 7) prolonged or excessive pain or distress observed as prostration, hunched posture, paralysis, paresis, distended abdomen, ulcerations, abscesses, seizures or hemorrhages.

[00253] The percentage of animal mortality and time to death were recorded for every group in the study.

6.3.2. Results

6.3.2.1 Animal body weights:

[00254] Animal body weights for non-irradiated controls were not recorded. Animal weights for all irradiated groups trended to decrease similarly to roughly 90% total body weight by Day 5 following radiation exposure. Animals exposed to radiation and treated with Homspera ^® (post-irradiation treatment) were observed to have a slight recovery in

lost body weight by Day 9. However, pre-irradiation treatment animals and irradiated control (vehicle control) animals continued to lose weight until moribund or sacrificed at Day 12. Irradiated control animals lost 16.1% (+/- 1.6%) body weight at Day 12. Pre- irradiation treatment animals lost 20.2% (+/- 2.4%) body weight at Day 12, while post- irradiation treatment animals lost 10.6% (+/- 1.9%) body weight at Day 12.

6.3.2.2 CBC (blood differentials):

[00255] Blood differentials evaluated white blood cell (WBC), lymphocyte (LYM), monocytes (MON), granulocyte (GRA), red blood cell (RBC), and platelet (PLT) levels. Results are reported as cells/liter and normalized to the non-irradiated control group values.

6.3.2.3 Six hours

[00256] White blood cell and lymphocyte levels trended to decrease significantly in irradiated animals. Animals pre-treated with Homspera ^® were observed to have lower lymphocyte counts than test non-irradiated control (vehicle control) and irradiated control animals. Monocyte and granulocyte counts in irradiated animals were observed to increase significantly for both the irradiated control group and the Homspera® pre-treatment group. Monocytes increased about 200% over non-irradiated controls and granulocytes increased about 300% over non-irradiated controls. Red blood cell and platelet levels in irradiated animals were not found to differ significantly from non-irradiated controls.

6.3.2.4 24 hours post exposure

[00257] White blood cell counts in irradiated animals continued to be lower than non- irradiated controls at 24 hours post-exposure. Animals treated with Homspera ^® (both pre- and post- irradiation treatment) had white blood cell counts higher than that of the irradiated controls. Animals treated with Homspera ^® post-radiation exposure were observed to have greater white blood cell counts than animals treated with Homspera ^® prior to radiation exposure (about 30% versus about 19%). This same trend was observed in both lymphocyte (about 17% versus about 4%) and monocyte counts (about 119% versus about 95%). However, monocyte counts in Homspera ^® -treated animals were similar to those seen in non-irradiated control animals while irradiated control animals were observed to have a nearly 5 fold decrease in monocyte levels (about 100% versus

about 20%). Granulocyte counts in animals treated with Homspera ^® post exposure were significantly greater (about 158%) than those observed in non-irradiated controls (100%) and animals exposed to Homspera ^® prior to radiation exposure (about 95%), while animals exposed to radiation and administered vehicle control were observed to have decrease granulocyte counts (about 75%). Again, red blood cell and platelet counts in irradiated animals were not observed to be significantly different from that seen in non-irradiated controls.

6.3.2.5 48 hours post exposure

[00258] White blood cell counts in irradiated animals at 48 hours post-radiation exposure were very similar to those observed at 24 hours post-exposure. Again, irradiated controls were observed to have WBC counts lower than non-irradiated controls (about 17% versus 100%), while the post-radiation subjects had about double that amount (about 38%). Lymphocyte counts at 48 hours post-exposure mirrored those observed at 24 hours post-exposure. Animals treated post-radiation exposure had the highest lymphocyte levels (about 18%) of the three irradiated groups. Monocyte and granulocyte levels followed this trend as well. Animals treated with Homspera ^® post-radiation exposure were observed to have monocyte and granulocyte levels greater than that of non-irradiated animals, control irradiated animals and pre-radiation treatment animals (about 120% vs. 100%, 38% and 75% for monocytes; 170% vs 100%, 78% and 102% for granulocytes). Red blood cell counts were not significantly different for animals exposed to radiation. However, animals exposed to radiation were observed to have significantly reduced platelet counts. Animals exposed to Homspera ^® , either pre- or post-radiation exposure were observed to have platelet counts lower than that of control irradiated animals (about 40%).

6.3.2.6 96 hours post exposure

[00259] White blood cell counts continued to decrease at 96 hours post-exposure. At this time point, irradiated animals treated with vehicle or Homspera ^® pre-radiation exposure had white blood cell counts roughly 1/2S* that of non-irradiated controls (about 4%). Animals treated with Homspera ^® following radiation exposure had white blood cell counts roughly 3 times greater (about 12%) than those observed in irradiated control animals. Interestingly, similar results were observed for lymphocytes, monocytes, and

granulocytes. Monocyte and granulocyte levels in irradiated animals fell dramatically at 96 hours post-exposure compared to 48 hour results. Again, animals treated with Homspera ^® following radiation exposure were observed to have substantially greater cell counts than those which were irradiated and treated with a vehicle control. Red blood cell counts continued to remain essentially unchanged. Platelet counts were observed to be similar to that seen at 48 hours post-radiation exposure. Nearly 50% reduction in platelet counts was observed in irradiated animals, and a small decreasing trend in Homspera ^® treated animals.

6.3.2.7 Flow cytometry

[00260] Flow cytometry was conducted to identify and quantify cell markers in the animal groups but the results did not reveal conclusive trends. For example, non-irradiated control animals were observed to have a significant variance in positive CD34 cells and positive Sca-1 cells over the 4 time points measured (6, 18, 24, and 48 hours). Non- irradiated controls were observed to have a decreasing trend in percent positive CDl 17 and CD9 cells. Similar results were observed in irradiated animals.

6.3.3. Discussion

[00261] A study was executed to evaluate the physiological effects of radiation and treatment with Homspera ^® (intranasally administered 25 μL of 300 μM solution) on mice. Mice were grouped into non-irradiated controls, irradiated controls, irradiated / treated preexposure, and irradiated / treated post-exposure. Irradiated animals were exposed to 4 Gy X-ray irradiation at a rate of 1/Gy per minute. Irradiated animals not treated with Homspera ^® were observed to have dramatic losses in body weight and significant decreases in CBC markers.

[00262] Animals treated with Homspera ^® for 8 days, beginning 1 day pre-radiation exposure, were observed to have weight losses greater than that of irradiated / non-treated animals. Alternatively, animals treated with Homspera ^® for 7 days following radiation exposure were observed to have a decreased weight reduction in comparison to irradiated controls.

[00263] Animals exposed to radiation were observed to have a significant reduction in white blood cell counts, lymphocyte counts, monocyte counts, granulocyte counts, and

platelet counts. Most of these effects were observed as early as 6 hours post-radiation exposure; however decreases in platelet levels were not observed until 48 hours postexposure. Treatment with Homspera ^® prior to radiation exposure (and 7 days thereafter) resulted in increases in white blood cell, lymphocyte, monocyte, and granulocyte counts when compared to irradiated controls. However, treatment with Homspera ^® for 7 days beginning after exposure to radiation was observed to have even greater effects on these same cell types. Animals treated with Homspera ^® following radiation exposure were observed to have greater monocyte and granulocyte counts than non-irradiated control animals for the first 48 hours following radiation exposure. However, these effects were not seen at 96 hours post-exposure, as monocyte and granulocyte counts were dramatically reduced.

[00264] Analysis of flow cytometry data did not reveal conclusive trends for any of the groups tested. Non-irradiated control animals were observed to have a level of variance similar to that seen in irradiated / drug-treated animals. This may be a product of biological variance, equipment variance, or both.

[00265] Animals exposed to radiation were observed to have a dramatic decrease in weight that continued until sacrifice or death. Animals treated with Homspera ^® daily for 7 days following irradiation were observed to have significantly less weight loss. The post- irradiation treatment group was also observed to have significantly increased levels of white blood cells, lymphocytes, monocytes and granulocytes when compared to irradiated control animals and pre-irradiation treatment animals. Platelet levels were observed to decrease significantly in all irradiated animals after 48 hours post-exposure. Treatment with Homspera ^® for 7 days following radiation exposure yielded the most efficacious method of maintaining animal weights and increasing vital CBC markers.

6.4 Example 4. The effect of an exemplary substance P analog, Homspera®, on cellular differentiation and proliferation

6.4.1. Introduction

[00266] In the study described below, human bone marrow-derived hematopoietic cell populations (or hematopoietic stem cells, HSCs) were cultured with or without

Homspera® to determine whether Homspera® affects proliferation or differentiation of the cells.

[00267] To assess proliferation, intracellular ATP (iATP) levels were measured. Increased levels of iATP correlate with increased cellular proliferation, because cells that are proliferating typically require high levels of energy, which is provided by iATP. [00268] To examine or assess proliferation, in this case, the experiment was designed to compare the effects of Homspera® on proliferation and differentiation after a 14 day incubation period. Although more or increased concentrations of cytokines or growth factors are typically added to support differentiation than proliferation alone- these concentrations are still effective for proliferation. Therefore proliferation was examined under the same conditions as those used for differentiation.

[00269] To induce differentiation "optimal" concentrations of growth factors and cytokines were used. Rich, 2003, Curr. Op. Drug Discovery Devel. 6:100-109, Rich and Hall, 2005, J. Tox. Sci. 87(2): 427-441. However, because substance P is known to stimulate hematopoiesis and promote the release of cytokines that contribute to differentiation, "suboptimal" concentrations of growth factors and cytokines were also used in the even the effects of Homspera® wouldn't be observed under saturating and therefore optimal cytokine conditions. Suboptimal concentrations were approximately one-fifth of optimal concentrations and were concentrations known to support colony formation. Rich, personal communications.

6.4.2. Methods

[00270] Homspera® (5 mg, Lot E844) was shipped as a solid compound. The compound was dissolved in 1 ml of Iscove's Modified Dulbecco's Medium (IMDM) and a serial dose response prepared in single log doses so that the final dose in culture ranged from 1 nM to 1 x 10 ^"16 M. All working dilutions were performed in IMDM. [00271] Starting with human bone marrow aspirate, the mononuclear cell (MNC) fraction was separated from the whole bone marrow using Ficoll-Paque density gradient centrifugation. The resulting MNC fraction had a cell concentration of 6.2 x 10 ⁶ cells/ml with a viability of 99.9%. The cell concentration was adjusted so that the final cell concentration in culture was 10,000 cells/well.

[00272] Human bone marrow MNC was dissolved in IMDM and cultured at a concentration of 10,000 cell/well in a CAMEO™-96 Master Mix (HemoGenix, Inc., Colorado Springs, CO). Master Mix is a HemoGenix proprietary cell culture media comprised of a serum mix (4 parts), a methyl cellulose mix (4 parts) and a growth factor mix (1 part) with the bone marrow target cells (1 part). See, Rich and Hall 2005, Toxicol. Sci. 87(2): 427-441.

[00273] Different combinations and concentrations of growth factors are used to induce differentiation of cells into specific cell types. The target cell populations were: High Proliferative Potential-Stem and Progenitor cell (HPP-SP), Colony-forming Cells- Granulocyte, Erythroid, Macrophage, Megakaryocyte (CFC-GEMM), Blast Forming Unit- Erythroid (BFU-E), Granulocyte-Macrophage- Colony Forming Cells (GM-CFC), Megakaryocyte- Colony Forming Cells (Mk-CFC), T-lymphocyte-Colony Forming Cells (T-CFC), B-lymphocyte-Colony Forming Cells (B-CFC).

[00274] Growth factors or cytokines used in the study were: erythropoietin (EPO), granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), Interleukins 3, 6, 2 and 7 (IL-3, IL-6, IL-2 and IL-7), stem cell factor (SCF), thrombopoietin (TPO) and soluble mutant flt3 ligand (Flt3-L). [00275] The concentrations of growth factors or cytokines used for each cell type assay are provided in Table 4 (Optimal Growth Concentrations) and Table 5 (Sub-Optimal Growth Concentrations).

Table 4. Optimal Growth Factor or Cytokine Concentrations for 7 Cell Lines (/ml)

Table 5. Sub-Optimal Growth Factor or Cytokine Concentrations for 7 Cell Lines (/ml)

U = Unit, ng = nanogram

[00276] The sub-optimal concentrations were about 50 fold less than the optimal concentrations used.

[00277] Eleven μl of the diluted Homspera® solution was added to each well followed by lOOμl of the master mix for each cell population detected. The cells were incubated in the absence or presence of Homspera® under sub-optimal and optimal stimulatory conditions for 7 target cell populations in 96-well plates for 14 days at 37° C in a fully humidified atmosphere comprised of 5% CO ₂ and 5% O ₂ . The total colony counts were manually enumerated under an inverted microscope, followed directly by processing the plates for bioluminescence to determine the intracellular ATP concentrations of the cells in each well. The study was concluded within 30 days of obtaining Homspera® and within 14 days of obtaining the human bone marrow aspirate.

[00278] Prior to processing all 96-well plates, the total colony counts/well were manually enumerated by microscopy. The mean, standard deviation and percent coefficient of variation was calculated for all groups, transposed and plotted. The intracellular ATP (iATP) concentration was measured after manual enumeration. The output of the luminometer is non-standardized Relative Luminescence Units (RLU). Prior

to measuring the samples, an ATP standard curve was performed. This allowed the RLU values to be automatically calculated into standardized ATP (μM) units. For both the RLU and ATP values derived from each well, the luminometer software calculated the mean, standard deviation and percent coefficient of variation.

6.4.3. Results

[00279] The data indicate Homspera® stimulates proliferation or differentiation of hematopoietic stem cells (HSCs) isolated from human bone marrow MNCs. Homspera® stimulated proliferation as indicated by the increase in iATP. Homspera® also stimulated differentiation as indicated by the increased colony forming units of hematopoietic progenitor cells.

[00280] Some background is helpful to understanding the results. A traditional CFC assay is usually performed in duplicate in 35mm Petri dishes. Sub-optimal growth factor studies cannot be performed with commercial media. Instead, the individual reagents have to be prepared and added individually. In this study, optimum growth factor/cytokine concentrations would be those that are normally used for the CFC assay. This assay is a functional differentiation assay, meaning that the assay relies on the functional ability of the target cells to divide and differentiate into colonies containing cells that identify the types of colonies being produced. Many lineage-specific growth factors, e.g. EPO, GM- CSF, TPO, are known to exhibit bifunctionality in that they will act as a proliferation factor for primitive cells in the series, but as a survival factor for the differentiating and maturating cells. Without the factors, the cells will enter into apoptosis. To induce proliferation, lower concentrations of growth factors or cytokines are required. These concentrations will, in many cases, be sub-optimal for the CFC differentiation assay. If proliferation only had been measured using the ATP assay at 7 days rather than 14 days, it is possible that a different response might have been observed using optimal and sub- optimal growth factor/cytokine concentrations. For the 7 cell populations detected in this study, the growth curve at 14 days indicates that proliferation decreased as differentiation increased. Therefore, the iATP concentration detected at 14 days represents residual proliferation within the colonies. However, since both the CFC and ATP assays were performed under the same conditions, it is possible to directly compare the results from the

two separate readouts. Taking these factors into account, the response between the 7 cell populations is probably best described as a percentage of the respective control.

6.4.3.1 Controls

[00281] The control values after 14 days in culture are shown in Table 6 and Table 7. In most cases, the control results are within the expected range of values with the cell populations falling into three categories: stem cell populations, myelopoietic populations and lymphopoietic populations. The 2 stem cell populations (HPP-SP and BFU-E) show the greatest proliferation and differentiation potential followed by the 3 myelopoietic populations (CFC-GEMM, GM-CFC and Mk-CFC) and the 2 lymphopoietic populations (T-CFC and B-CFC).

Table 6. iATP Proliferation Assay Controls

Table 7. CFC Differentiation Assay Controls

6.4.3.2 Effect of Homspera® on differentiation

[00282] In the presence of optimal growth factors, all cell populations, with the exception of BFU-E, exhibited enhancement or potentiation in differentiation potential. The response of BFU-E was significantly lower than the controls at all compound doses, although a slight increase from the lowest dose at 10 ^'16 M to 10 ^"13 M was observed prior to a decrease to the highest dose used (1 nanoMolar (nM)). For B-CFC, the dose response was a bell-shaped curve, beginning below control values at 10 ^"16 M, but peaking at 1 picomolar (pM) at about 163%, prior to decrease to control values. The Mk-CFC and CFC-GEMM populations also increased from control values at the lowest dose to reach peak between 10 ^'14 M and 10 ^"13 M respectively before decrease to control values at the highest dose of Homspera®. HPP-SP, T-CFC and GM-CFC all started at values significantly higher than control at the lowest dose. The T-CFC produced an approx. plateau between 10 ^"16 M and 10 ¹³ M before decreasing to control levels. The HPP-SP population peaked at 10 ^"15 M and decreased thereafter to control values. The GM-CFC produced the greatest potentiation of all cell populations peaking at 10 ^"14 M.

[00283] Under sub-optimal growth factor/cytokine conditions, both GM-CFC and CFC- GEMM produced a dose response that peaked at 10 ^"14 M and decreased thereafter, although at the highest dose of InM, the values from these two population did not fall below control values. In contrast, the B-CFC and HPP-SP populations, produced a very gradual increase with a slight decrease at InM. The T-CFC hovered around control values and exhibited a decrease to below control values after 10 ^"13 M. Both the BFU-E and Mk-CFC were below control values for essentially the whole dose response, although a peak did occur at 10 ^"14 M for Mk-CFC and 10 ^"13 M for BFU-E. For those populations that exhibited values greater than control at the lowest dose used, the dose response could be extended to doses lower than 10 ^"16 M.

6.4.3.3 Effect of Homspera® on proliferation

[00284] The ATP proliferation assay shows a different profile to that of the differentiation assay for all cell populations. Like the CFC assay at optimum growth factor/cytokine conditions, BFU-E exhibited a dose response below control values, with a gradual increase to control values at the highest dose used. The dose response for T-CFC was essentially flat at control levels. The B-CFC exhibited a flat dose response over the complete dose range, but at approx. 200% of control values. All other populations, (HPP- SP, CFC-GEMM, GM-CFC and Mk-CFC) exhibited an unusual U-shaped dose response curve, decreasing from the lowest Homspera® dose to about 10 ^"14 M and increasing again from about 1OfM to InM.

[00285] At sub-optimal growth factor/cytokine concentrations, only the lymphopoietic cell populations (T-CFC and B-CFC) exhibited a potentiation between 200 and 300% above control values. However, for both of these cell populations, the dose response was essentially flat. The Mk-CFC population exhibited essentially no response, while HPP-SP, CFC-GEMM, BFU-E and GM-CFC exhibited dose responses that were below control levels for most of the doses used.

[00286] However, although B-CFC are enhanced under optimal and sub-optimal condition in the ATP proliferation and T-CFC are enhanced under sub-optimal conditions also in the ATP proliferation assay, this enhancement effect is not dose-dependent, at least over the dose range used. The absence of a dose response indicates that the response

observed may actually be a plateau effect and that the cell populations are sensitive to the compound at much lower doses than were tested in this study. In addition, these cells do not demonstrate toxicity at the levels tested.

6.4.4. Discussion

[00287] After 14 days of incubation, Homspera® exhibited its maximum effect on the differentiation, rather than the proliferation process. Notable, for most of populations, was the apparent absence of distinct cytotoxicity. For both the CFC differentiation and ATP proliferation assays, the BFU-E population was the only population that was suppressed under optimal and sub-optimal conditions. However, see Example 5, below, where BFU-E differentiation and/or proliferation was enhanced.

[00288] The GM-CFC exhibited the greatest enhancement in the CFC differentiation assay, a result which is in accordance with published data for substance P. The T-CFC and B-CFC exhibit a dose response in the CFC differentiation assay under optimal and sub- optimal conditions. However, although B-CFC are enhanced under optimal and sub- optimal condition in the ATP proliferation and T-CFC are enhanced under sub-optimal conditions also in the ATP proliferation assay, this enhancement effect is not dose- dependent, at least over the dose range used. The absence of a dose response indicates that the response observed may actually be a plateau effect and that the cell populations are sensitive to the compound at much lower doses than were tested in this study. In addition, these cells do not demonstrate toxicity at the levels tested.

[00289] Several of the effects observed using the CFC differentiation readout have also been found for substance P and published in the literature. The difference between results of the ATP proliferation assay and the CFC differentiation assay is noteworthy. Firstly, higher doses of Homspera® enhance differentiation rather than proliferation, an effect known for lineage-specific growth factors, for example, erythropoietin. Secondly, for most lympho-hematopoietic cell populations exposed to the present dose range, the primary effect of Homspera® is during differentiation or maturation.

[00290] For those populations that exhibited values greater than control at the lowest dose used, the dose response could be extended to doses lower than 10 ^'16 M. In evaluating the colony numbers prior to detecting iATP, it did appear that the change in colony

numbers was due to a change in the size of the colonies as the compound dose increased. This was particularly the case for the HPP-SP population. [00291] Homspera® was effective at stimulating differentiation of several hematopoietic progenitor cells under both optimal and sub-optimal growth factor conditions. Under sub-optimal conditions HPP-SP, GM-CFC, CFC-GEMM and B-CFC were noticeably stimulated to differentiate. Under optimal conditions, HPP-SP, GM-CFC, T-CFC, Mk-CFC, CFC-GEMM and B-CFC progenitor cells were stimulated to differentiate.

[00292] Granulocyte/macrophage progenitors were the most responsive to Homspera® and were stimulated approximately 250% and 200% above controls lacking Homspera® treatment for both optimal and sub-optimal growth factor conditions respectively. CFC- GEMM cells were also stimulated to surprising levels above controls at about 175% in optimal conditions and about 225% in sub-optimal conditions. HPP-SP and T-CFC cell numbers were both about 175% above control values for optimal conditions. Under sub- optimal conditions, T-CFC populations did not change much from control values, whereas HPP-SP populations were enhanced roughly 125% from the population controls. In optimal conditions, B-CFC was 160% above control populations at Homspera® concentration of about 10 ^"12 M. The effects of Homspera® on B-CFC cells was not as pronounced under sub-optimal conditions, and were only stimulated 125% from the control population.

[00293] Homspera® was also effective at stimulating proliferation as measured by iATP levels using a fluorescent read-out. The most notable effects of Homspera® were on B- CFC and T-CFC progenitors cultured under sub-optimal cytokine levels. B-CFC iATP levels increased nearly 300% from control populations lacking Homspera® and T-CFC iATP levels increased 200% from controls. Furthermore Homspera® is effective at the lowest dose tested, 10 ^'16 M, suggesting biological activity for proliferation at sub- femtomolar concentrations. The results for the optimal growth factor conditions are similar to the differentiation assays in that the same cell types were stimulated with Homspera® (HPP-SP, GM-CFC, T-CFC, Mk-CFC, CFC-GEMM and B-CFC progenitor cells). B-CFC iATP levels were again significantly higher than controls (200%).

6.5 Example 5. The effect of an exemplary substance P analog, Homspera®, on cellular differentiation and proliferation (multi-donor study)

[00294] The study was undertaken to further illustrate lympho-hematopoietic differentiation in response to Homspera® using human-derived hematopoietic cell populations from three different bone marrow donors.

[00295] Homspera® (5mg, Lot F209) was shipped as a solid compound and stored at 4°C upon arrival. Compound was dissolved in ImI of Iscove's Modified Dulbecco's Medium (IMDM) and a serial dose response was prepared in single log doses so that the final dose in culture ranged from 1 nanoMolar (nM) to 1 x 10 ^"16 M. All working dilutions were performed in IMDM. Cell cultures from each donor were started at different times and a fresh solution of Homspera® was prepared for each individual experiment. [00296] Starting with human bone marrow aspirate, the mononuclear cell (MNC) fraction from each aspirate was separated from whole bone marrow using Ficoll-Paque density gradient centrifugation.

[00297] The colony-forming cell (CFC) assay was performed using optimal growth factor/cytokine concentrations. The reagents and conditions were similar to those used in Example 4, except that no ATP measurements were performed. The MNC fraction from each bone marrow donor was dissolved in IMDM and cultured at a concentration of 5,000 cells/well in a Culture Master Mix (HemoGenix, Inc., Colorado Springs, CO). [00298] The target cell populations were: High Proliferative Potential-Stem and Progenitor cell (HPP-SP), Colony-Forming Cells-Granulocyte, Erythroid, Macrophage, Megakaryocyte (CFC-GEMM), Blast Forming Unit-Erythroid (BFU-E), Granulocyte- Macrophage-Colony Forming Cells (GM-CFC), Megakaryocyte-Colony Forming Cells (Mk-CFC), T-lymphocyte-Colony Forming Cells (T-CFC), B-lymphocyte-Colony Forming Cells (B-CFC).

[00299] Growth factors or cytokines used in the study were: erythropoietin (EPO), granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), Interleukins 3, 6, 2 and 7 (IL-3, IL-6, IL-2 and IL-7), stem cell factor (SCF), thrombopoietin (TPO) and soluble mutant Flat 3 ligand (Flt3-L). [00300] The concentrations of growth factors or cytokines used for the CFC assay are the same as those provided in Table 4 of Example 4, e.g., Optimal conditions.

[00301] The assay was performed in a 96-well plate. To each well, 11 μl of the test compound (Homspera®) dilution was added followed by 100μl of the Culture Master Mix for each cell population detected. Cultures were incubated for 14 days at 37 ⁰ C in a fully humidified atmosphere containing 5% CO ₂ and 5% O ₂ . Thereafter, the total colony counts were manually enumerated under an inverted microscope.

[00302] The mean, standard deviation and percent coefficient of variation was calculated for all groups, transposed and plotted as a function of donor. The percent from control values were calculated and also plotted as a function of donor. In addition, results were compared from each individual cell population from all donors. All results were plotted using Prism Version 5 for Mac.

6.5.1. Results: Response from Individual Cell Populations

6.5.1.1 Stem Cells:

[00303] For all three donors, the primitive HPP-SP stem cells exhibited a greater response than the more mature multi-potential CFC-GEMM stem cells. Variations with respect to the level of potentiation did occur; and at the highest doses, a decrease in colony counts was usually observed. For example, with the HPP-SP cells, donor 1 was stimulated to approx. 300% from controls at Homspera® concentrations of 10 ^'12 M and 10 ^" " M, while donors 2 and 3 were approx. 200% and 175% from controls respectively. Both donors 2 and 3 were maximally stimulated at 10 ^"15 M Homspera®, a concentration lower than that for donor 1.

[00304] The variation between donors for CFC-GEMM was less pronounced and the trend was similar to levels previously observed with optimal growth factors in Example 4. Donor 1 exhibited the greatest potentiation to greater than 200% from controls, while donors 2 and 3 were both around 175% from controls. For all three donors, the maximum effects of Homspera® were observed at the 10 ^'14 M dose.

6.5.1.2 Hematopoietic Lineage Cells:

[00305] For the GM-CFC population, donors 1 and 2 demonstrated responses above background, while donor 3 was very close to control levels for nearly all the Homspera® concentrations tested. Both donors 1 and 2 were maximally stimulated to 200% and 150% relative to controls at 10 ^'13 M Homspera®, respectively. The trend observed for GM-CFC

is similar to that from the single marrow donor study. Additionally, the two-fold enhancement of colony forming activity and effectiveness at low Homspera® concentrations is consistent with the multi-donor study study of Example 6. [00306] This study showed that for all three donors, BFU-E exhibited a response that was in most cases significantly greater than the control over the entire Homspera® dose range. Indeed BFU-E showed the greatest response of all cell populations from the second donor at 10 ^'11 M Homspera®. Colony numbers were enhanced to approx. 150-300% relative to controls, depending on the donor. These levels of stimulation are consistent with the multi- donor study of Example 6, examining the effects of Homspera® on BFU-E colony formation.

[00307] The Mk-CFC population also exhibited a varied dose response to Homspera®. However, whereas donor 1 demonstrated an overall increase in potentiation with increasing compound dose, the response of donors 2 and 3 was relatively flat, and that from donor 3 was either below or near control levels over the Homspera® doses examined. While the concentration for maximal stimulation varies between donors, donors 1 and 2 both stimulated megakaryocyte colony formation greater than 200% from controls.

6.5.1.3 Lymphopoietic Cell Populations:

[00308] These two populations demonstrated the greatest effects of Homspera® with donor 1. The T-CFC response was greater than 300% from controls and greater than 200% for B-CFC for donor 1. However, the maximum effect of Homspera® on donors 2 and 3 was less than 150% from controls. T-CFC for donors 2 and 3 demonstrated a gradual increase with increasing compound dose, but the B-CFC demonstrated a slight decrease with increasing compound dose. The T-CFC population for Donors 2 and 3 exhibited a maximum at 0.1 picoMolar (10 ^"12 M), slightly above control values. For Donors 2 and 3, the peak value occurred at 1 x 10 ^"15 M.

6.5.2. Conclusions

[00309] The overall response of the three donors was that Homspera® potentiates differentiation of all seven cell populations tested when stimulated with optimal growth factor and cytokine concentrations. There was no apparent toxicity at the highest doses.

[00310] Homspera® is effective at increasing colony formation of the multi-lineage progenitor CFC-GEMM, and consistently acts to stimulate the colonies produced from CFC-GEMM.

6.6 Example 6: An exemplary substance P analog, Homspera®, stimulates proliferation of exemplary adult stem cells, human bone marrow cells (HBMCs)

[00311] The objective of this study was to compare stem cell hematopoiesis of Homspera ^® with native, C-terminally amidated substance P using human bone marrow cells in vitro.

6.6.1. Materials and Methods

[00312] Colony forming unit (CFU) assays were used to examine the effects of Homspera ^® in stimulating hematopoietic stem cells isolated from human bone marrow to differentiate into lineage-specific progenitor cells. As stem cells differentiate in response to growth factors, they form a colony of cells with distinct morphologies that can be visualized using a microscope. This study examined the formation of three different progenitor cell populations, erythrocytes, platelets and granulocytes/macrophages. [00313] Bone marrow aspirates were obtained from three healthy donors between 18 and 35 years of age following appropriate guidelines and protocols. Bone marrow mononuclear cells were isolated using a Ficoll-Hypaque density gradient, separating red blood cells from the others. Cells from each donor were processed independently and used for setting up individual experiments to assess the effects, if any, of donor variability. [00314] Cells (1 X 10 ⁵ ) were plated in duplicate onto 35 mm tissue culture plates for each condition tested and set up as described in Rameshwar et al. 1993, Blood. 81:2, 391- 398. Erythroid and granulocyte/macrophage cultures were plated using methylcellulose and megakaryocyte cultures (platelet precursors) used a collagen-based support (StemCell Technologies, Vancouver, Canada, catalogue #04973). Cells were cultured in the presence of either Homspera ^® or substance P at various concentrations. In the platelet study, neurokinin receptor antagonists were used to demonstrate receptor-specific effects. CP 99,994 (Pfizer) was used as a neurokinin- 1 receptor antagonist and SR 48968 (Sanofi) was used as a neurokinin-2 receptor antagonist.

[00315] The cytokines and growth factors added to Blast Forming Unit -Erythroid (BFU-E) cultures, Colony Forming Unit - Erythroid (CFU-E) cultures, and Colony Forming Unit - Granulocyte/Macrophage (CFU-GM) cultures were added as defined by proliferative units. BFU-E cultures contained 2 Units human interleukin-3 (hIL-3) and 2 Units recombinant human erythropoietin (rhEpo). Two Units IL-3 is about 0.1 ng/3ml. Rameshwar, private correspondence. CFU-E cultures contained 2 Units rhEpo, and CFU- GM cultures contained 2.6 Units recombinant human granulocyte macrophage- colony stimulating factor (GM-CSF). The Units of GM-CSF (2.6) is about 2ng/3ml. Rameshwar, private correspondence.

[00316] The biological activity in proliferative units for Epo was characterized by R&D Systems, Inc. in a cell proliferation assay using TF-I cells, a factor-dependent human erythroleukemic cell line. Kitamura, et al., 1989, J. Cell. Physiolλ40: 323-334. The units for hIL-3 and GM-CSF were defined using an IL-3/GM-CSF-dependent cell line, M-07e, a subline of the M-07 human megakaryoblastic leukemia cell line. Avanzi, G et al., 1990, J Cell. Physiol, 145:458-464. Standard growth curves were established using serial dilutions of rhIL-3 (50 ng/ml) or rhGM-CSF (1 ng/ml). One cytokine proliferative unit was defined as the amount required to stimulate one-half maximal growth of the M-07e cells.

[00317] Cultures were set up with limited cytokines and growth factors which would promote differentiation of one progenitor per plate. Growth factors and cytokines added to Colony Forming Unit — Megakaryocyte (CFU-Mk) cultures used weight/volume ratios and included 50ng/ml recombinant human thrombopoietin (rhTpo), 10 ng/ml recombinant human interleukin-6 (rhIL-6), and 10 ng/ml recombinant human interleukin-3 (rhIL-3) as suggested and supplied by StemCell Technologies.

[00318] Cells were cultured in the presence of Homspera ^® or substance P over the following molar concentrations: 10 ^'7 , 10 ^"8 , 10 ^"9 , 10 ^'10 , 10 ^'n , lO ^"12 , 10 ^'13 _, and 10 ^"14 M. Each compound was dissolved in endotoxin- free water to a final concentration of 0.1 mM. After reconstitution, the solutions were aliquoted into siliconized microcentrifuge tubes and exposed to nitrogen gas, eliminating oxygen in the head space of the enclosure. All aliquots were stored at -20 ⁰ C until use and used within one month of reconstitution. See, Rameshwar, et al. 1997, J. Immunol. 158:3417-3424.

[00319] Control plates without Homspera ^® or substance P were used to assess the baseline for colony growth. Cultures were incubated at 37°C with 5% CO ₂ for approximately 14 days, after which colonies were manually enumerated using a microscope. To count megakaryocyte colonies, the cells were fixed followed by a staining procedure using the following antibodies: primary - mouse anti-human GPl lb/111a, isotype control - mouse anti-trinitrophenyl, secondary - biotin-conjugated goat anti-mouse IgG, detection - avidin-alkaline phosphatase conjugate.

[00320] The results are expressed as percent colonies of control cultures (without Homspera® or substance P). The number of control colonies were normalized to 100% and represented as a zero level on the Y-axis.

6.6.2. Results

[00321] Homspera® was more effective than substance P at increasing colony counts for all stem cell progenitors examined. Two different red blood cell progenitor types were examined, BFU-E and CFU-E, which derive from a common progenitor, colony forming unit- granulocyte erythrocyte macrophage megakaryocyte (CFU-GEMM). BFU-E mature into CFU-E, which ultimately develop into functional red blood cells. (Figs. 1 & 2) [00322] Homspera® was more effective than substance P at enhancing stem cell differentiation. Homspera® enhanced BFU-E colony formation 2-fold (or 100%) relative to controls, whereas substance P increased colonies about 1.5 -fold (or 60%) from control values (Fig. 1). These effects are similar to those of Example 5.

[00323] For CFU-E, the difference between Homspera® and substance P treatment was less pronounced. For example, Homspera® enhanced colony formation to greater than 80% from control values, while substance P enhanced colony formation to about 70% from control values (Fig. 2).

[00324] This study demonstrated greater than a 2-fold increase in granulocyte/macrophage precursors when cultured with several different Homspera® concentrations from 10 ^"13 tolO ^"9 M (Fig. 3), and Homspera® was twice as effective as substance P at stimulating differentiation of human stem cells into CFU-GM. These results suggest that Homspera® could increase circulating levels of granulocytes and macrophages in vivo, possibly acting to mobilize the progenitors from the bone marrow or

through a combination of differentiation and mobilization. The two-fold stimulation is similar to levels observed in Example 5.

[00325] Homspera® and substance P treatments each demonstrated approximately a 2- fold stimulatory effect above controls for platelet precursors. However, Homspera® was effective at a concentration one log unit below substance P (10 ^"9 M vs. \0 ^'s M). To demonstrate that the effects of substance P were occurring through activation of the neurokinin- 1 receptor, two different receptor antagonists were used in the presence of substance P. CP-99,994 is a neurokinin- 1 receptor antagonist which blocks the stimulatory activity of substance P. Additionally, a neurokinin-2 receptor antagonist SR48968 was used, which showed no effect on substance P activity in enhancing platelet colony formation indicating that the effects of substance P on Mk colonies are via the neurokinin- 1 receptor. The effects of Homspera® on platelet precursors is similar to the two-fold levels observed in Example 5.

6.6.3. Conclusion

[00326] These data indicate Homspera® can stimulate hematopoiesis of all 3 major blood cell types. Homspera® was effective at concentrations ranging from 10 ^'7 M to 10 ^{' 14} M. In addition, Homspera® was more potent than substance P in enhancing colony formation of BFU-E, CFU-E, CFU-GM and CFU-Mk.