TW200808341A - Use of TPO peptide compounds and pharmaceutical compositions in the treatment of anemia - Google Patents

Abstract

A method of treating anemia is disclosed. The method includes administering a TPO peptide compound to a subject. Pharmaceutical compositions including a TPO peptide compound and a pharmaceutically acceptable carrier as well as diagnostic methods employing a labeled TPO peptide compound are also disclosed.

Description

200808341 IX. INSTRUCTIONS: This application is part of a continuous case and is requested to be filed on July 14, 2005. Please refer to US Application No. 11/181,049 and August 16, 2004. The priority of U.S. Application Serial No. 60/601,921, the entire disclosure of which is incorporated herein by reference. TECHNICAL FIELD OF THE INVENTION The present invention provides for the binding and activation of thrombopoietin receptor (c-mpl or TPO-R) or as a thrombopoietin (, TP〇) promoter. Ίο胜化合物. The invention can be used in the fields of biochemistry and medicinal chemistry and in particular to provide a sputum promoter for the treatment of human diseases. The compound of the invention can be used for treating anemia and/or preventing the occurrence of anemia and/or maintaining normal red blood cells. Generated. 15 [Prior Art] The gene encoding the sputum has been copied and characterized. See & to hit et al., Proc. Natl. Acad Sci USA 91:11104-11108 (1994); Barley Celebrity Cell 77: 1117-1124 (1994); Kaushansky et al, Nature 369: 568-571 (1994); Wendling et al, Nature 369: 571-574 20 (1994); and Sauvage et al, Nature 369: 533-538 (1994) TPO is a glycoprotein having at least two molecular weights of 25 kDa and 31 kDa with a common N-terminal amino acid sequence. See Bartley et al., Cell 77: 1117-1124 (1994). A distinct region separated by the potential Arg-Arg splitting position. The amine-based end of human and mouse is a highly reserved region, 5 200808341 and has some homology with red erythropoietin and interferon-a and interferon 7. The carboxy-terminal region shows a wide range of differences. ·: The DNA sequence and coding peptide sequence of human TP0-R (also known as c-mpl) has been described. See Vigon et al., Proc. Natl. Acad. Sci. USA 5 89:5640-5644 (1992) TPO-R is the growth factor of hematopoietin

A member of the subreceptor family, characterized by a common structural design of the extracapsular region, consisting of four remaining c residues in the N-terminal portion and a WSXWS • motif (SEQ ID NO: 1) near the transmembrane region. See Bazan Proc. Natl. Acad. Sci. USA 87:6934-6938 (1990). Evidence that this receptor plays a functional 10 role in hematopoiesis is observed in the spleen, bone marrow or fetal liver of mice (see Souyri et al., Cell 63: 1137-1147 (19903⁄4 performance and megakaryocytes in humans) The performance of cells, platelets, and CD34+ cells (see Methia et al., Blood 82: 1395-1401 (1993)) is limited. Some studies suggest that the receptor function is a homogenous binary, with G-CSF and erythropoietin. The situation is similar. The available replication gene of TPO-R helps to study this important receptor. The recombinant receptor protein obtained by Coru makes the receptor-ligand interaction in various random and semi-random peptide differential generation systems. The study of the effects is carried out. These systems are disclosed in U.S. Patent Nos. 6,251,864, 6,083,913, 6,121,238, 5,932,546, 20 5,869,451, 6, 5, 6,362, and 6,465,430, and also to 1*〇(:.

In the description of Natl. Acad. Sci. USA 87: 6378-6382 (1990), the foregoing is incorporated herein by reference. These morphologically identifiable and functional cells circulating in the blood include red blood cells, neutrophils, eosinophils and basophils, B-, T-, 6 200808341 non-B-, non-T- Lymphocytes and platelets. These mature hematopoietic cell lines are required, derivatized, and replaced by morphologically identifiable dividing progenitor cells of different lineages, such as red blood cells, red blood cell lines, bone marrow mother cells, pre-myeloid cells, and bone marrow cells. In the granulocyte series, and megakaryocytes to platelets. These rain-swept cells are derived from more primitive cells, which can be simply divided into two major subpopulations: stem cells and progenitor cells (see Broxmeyer, Η. Ε., 1983, ffColony Assays of Hematopoietic Progenitor Cells and Correlations to Clinical Situations/1 CRC Critical Review in

Oncology/Hematology 1:227-257). The definition of stem cells and progenitor cells is an operational criterion and is based on functional rather than morphological criteria. Stem cells have a tight self-renewal or self-maintenance ability (Lajtha, Differentiation, 14:23 (1979)), a necessity, because the lack or consumption of these cells may cause one or more cell lineages to be completely depleted' and this event can be Causes illness and death in a short time. Some stem cell lines differentiate as needed, but some stem cells make other stem cells to maintain these cell populations. Therefore, in addition to maintaining its own species, multi-functional stem cells can differentiate into several sub-progenitor cell lines with more restrictive self-renewal ability or no self-renewal ability. These progenitor cells eventually produce morphologically identifiable precursor cells. These progenitor cells can proliferate and differentiate through one or more myeloid differentiation pathways (Lajtha, Blood Cells, 5:447 (1979)). Various virulence factors, genetic abnormalities, and environmental factors can cause defects in one or more hematopoietic cell types. In addition, chemotherapy and radiation therapy for the treatment of cancer and certain immune disorders can cause pancytopenia and or anemia, leukocytosis or blood count reduction. Therefore, the addition and replacement of hematopoietic cells is usually an important success factor for these treatments in 200808. (For a general discussion of blood disorders and their causes, see, for example, "Hematology" in Scientific American

Medicine, E. Rubenstein and D. Federman, eds., Volume 2,

Chapter 5, Scientific American, New Y〇rk (1996)). 5 Currently available treatments for many blood disorders and endogenous hematopoietic cell destruction caused by chemotherapy and radiation therapy are bone grafts. However, because there are few donors who are fully compliant (gene-identical), bone marrow transplantation is performed in addition to the identical twins or the bone-riding cells of the symptom-reducing patients stored in a viable frozen state. Strictly restricted. In addition to the fact that the inevitable genes have some degree of non-conformity in the case of autologous sources, they suffer from serious and sometimes fatal complications. These complications are two-stage. First, patients are pre-vaccinated with drugs to avoid immune rejection of foreign bone marrow cells (host versus graft response). Second, when or if donated bone marrow cells begin to build, they will identify the patient as an alien and attack the patient (transplant 15 anti-host response). Even in the case of highly eligible family donors, these complications, which are directly due to genetically distinct individuals transplanted into the bone marrow, are responsible for the actual mortality and morbidity. Peripheral blood has also been assessed as a source of stem cells for hematopoietic reconstitution (Nothdurtt, W) et al., 1977, Scand J. Haematol 19: 470-481; Sarpel, S. C., 20 et al., 1979, Exp Hematol. 7:113-120; Ragharachar, A., et al., 1983, J. Cell. Biochem·Suppl·7A:78; Juttner, C. A., et al., 1985, Brit. J. Haematol· 61:739- 745; Abrams, R. A., et al., 1983, J. CelL Biochem. Suppl. 7A: 53; Prummer, 0, et al., 1985, Exp. Hematol. 13: 891. 898. In some studies In the middle, 7 豕 已 has got the results of various 8 200808341 leukemia patients ^^^ plus 5,]·, ^^·, 1986, ^?·1^11^01· 14:312-315 ; Goldman, J· M ·, et al., 1980, Br·J. Haematol· 45:223-231; Tilly, H., et al., Jul 19, 1986, The Lancet, ρρ· f 154-155; see also LB and Juttner, C. A., 1987, Brit λ 5 Haematol. 66: 285_288 and references cited therein; and results of lymphoma patients (Korbling, M., et al., 1986, Blood 67: 529-532) However, other studies using peripheral blood are heavy Failure (Hershko, C., et al., 1979, The Lancet 1:945-947; Ochs, H. D., * et al., 1981, Pediatr Res 15: 601). The study also evaluated the use of fetal liver ίο. Cell transplantation (Cain, G. R., et al., 1986, Transplantation 41: 32-25; Ochs, HLD, et al., 1981, Pediatr Res 15: 601; Paige, C. J. et al., 1981 J. Exp. Med. 153: 154-165; Touraine, J. L., 1980, Excerpta Med. 514:277; Touraine, J. L., 1983, Birth Defects 19: 139; see also Good, RA, Et al., 1983, Cellular 15 Immunol. 82:44-45 and references cited therein) or spleen cell transplantation in neonates (Yunis, E. J., et al., 1974, Proc. Natl. Acad. Sci · 10 USA· 72:4100) as a source of stem cells for hematopoietic reconstitution. Neonatal chest cells are also transplanted in immunoreconstruction experiments (Vickery, AC, et al., 1983, J. Parasito L 69(3): 478-485; Hirokawa, K., et al., 1982, Clin. 2 〇Immunol·Immunepathol. 22:297-304). Clearly, there is a great need for a method of expanding blood cells in vitro or in therapy that increases the production of hematopoietic cells in a living body. A poor company is defined as a decrease in hemoglobin concentration in the blood, usually associated with a decrease in the total circulating red blood cell volume. Regardless of the cause, anemia reduces the ability of the blood to carry oxygen and, when severe enough, causes clinical signs and symptoms. Clinically, anemia is characterized by pale skin and mucous membranes and hypoxia, the most common weakness, fatigue, lethargy or dizziness. Myocardial hypoxia can produce a high power cycle that increases heart rate and stroke volume. A projecting heart murmur is developed, and if the anemia is severe enough, heart failure may follow. Poor gold is generally categorized in one of two ways: by etiology (mainly for reasons) or by morphological classification (mainly by shape and size). More commonly used is the etiology classification. When a person's antibodies interact with another person's red blood cells (RBC), the same immune hemolytic anemia occurs. Immune hemolytic anemia typically occurs after blood that enters ABO blood group incompatibility and neonatal rheus disease. It also occurs after xenotransplantation (Hoffbrand, AV in Essential Hematology, 3rd. ed) Blackwell Scientific Publications, 1993, p. 9〇). 15 Administration of certain drugs may cause anemia in transitional drug bows. It can occur by three mechanisms: 1) antibody anti-drug-erythrocyte membrane complex (eg _ such as penicillin or cephalothin); 2) via drug protein (antigen complex) Depositing on the surface of red blood cells (such as quinidine or chloropropamide); or 3) autoimmune hemolytic anemia in which the role of the drug is unknown (eg methyldopa ( Methyl dopa)) In each case, anemia will only disappear after the drug has been interrupted (however, in the case of 曱 多 多 巴, the antibody can last for several months) (Hoffbrand, V. in Essential Hematology, 3rd. Ed, Blackwell Scientific Publications, 1993, p. 90-1) 0 200808341 Regenerative blood, defined as whole blood cell reduction due to dysplasia of bone mineral cells (anemia, leukopenia, and Small plate reduction). The system is classified into the following main types: congenital form (Fanconi anemia) and acquired form, no obvious cause (idiopathic). The second factor may be 5 industries, medical and The next factor is obviously that the number of hematopoietic pluripotent stem cells is low and the remaining stem cells are defective, or against their immune response, making them unable to effectively divide or differentiate to assemble bone marrow cells _ (Hoffbrand, Α· V· in Essential Hematology, 3rd· ed

Blackwell Scientific Publications, 1993, p. 121). In some cases 10 it has been demonstrated that inhibitory T-cells and immunoglobulins inhibit erythropoietin or block differentiation of hematopoietic stem cells in vitro (Andre〇li, Τ, in

Essentials of Medicine, W· Β· Saunders, 1986, ρ· 349) 〇

Neelis et al., Blood, 90(1): 58-63 (1997) revealed that human recombinant TPO stimulated the restoration of the erythrocyte lineage in rhesus monkeys exposed to 5 Gy whole body radiation (300-kV X-ray). Red blood cell regeneration occurred 10 days earlier than placebo-treated animals. Neelis et al. also revealed an increase in hemoglobin and hematocrit values compared to the control group.

Basser et al., Blood, 89(9): 3118-3128 (1997) discloses that administration of PEG-rHuMGDF and filgastrim enhances exposure to carboplatin 600 mg/m2 and cyclophosphamide. (cyclophosphamide) Peripheral blood-derived progenitor cells from patients in 1,200 mg/m2.

Papayannopoulou et al., Exp. Hematol., 24(5): 660-669 (1996) reveal the utility of EPO and TPO in the differentiation of erythrocytes and platelet production in vitro. π 200808341

Kaushansky et al., J. Clin Invest., 96(3): 1683-1687 (1995) revealed that TPO synergizes with EPO to amplify red blood cell progenitor cells. Kaushansky et al., Exp. Hematol., 24(2): 265-269 (1996) revealed that TPO amplifies BFU-E, CFU-GM and CFU-Mk 5 progenitor cells in bone marrow-suppressed animals. Anemia is a serious problem and research into blood growth factor promoters that prevent anemia, treat anemia, increase RBC precursor cell survival, and/or maintain normal red blood cell production becomes urgent. The present invention provides such an accelerator. SUMMARY OF THE INVENTION The present invention relates to the use of a defined low molecular weight peptide compound for the treatment of anemia. Compared with the known TPO promoter, the defined low molecular weight peptide compound has strong binding to TPO-R and can activate τρο-R, which can reduce side effects and in vitro and in vivo. It has the ability to stimulate red blood cell production. The low molecular weight peptide compound may be in various forms such as a monomer, a polymer and an oligomer and/or may be derived from a hydrophilic polymer. Therefore, the peptide compound can be used for the therapeutic purposes of treating and/or preventing anemia and for the purpose of studying the diagnosis of poor jk. -0 peptide compounds suitable for the treatment and/or for the purpose of the assay, for example as determined by the Baf/3 binding assay (discussed hereinafter), having an ic% of about 2 mM or less, and more preferably 2 nM or more. Low, where the lower IC5 有关 is related to the reluctance of Tp〇_Ri. For pharmaceutical purposes, the peptide compound preferably has an IC% of not more than about ΙΟΟμΜ, more preferably no more than about 5〇〇nM, ^ 12 200808341 preferably no more than about i 〇 0 pm, and more preferably It is no more than about 5_. A peptide compound suitable for therapeutic and/or diagnostic purposes, for example, using well known techniques in well known assays, such as the Baf/3 binding assay (discussed hereinafter), = EQo of about 2 mM or less, and more preferably It is 2 nM or lower, and the lower of 8 and 5 is related to the stronger binding force to TP〇-R. Preferably, the peptide compound has a ratio of not more than about 1 〇〇 ] ] 之 : ( 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不 不〇〇 pm, and more preferably, is no greater than about 5 pm. The molecular weight of the 1 peptide peptide ranges from about 500 to about 8,000 Daltons, and 10 more preferably from about 900 to about 200 Daltons. If the peptide compound is oligomerized, dimerized, and/or derived from a hydrophilic polymer as described herein, the molecular weight of the peptide will be greater than or from about 1500 to about 12 Torr. The range of liters, more preferably from about 3,000 to about 8, from Dalton, and more preferably from about 3, 0 to about 50,000. 15 Suitable hydrophilic polymers include ( But not limited to) polyalkyl ethers, exemplified by _ polyglycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkylene, polyvinyl alcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, Portuguese Glycans and dextran derivatives and the like are described in U.S. Patent No. 5, the disclosure of which is incorporated herein by reference. When a peptide compound is derived from a hydrophilic polymer, its solubility and circulating half-life are increased and its immunogenicity is masked. The foregoing may be carried out in a small amount (right) to detract from its binding activity. The polymer has an average molecular weight ranging from about 5 Torr to about 1 Torr, preferably from about 2,000 to about 40, and more preferably from about 4,000 to about 40. About 5, 〇〇〇 to about 13 200808341 20,000 Daltons. In a preferred embodiment, the hydrophilic polymer has an average molecular weight of about 5,000 Daltons, 10,000 Daltons, and 20,000 Daltons. The peptide compounds of the invention may be derivatized or coupled by such polymers using any of the methods described in 5 below: Zallipsky, S., Bioconjugate Chem., 6: 150-165 (1995); Monfardini, C, et al., Bioconjugate Chem., 6:62-69 (1995); U.S. Patent No. 4,640,835; U.S. Patent No. 4,496,689; U.S. Patent No. 4,301,144; U.S. Patent No. 4,670,417; U.S. Patent No. 4,791,192; 4,179,337 or WO ίο 95/3432 6. The text is hereby incorporated by reference in its entirety. In the presently preferred embodiment, the peptide compound of the present invention is derived from polyethylene glycol (PEG). PEG is an ethylene oxide repeat. Units, linear, water-soluble polymers with two terminal hydroxyl groups. PEG is classified by its molecular weight, typically ranging from about 500 Daltons to about 40,000 Daltons. In a preferred embodiment of the invention, the PEGs used have a molecular weight in the range of from 5,000 Daltons to about 20,000 Daltons. The PEG coupled to the peptide compound of the present invention may be a bond or a non-branched bond (see, for example, Monfardini, C., et al.

Bioconjugate Chem., 6: 62-69 (1995)). PEG is commercially available from Nektar Therapeutics (San Carlo, CA), Sigma Chemical Corporation 20 and others. Such PEGs include, but are not limited to, monomethoxypolyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol succinate (MepEG-S), monodecyloxy polyethylene glycol Amber succinimide succinate (MePEG-S-NHS), monodecyloxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-trifluoroethanesulfonic acid cool (MePEG- TRES) and monomethoxy polyethylene glycol </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; It is covered with a non-reactive group such as a methoxy group or an ethoxy group at one end. Thereafter, at its other end by reaction with a suitable activator, such as a melamine halide (eg, cyanide, melamine or melamine), a diimidazole and an anhydride reagent (eg, a bidentate) Succinic anhydrides such as dibromosuccinic anhydride), mercapto nitrogen, p-azobenzylmethyl, 3-(p-diazo-phenoxy)-2-ylpropyl hydrazide and the like . The activated polymer is then reacted with a peptide compound of the invention to produce a polymer-derived peptide compound. Alternatively, one of the functional groups on the peptide compound of the present invention can be activated to react with the polymer, or a single group can be added to a synergistic coupling reaction using known coupling methods. It will be appreciated that the peptide compounds of the present invention may be derived from PEG using a myriad of other reaction schemes known or used by those skilled in the art. When used for diagnostic purposes, the peptide compound is preferably labeled with a detectable label, and thus, the unlabeled peptide compound can be used as an intermediate for the preparation of the labeled peptide compound. Preferred peptide compounds are those having: (1) a molecular weight of less than about 5,000 Daltons, whether a monomer, a dimer or an oligomer, and (2) a butyl group? 0_11 binding affinity is represented by 1 (:5{) is not more than about 1〇〇111]^, wherein zero or several peptides [-C(0)NR-] chains (bonds) are via non-peptide chains, for example -CH2-carbamate chain [„CH2„〇c(〇)NR—]; phosphonic acid chain; —CH2-amine sulfonamide [„Ch2—S(0)2NR-] chain; urea [-NHC (0) NH__] chain; -CH^ secondary amine chain; or alkylated peptide chain 15 200808341 [—C(0)NR6—where R6 is a lower carbon group]; peptide, wherein N-terminal Derived into an NRR1 group; -NRC(0)R group; -NRC(0)0R group; -NRS(0)2R group; -NHC(0)NHR group wherein R and R are hydrogen or low carbon alkyl group, the limitation The conditions are that r and r1 are not both 氲; amber quinone imine; phenyl hydrazinocarbonylcarbonyl-NH-(CBZ--NH-) group; or phenylhydrazino oxycarbonyl group - phenyl group in benzene The base ring has 1 to 3 substituents selected from the group consisting of low carbon alkyl, low carbon alkoxy, chlorine and bromine; or a peptide wherein the C terminal is derived as -C(0)R2, Wherein r2 is selected from the group consisting of low carbon alkyl, low carbon alkoxy and one, wherein R3 and R4 are independently selected from the group consisting of hydrogen and low carbon. Groups.

The core peptide compound has been found to comprise an amino acid sequence (SEQ ID N〇; 2):

Xi X2 x3 x4 X5 x6 x7 where ST^V, X3 is C, F, I, L, M, R, S, \^w; X4g Any 20 genes encode L-amino acid; X5*A, D, b, g, k, M, QR, S, T, V or Y, X6 is (3-(2-naphthyl)alanine (2_Na!); and X7 is C, G, I, K, L, M , N, R* V. In a preferred embodiment, the core peptide compound comprises an amino acid sequence (SEQ ID N0.3): 3 soil

Xb GX! X2 X3 X4 X5 (2-Nal) X7 16 200808341 wherein \ to X7 are as defined above; and each Xs residue is independently selected from the group consisting of a gene encoding L-amino acid, its stereoisomer D-A Base acid; and non-natural amine acid. In another preferred embodiment, the core peptide compound comprises a monoamine 5-acid sequence (SEQ ID NO: 4): X9 Xg G Χι X2 X3 X4 X5 (2-Nal) X7 i〇 and 乂8 are Eight, 0 0, £, 〖,! ^, (), ;^, 3, 1 [or 乂. More preferably, X9 is A or I; and X8 is D, E or K. A particularly preferred peptide compound is (SEQ ID NO: 5): I E G P T L R Q (2-Nal) L A A R (Sar) 15 wherein (Sar) is sarcosine. In another embodiment, the peptide compound is dimerized or oligomerized to enhance the affinity and activity of the peptide compound. A particularly preferred peptide compound is the 29-mer peptide which has two identical 14-mers linked to the amine amine residue. Therefore, a particularly good peptide compound is (SEQ ID NO: 6, also referred to herein as TPO compound number 1): IEGPTLRQ(2-Nal)LAAR(Sar) \ 25 K(NH2) / IEGPTLRQ(2-Nal)LAAR (Sar) 17 200808341 A preferred peptide compound is a pegylated TPO compound i. The polyethylenated form may comprise a 20,0 MPEG & base group covalently linked to each N-terminal isoleucine. The complete molecular structure of this compound example is detailed below:

(This compound refers to the PEGylated TPO Compound 1 herein). PEGylated TP0 compound 1 chemical full name: oxime ethoxylate polyethylene glycol 2 〇〇〇〇 醯 醯 - - L - L L _ _ _ _ _ _ _ _ _ _ _ _ _ _醯基丄_苏胺醯1〇-L-Acetylhydrazone-Spermine Amine-L-glutamine Amidinoquinone-2-naphthylpropylamine Alkylamine Alkylamine Aminoamine Alkyl L_Alanamine-L-spermine 醯_Adenine-based-Ne-(曱 ethoxylate polyethylene glycol 20000 醯 醯 醯 丄 异 异 异 _ _ _ L_ glutamic acid-glycine 醯丄 丄 脯 脯 苏 苏 苏 苏 - - - - - - - - L - - - - - - - - - - - - - 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘 萘Spermine 醯 - myosin _ _) _ off-amine amide. Polyallylated TP0 compound 1 is linked by two amine amine residues and linked at each N-terminus to a molecular weight of about 2 Å. The same is true for the polyethylene glycol (PEG) chain of 〇〇〇Dalton. It consists of 14 amino acid peptide chains. The original peptide without peg has a molecular weight of 3,295 Daltons and has two PEG chains of approximately 2 〇 43,295 遒. The short molecular structure of PEGylated TPO Compound 1 is (MPEG-He-Glu- 18 200808341)

Gly-Pro-Thr-Leu-Arg-Gln-(2-Nal)-Leu-Ala-Ala-Arg-(Sar))2-L ys-NH2; wherein (2_Nal) is (3-(2-naphthyl) Alanine, (Sar) is sarcosine and MPEG is decyloxy poly(ethylene glycol) (MW about 20,000 Daltons). One or more peptide compounds and especially PEGylated peptide compounds, including a pharmaceutically acceptable equivalent thereof (herein collectively referred to as "peptide compound", "TPO triumphant compound", "winning compound of the present invention") for the prevention and treatment of diseases mediated by TPO, and In particular, it is used for the treatment and/or prevention of anemia. Therefore, the present invention provides a method for treating and/or preventing anemia, wherein a patient suffering from anemia or a patient who is expected to develop a poor family receives or is administered a therapeutic or prophylactically. An effective amount or amount of a peptide compound of the invention. The invention also provides a pharmaceutical composition comprising one or more peptide compounds as described herein and a physiologically acceptable carrier. The pharmaceutical compositions can be in a variety of forms including Oral dosage forms as well as inhalable powders and solutions and injection and infusion solutions. Specific examples illustrate the following The definitions are used to clarify and define the meaning and scope of the various terms used to describe the invention. "Accelerator" refers to a physiologically active ligand that binds to its complementary physiologically active receptor and Activation to cause physiological reactions of the receptor or to promote the physiological activity of the receptor in advance. "ECso" and "50% effective concentration" refer to the concentration of the promoter which produces the maximum possible effective response of 5 〇〇/〇 to the promoter. "IC%" and "50% inhibitory concentration" refer to the 50% specific binding promoting 19 competitive concentration of the drug. "Pharmaceutically acceptable equivalents" include (not limited to) pharmaceutically acceptable salts, acids Addition salts, esters, guanamines, hydrates, metabolites, prodrugs and electricity; isosteres. Many pharmaceutically acceptable equivalents are expected in vivo with the peptide compounds of the present invention. The same or similar activity.

10 15 20 "Medically acceptable salts" means non-toxic alkali metals, soil-checking metals and ammonium salts commonly used in the pharmaceutical industry, including sodium, potassium, lithium, calcium, magnesium, strontium, ammonium and protamine zinc. Salts which can be prepared by methods well known in the art. The term one includes non-toxic acid addition salts which are prepared by reacting a peptide compound of the invention with a suitable organic or inorganic acid. Representative salts include hydrochlorides, hydrochlorides, sulfates, hydrogen sulfates, acetates, oxalates, valerates, oleates, laurates, borates, lactic acid, lactic acid Salts, phosphates, toluenesulfonates, citrates, maleates, fumarates, succinates, tartrates, naphthalenesulfonates and the like. "Pharmaceutically acceptable acid addition salt" means the biological utility and characteristics of such free bases, and salts which are not biologically or otherwise undesirable, and inorganic acids such as hydrochloric acid, hydrochloric acid, and hydrochloric acid. Acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartrate, lemon It is formed by acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable acid Addition salts are described as drugs, see Bundgaard, Η, Yang (4). "Pharmaceutically acceptable esters" means those which are hydrolyzed by ester bonds, retain the biological utility and properties of the carboxylic acid or alcohol, and are not biologically or otherwise undesirable. For a description of pharmaceutically acceptable esters as prodrugs, see

Bundgaard, H., ed., Design of Prodrugs, Elsevier Science Publishers, Amsterdam (1985). These esters are typically formed from the corresponding carboxylic acid and alcohol. In general, the formation of esters can be accomplished by conventional techniques (see, for example, March Advanced Organic Chemistry, 3rd Ed) John Wiley &amp; Sons, New York (1985) ρ 1157 and cited therein. References, and Mark et al., Encyclopedia of φ Chemical Technology ^ Wiley &amp; Sons, New York (1980). The alcohol component of the ester generally comprises (1) 乂 脂 脂 脂 醇 醇 醇 醇 醇 醇One or more double bonds and may or may not contain branched carbon, or (iiJCH^2 aromatic or heteroaromatic alcohol. The invention also encompasses these as vinegar as described herein and at the same time pharmaceutically acceptable Use of a combination of both acid addition salts. "Pharmaceutically acceptable guanamine" means the biological utility and properties of the carboxylic acid or amine retained by the hydrolysis of the guanamine bond, and biologically or Other aspects are not unwanted amines. For a description of pharmaceutically acceptable guanamine as a prodrug, see Bundgaard, H., ed, Design of Prodrugs, Elsevier Science

Publishers, Amsterdam (1985). These guanamines are typically formed from the corresponding carboxylic acids and amines. In general, the formation of guanamine can be accomplished by conventional synthetic 20 techniques (see 'eg March Advanced Organic Chemistry, 3rd Ed') John Wiley &amp; Sons, New York (1985) ρ 1152 and Mark et al., Encyclopedia of Chemical Technology, John Wiley &amp; Sons, New York (1980). The present invention also encompasses these compositions which are guanamine as described herein and which are both pharmaceutically acceptable acid addition salts thereof. "on-market or therapeutically acceptable carrier" means a carrier vehicle that does not interfere with the active ingredient's activity and is not toxic to the host or patient. 5

10 15 20 "Stereoisomers" means compounds having the same molecular weight, chemical composition, and different atomic clusters. That is, some of the same chemical groups have different orientations, and therefore, when they are pure stereoisomers, they have the ability to rotate the polar surface. However, some pure stereoisomers may only have a slight knob, and the current system cannot be compensated. The peptide compound of the present invention may have - or a plurality of asymmetric post-atoms, and thus include various stereoisomers. All stereoisomers are included within the scope of the invention. "Therapeutically or pharmaceutically effective amount" as used in the compositions of the present invention means that the amount of the composition is sufficient to elicit the desired biological result. The result can be a reduction in the cause of the symptoms, symptoms or disease, or any other desired biological system change. In the present invention, this result generally relates to increasing red ball generation. The amino acid residues in the peptide are abbreviated as follows: phenylalanine is phe4F; leucine is Leu or L; isoleucine is lie or I; methionine is Met or ]VI; proline is Val or V; serine is Ser or S; proline is pro or p; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or Η; Proline is Gin or Q; asparagine is Asn or N; aminic acid is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C ; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G. In addition, Bu is Butoxy, Bzl is benzyl, CHA is cyclohexylamine, Ac is ethyl thiol, Me is sulfhydryl, Pen is penicillamine, Aib is aminoisobutyric acid, and Nva is n-proline. Abu 22 200808341 Aminobutyric acid, Thi is thienyl alanine, OBn is 〇-benzyl and hyp is hydroxyproline. Peptidomimetics or peptide analogs are also provided in addition to peptides consisting only of naturally occurring amino acids. The peptide is usually used in the pharmaceutical industry as a non-peptide drug with similar peptide properties. These types of non-peptide compounds are referred to as "peptide analogs" or "peptidomimetics" (Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger TINS ρ. 392 (1985); Evans Lu et al., J. Med. Chem. 30: 1229 (1987), which is incorporated herein by reference. The peptide mimetic structure is structurally similar to the peptide used in therapy and can be used to produce an equivalent or to promote therapeutic or prophylactic utility. In general, a peptidomimetic is structurally similar to an exemplary polypeptide (ie, a biologically or pharmacologically active polypeptide), such as a naturally occurring receptor binding polypeptide, but having one or more of those known in the art as desired. The method replaces the peptide chain with a chain selected from the group consisting of: -CH2NH-, -CH2S-, -CH2--CH2-, 15-CH=CH-(cis and trans), -c〇CH2 —, —CH(OH)CH2- and a CH2SO—, and are further described in the following references: Spatola, A. R _ Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds·, Marcel Dekker, New York, p. 267 (1983); Spatola, A·F” Vega Data (March 1983), Vol· 1, Issue 3, 20 Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) Pp. 463-468 (general review); Hudson 5 D· et al., Int J Pept Prot Res 14 : 177-185 (1979) (—CH2NH—, CH2CH2-); Spatola et al., Life Sci 38: 1243-1249 ( 1986) (-CH2-S); Hann J. Chem. Soc Perkin Trans. I 307-314 23 200808341 (1982) (-- CH—CH—,cis and trans) ; Almquist et al., J. Med·Chem. 23 * 1392-1398 (1980) (-OOCH2 —) i Jennings-White et al., Tetrahedron Lett 23 : 2533 (1982) (— COCH2 —); Szelke et al., European Appin. EP 45665 CA (1982) : 97 : 39405 5 (1982) (—CH(OH)CH2 —); Holladay et al., Tetrahedron Lett 24 : 4401-4404 (1983) ( -C(OH)CH2 and Hruby Life Sci 31: 189-199 (1982) (-·〇Η2-8-); each of which is incorporated herein by reference. The best non-winning key is -CH2NH--. Such winning mimetics can have significant advantages over polypeptide embodiments, including, for example, greater yield, greater chemical stability, increased pharmacological properties (half-life, absorbency, strength, efficacy, etc.), Specificity changes (eg, broad-spectrum biological activity), decreased antigenicity, and others. The peptidomimetic calibration is usually by covalently attaching one or more labels directly or by a spacer (eg, amidino group) to a non-interfering position of the peptidomimetic, which can be quantified by quantitative structural activity data and/or Or a molecular model to predict. These non-interfering positions are generally in a position that does not directly contact a macromolecule (e.g., an immunoglobulin superfamily molecule), which binds to the peptidomimetic to produce a therapeutic effect. Derivatization of the peptidomimetic (e. g., calibration) should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic. In general, a peptidomimetic peptide that binds to a peptide with a high affinity and a receptor moiety has a detectable biological activity (ie, promotes or antagonizes the phenotypic changes transmitted by one or more receptors). Sex). Systematic substitutions of homologous sequences of one or more amino acids of the D-amino acid of the same type (e.g., D-isoacid substitution of L-lysine) can be used to produce more stable peptides. In addition, homologous sequences or substantially identical homologous sequence variants are included 24 200808341

The constrained peptide can be produced by methods known in the art (RiZO and Gierasch Ann. Rev. Biochem 61: 387 (1992), incorporated herein by reference); for example, by adding internal The cysteine residue of the intramolecular disulfide bridge can be formed to cyclize the peptide. 5 "Detectable label" means a substance which, when covalently linked to a peptide compound of the present invention, is capable of detecting a peptide compound in a patient to which a peptide compound has been administered. Suitable detectable labels are well known and include, for example, φ for radioisotopes, fluorescent labels (e.g., luciferin), and the like. The particular detectable label used is not critical and is selected based on the amount of label used and the labeling toxicity at the amount of label used. Relative to these factors, the selection of the target has been well known to the technology. ^ The covalent attachment of a detectable label to a peptide compound can be accomplished by the methods employed in the art. For example, when a radioisotope is used as a detectable label, the covalent attachment of ΐ25ί to the peptide compound can be achieved by combining the amino acid and the peptide compound, and then the peptide compound. Similarly, using the chemistry of driving, you incorporate 32 Å into the peptide compound as a phosphate group, for example, via a radical group on the peptide compound. The present invention provides binding and activation of TP0-R or otherwise as τρ(10);

r is different. The invention also provides a peptide compound comprising an effective amount of Τρ〇 and more specifically for the treatment of the poor 25 200808341 ^ Τ 7 胜 the peptide compound of the invention can also be administered to warm-blooded animals, including humans, to activate TPO-R in vivo. Accordingly, the present invention encompasses a method of treating ash depletion comprising administering an effective amount of a peptide compound of the present invention to mimic the effect of 5 TPO-R in vivo. The activity of the peptide compound of the present invention can be evaluated in vivo or in vitro, for example, one of several modes described in McDonald Am. J. of Pediatric Hematology/Oncology 14: 8-21 (1992), which is incorporated by reference. The manner of this is incorporated herein, or the analysis disclosed therein. According to one embodiment, the composition of the invention is used to treat anemia associated with bone marrow infusion, radiation therapy, and chemotherapy. The peptide compound is typically administered prophylactically prior to chemotherapy, radiation therapy or bone marrow transplantation, or after administration. Accordingly, the present invention also provides a pharmaceutical composition comprising an active ingredient, at least one of the present invention, in combination with a pharmaceutical carrier or diluent. The peptide compound of the present invention can be administered orally, pulmonaryly, parenterally (intramuscularly, intraperitoneally, intravenously (IV) or subcutaneously), inhaled (via fine powder formulation), skin penetration, intranasal, vaginal The rectal or sublingual route of administration is administered and can be formulated into a dosage form suitable for each route of administration. See, for example, Bernstein et al., PCT Patent Application Publication No. WO 93/25221; Pitt et al., PCT Patent Publication No. WO 94/17784; and Pitt et al., European Patent Application No. 613,683, Each is incorporated herein by reference. Solid dosage forms for oral administration include capsules, lozenges, tablets, powders and granules. In this solid dosage form, the active peptide compound can be combined with at least one inert carrier, such as a sugar, a milk mask or a temple powder. This dosage form may also contain 3 (in general practice) other than inert diluents, such as the town of stearic acid. In the case of a capsule or a tablet, the dosage form may also include a buffer. Tablets and tablets can also be prepared by using a casing film. 5 — Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents (e.g., water or saline) conventionally employed in the art. In addition to such diluents, the compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents and perfuming agents. The preparations of the invention for administration (iv) include g-free solutions or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as eucalyptus oil and corn oil, animal glue, and injectable organic vinegars such as oleic acid vinegar. These dosage forms may also contain adjuvants such as preservatives, elixirs, emulsifiers and dispersing agents. It can be sterilized by, for example, passing the bacteria 15 through the bacteria, treating the composition, irradiating the composition with radiation, or heating the sigma. It can also be prepared by using sterile water or _ some other sterile injectable vehicle. The composition for direct or vaginal administration is preferably a suppository which may contain, in addition to the active ingredient, an excipient such as cocoa butter or a plug. Compositions for intranasal or sublingual administration are also prepared using standard excipients well known in the art. '〇 The composition of the present invention can also be microgelled by, for example, Tiee and Bibi (Treatise on Controlled Drug Delivery, ed·A·Kydonieus,

Marcel Dekker, RY· (1992), pp 315_339). Compositions containing a peptide compound can be administered for prophylactic and/or therapeutic treatment. For therapeutic use, the composition is administered to a patient already suffering from a condition as described above, in an amount sufficient to treat or at least inhibit the symptoms of the disease and its complications. A quantity sufficient to accomplish this is defined as "a therapeutically effective agent". The effective amount for this purpose will depend on the severity of the disease and the weight and general condition of the patient. For prophylactic applications, a composition containing a peptide compound of the present invention is administered to a patient susceptible to or at risk of having a particular disease. This amount is defined as "preventive effective dose." For this purpose, the exact amount will still depend on the health and weight of the patient. The amount of TPO promoter required for effective treatment will depend on a number of different factors, including the mode of administration, the location of the subject, the physiological condition of the patient, and other pharmaceuticals administered. Therefore, it should be titrated into a therapeutic agent that is optimized for safety and efficacy. Typically, the dosages used in vitro provide useful guidance in the amounts used to administer these agents in situ. Animal testing for an effective dose for the treatment of a particular condition will further provide predictive guidance for human dosage. Various considerations are described, for example, in Gilman et al. (eds), Goodman and Gilman, s: The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press (199 Ό); and Remingt〇n's Pharmaceutical Sciences, 7th ed., Mack Publishing. Co., Easton, Pa. (1985); each of which is incorporated herein by reference. The peptide compound of the present invention is effective for treating anemia when administered in a dose ranging from 1 ug to about 300 ug/kg body weight per day. The particular dose used will be adjusted according to the route of administration and by the participating physician in accordance with, for example, the severity of the patient's symptoms, age, and general condition. 28 200808341 [Embodiment] Example Animal model The utility of the PEGylated TPO compound 1 in mice treated with carboplatin 5 was observed. For all of the examples herein, a storage solution of 1 〇 mg/ml PEGylated τρ〇 No. 1 compound was prepared in sterile saline. The preparation was placed in a rotary shaker at 200 rpm for 15 minutes. This oxime method is used to dissolve the PEGylated TPO Compound 1 without foaming. This solution was filtered using a GVMillex ((X22um) filter. The solution was then prepared from the stock solution using no sputum saline. The stock solution and the solution were freshly prepared on the day of use. Example 1 After treatment with carboplatin, the effect of PEGylated TPO compound 1 on lean tolerance and severity was measured by hemoglobin, red blood cell count and hematocrit 15. In this study, a dose of carboplatin was given in one day. Thereafter, the amount of poly-glycolated TPO compound 1 administered to the mice was increased, and a dose-dependent effect on various red blood cell parameters was determined. The mice were administered intraperitoneally on days -2 and -1 as follows. Carboplatin 20 or vehicle (phosphate buffered saline, PBS). The optimal dose of carboplatin used to induce thrombocytopenia in BALB/c mice is a separate total dose of 120 mg/kg as described above for two consecutive days. Injectable for administration (ie 2x60 mg/kg). One day after the second dose of carboplatin, the petrol group was injected IV (-) with pegylated TP0 compound! or vehicle (sterile saline, 29 200808341) SS, no preservatives 〇·9% sodium chloride), Table 1 in the weight-based dose is administered as a reference (looumog weight) ϋ treatment group.:

Gp 10 20 20 20 20 Pre-treatment (9)), Day-2 &amp; -1 Day Test Item Admixture (iv) Day 0 Collection of Blood Wave Agent (PBS) Vehicle (SS) Pseudo Agent (Sham) Card Circulator (SS) pseudo-agent (Sham) on 5th, 7th, 9th and 11th day Eut5 mice one-one - carbomer PEGylated TPO compound 1 300ug/kg on the 5th, 7th, 9th and 11th day Eut5 Mouse card PEGylated TPO compound 1 lOOOug/kg on day 5, 7, 9 & 11 days Eut5 mice PEGylated TPO compound 1 3000ug/kg on days 5, 7, 9 & 11 Eut5 mice ^——---

Gp=group; Exit = euthanasia

On days 5, 7, 9, and 11, five mice from each of the test groups were weighed and then euthanized using C02-asphyxiation and bled by cardiac puncture. The gold sample was transferred to a separate EDTA (lavender-top) microcontainer for blood evaluation. Mice in the control group (5) were treated on days 5 and 11. The results are shown in Figure 1-4. The data is shown as a graph of group average + SEM. Mice treated with carboplatin alone caused about a 20% reduction in hemoglobin by day 11. This reduction can be inhibited by treatment with all doses of PEGylated TPO 30 10 200808341 1:1. The decrease in the RBC number and the specific volume of the specific volume is also related to the treatment of platinum, which can be inhibited by chemotherapy with PEGylated τρ〇 compound i; however, there is no statistical assessment of this effect. . Single 5, carboplatin or carboplatin plus various doses of PEGylated TPO compound 1 = mice in all groups treated, compared to the body weight collected on day 0 'in the 5th, 7th and 9 days experienced weight loss. During the η-day study, analysis of body weight measurements in a subgroup of mice showed that treatment with carboplatin alone caused a % weight loss&apos; and in all dose trials, PEGylated hydrazine compound 1 promoted recovery from lost body weight. · 10, mice treated with carboplatin began to appear appearance and behavior changes before the fifth day. Some mice have a back bow posture and appear to be weak. Many mice also have areas of genital contamination. Treatment with the polyethylene glycol tTp〇 compound 降低 reduced the occurrence, frequency and severity of these phenomena in a dose-dependent manner. 15 Example 2 Lu test PEGylation Ο Compound 1 should be sensitized to the possibility of carboplatin-treated virulence mice. In this study, a dose of f-polyethylene glycolated TPO Compound 1 was administered seven days prior to treatment with cardin or immediately after cardinal therapy. The other group was treated with poly 20 PEGylated TPO Compound 1 before and after carboplatin administration. The effects of these doses on blood parameters were also observed. Groups of mice were treated by ip administration on a 7th and 8th day with a cardin or vehicle (lindic buffered saline, PBS). The optimum dose of carboplatin used to induce thrombocytopenia in BALB/c糸 mice is as described above as a separate total dose of 12〇mg/kg 31.200808341, administered as two consecutive days of injection (ie 2x6〇mg/kg) ). The mice were treated with IV (once) injection of pegylated TPO Compound 1 or vehicle (septic saline, SS) seven days before the first carboplatin administration or one hour after the second carboplatin dose. , no preservatives 0.9% sodium chloride) treatment, as described in Table 2. Another group was treated with pegylated τρ〇 Compound 1 before administration of carboplatin (days) and after administration (day 8, t 2 hours). All doses were based on body weight (100 ul/l 〇 g body weight). Table 2 Research

Gp N giver (iv) Day 0 carboplatin (CBPL) [60mg/kg, q2d], (ip), 7th & 8th day of administration (iv) Day 8 (2ndCBPL after lh) Collect blood But 5 Mouse a 1 10 : vehicle (*ss) vehicle (PBS) vehicle (*SS) 14 &amp; 26 2 20 vehicle (*ss) card uranium vehicle (*ss) 14,18,22 &amp 26 3 20 PEGylated TPO compound 300ug/kg uranium vehicle (*ss) 14,18,22 &amp; 26 4 20 vehicle (*ss) card uranium PEGylated TPO compound 1 300ug/kg 14 , 18,22 &amp; 26 5 20 PEGylated TPO compound 300ug/kg carboxylated pegylated TPO compound 1 300ug/kg 14,18, 22 &amp; 26 32 10 200808341 on 14, 18, 22 and 26 On the day, five mice from each experimental group were weighed and then euthanized using C02-asphyxiation and gold was delivered via cardiac puncture. The jk fluid sample was transferred to a separate EDTA (lavender-top) microcontainer for evaluation. Mice in the control group (5) were treated on days 14 and 26. 5 The results are shown in Figure 5-8. The data is shown as a graph of group average + SEM. The mice treated with carboplatin alone caused hemoglobin, RBC number and hematocrit drop (lower than about 18%) in surviving mice compared to the control group on days 18 and 22. These reductions were inhibited by administration of pegylated TPO Compound 1 on Day 8 (1 hour after administration of the second dose of carboplatin), with or without additional administration of pegylated TPO Compound 1 on Day 10; However, the administration of PEGylated TPO Compound 1 only on Day 3 did not affect the changes in red 1 blood bath parameters caused by these carboplatin. All control mice underwent normal weight gain between day 7 and day 26, while in the same period, all mice treated with carboplatin alone had a small decrease in body weight (average about 4%). . All mice in the co-treatment group with carboplatin and PEGylated τρ〇 compound 1 maintained their original body weight or experienced normal weight gain between days 7 and 26. During the study period, analysis of body weight measurements showed that carboplatin treatment was the main cause of weight loss, while = pegylated bismuth compound 丨 synergistic treatment prevented weight loss, however, there was no statistical effect on this effect. evaluation of. Differences in body weight observed on day 7 (administration of carboplatin, dentin and day 26 (end of study), such as "w丄, w Λ _ δ". All controls were normal during the study period. The mice with carboplatin alone began to appear back-bow and appear rough and behavioral changes as early as the 12th day. Many mice receiving uranium (no or PEGylated 33 200808341 TPO Compound 1 treatment) In the second half of the study, the back arched posture appeared and appeared rough. On the 8th day, treatment with PEGylated sputum compound 1 did not seem to have any additional treatment on day 0, which seemed to delay the onset of these phenomena. Treatments on days 0 and 8 reduced the severity and duration; however, a detailed analysis of the efficacy of the full 5-body observation was not performed. Example 3 PEGylation at various times after carboplatin treatment After the dose therapy of bismuth compound, the effect of PEG compound 1 on the duration and severity of the poor drug was observed. In this study, one (1) hour, one (1) after the dose of carboplatin was administered. Day or four (4) days, given A certain amount of PEGylated hydrazine Compound 1. Groups of mice were treated with carboplatin or vehicle (phosphate buffered saline, PBS) by ip administration as described below. The optimal dose of carboplatin to induce a reduction in the platelet of BALB/c 15 mice is as described above as a separate total dose of 120 mg/kg, administered as two consecutive days of injection (ie _ 2 x 60 mg/kg). One hour (day 0), one day (day 0) or four days (day 4) after the carboplatin dose, IV (-) injection of pegylated TP0 compound l (300 ug / kg) or The vehicle (sterile saline, SS, no preservative 0.9% 20 sodium chloride) was treated as described in Table 3. All the doses were based on body weight (100 ul/10 g body weight). 34 200808341

Table 3

Gp N pre-treatment [2x60mg/kg], (ip), -1 &amp; day test project (iv) collection of blood Eut 5 mice 曰 1 10 vehicle (PBS) vehicle (SS) pseudo-agent 6th & 12th day 2 20 card uranium vehicle (SS) pseudo-agents 6,8,10 & 12 days 3 20 card PEGylated TPO compound 1 No. 300ug/kg, day 0, sixth, 8, 1〇&amp;12 days 4 20 card start PEGylated TPO compound 1 No. 300ug/kg, day 1 6th, 8th, 10th &amp; 12 days 5 20 carboplatin PEGylated TPO compound 1 300ug/kg, Day 4, 6, 8, 10 & 12 days - ^

Gp = cohort; Eut = euthanasia 5 On days 6, 8, 10 and 12, five mice from each experimental group were weighed' and then euthanized using C02-asphyxiation and bled via cardiac puncture. Blood samples were transferred to separate EDTA (lavender-top) microcontainers for blood evaluation. Mice in the control group (5) were treated on days 6 and 12. The results are shown in Figure 1-4. The data is shown as a graph of group average + SEM. Mice treated with carboplatin alone caused a significant decrease in hemoglobin, RBC, and hematocrit (about 47%) in surviving mice (2 mice) compared to the control group. These reductions can be inhibited by administering pegylated butyl PO compound 1 on day ( (after treatment with carboplatin! 35 10 200808341 hours) and day 1; however, PEGylated TPO compounds were administered on day 4 One, can not affect the changes in red blood cell parameters caused by these carboplatin. Mice in all groups with uranium or uranium plus various doses of PEGylated TPO Compound 1 / Oral Treatment, compared to the body weight collected on Day _1, in Brothers 6, 8, It took 10 and 12 days to lose weight. Analysis of body weight measurements during the study showed that carboplatin was a major cause of weight loss. Administration of a pegylated TPO compound on Days, Days 1, or Day 4 did not appear to cause weight loss associated with carboplatin treatment, however, no statistical analysis was performed. The decrease in body weight observed between day -1 and day 10 is shown in the figure. 15 20 All mice in the control group looked normal during the study period. The mice treated with carboplatin alone were on the 12th day. Beginning with frequent occurrence of dorsal arches and appearance of rudeness, it was expected that the mice that received the card (no or with PEGylated sputum compound 1) showed a back arched posture in the second half of the study. There is no domain. There is also a _ filth area in the bronchitis. Others are less: the phenomenon of appearance includes weakness, eye drop and abnormal control. To: 乙二醇 乙二醇 ΤΡΟ ΤΡΟ [ [ 冶 ' 似乎 似乎 似乎 似乎 似乎 似乎 似乎 似乎 似乎Or the severity does not have the effect of (4); the money is not analyzed. The anemia caused by the pegylated τρ〇 compound _ cardogen is given within 24 hours of the animal chemotherapy. 5 The substance has a bone ship protection effect not limited to the megakaryocyte lineage. Fantasy 4 Alcoholized TPO compound 1 Measurement of polyethylene 36 by observing changes in gold liquid parameters 200808341 As a megakaryocyte and erythrocyte lineage in mice treated with uranium The ability of the factor. In the previous study, a polyethylated TPO compound 1 at a dose as low as 300 ug/kg has been found to prevent anemia caused by carboplatin. In this study, a low dose of a pegylated TPO compound was detected. The dose response of ι (ie 3〇, 100 and 300 ug/kg) in the erythrocyte lineage to characterize this effect. The mice group was administered by ip on days -1 and 〇 day as follows Carboplatin or vehicle (phosphate buffered saline, PBS). The optimal dose of carboplatin used to induce the reduction of platelets in BALB/C mice is the continuous total dose of 120 mg/kg as determined above. Two days of injection (ie 2x60mg/kg). About one hour after the second dose of carboplatin, iv (once) injection of pegylated TPO compound 1 or vehicle (sterile saline, SS, no preservative) Treatment with 0.9% sodium chloride), as described in Table 4. All formulations were based on body weight (100 ul/10 g body weight). 15 Table 4 &amp; Group 1

Gp N pre-treatment (iP), day -1 and day 0 test item giver (iv) day 10 collect blood 1 10 vehicle (PBS) vehicle (SS) pseudo-agent on day 6 &amp; 12 days Eut5 only ' ^ 2 15 Carboagent (SS) pseudo-agent on day 6, 8 & 12 days Eut5 mice 37 200808341 3 15 Card uranium polyethylene glycol 彳匕 TPO compound 1 Compound 30ug/kg on 6,8&amp;12 Day Eut5 mouse 4 15 card PEGylated TPO compound 1 Compound 100ug/kg on the 6th, 8th & 12th day Eut 5 mice 5 15 card PEGylated TPO compound 1 300ug/kg 6, 8 &amp; 12 days Eut 5 mice

Gp = group; Eilt euthanasia on the 6th, 8th and 12th days of the brothers. Five of the experimental groups were weighed and then euthanized using C〇2-choking and bled by cardiac puncture. Blood samples were transferred to separate EDTA (lavender-top) microcontainers for blood evaluation. Mice in the control group (5) were treated on days 6 and 12. The results are shown in Figure 13-14. Mice treated with carboplatin alone caused a greater than 25% reduction in the amount of jk pigment in mice by day 11. This reduction is completely inhibited by treatment with all doses of polyethylene glycol 10 TPO Compound 1. The PEGylated TPO compound 1 ίο can also effectively inhibit the number of RBC and hematocrit caused by carboplatin treatment. Mice in all groups treated with carboplatin alone or with carboplatin plus various doses of PEGylated guanidine compound 1 were measured at 6, 8, and 12 relative to body weight collected on Day-Day. The sky has been weight loss. Analysis of body weight measurements during the 13-day 15 study period showed that significant weight loss was achieved with Kaming treatment alone. In this study, the PEGylated τρ〇 compound 丨 did not appear to affect weight loss and recovery in 2008 200841. Treated with carboplatin alone, &amp; ^ ^ change. Some mice present a soft appearance in appearance and behavior. A few animals two! = Potential: obvious f绮. Xu-diol τ τ complex u ^ rr ^ · ^ number of bloody stools. In order to gather the occurrence, frequency and severity of these phenomena. It is low and polyethylation TPO is determined by the number of platelets in the surrounding gold liquid and other blood parameters. The substance 1 functions as a means of maintaining the red blood cell spectrum in the pet gas by the card. All doses of PEGylated τρο compounds have been found to completely prevent anemia caused by card turnover. These results show that 桉1 = and erythrocyte lineages are PEGylated τρο compound k "maintaining the heart, and the field should be differentially sensitive / reactive. /" Political example 5 as follows: the small gas group is separated by ten days The second round of chemotherapeutic agent (spinning) treatment "mother wheel" consists of two consecutive days of cardinal (ie mg/kg/day, administered on days -1 &amp; 0 and 10 &amp; 11 days). The maximum tolerated dose of the small amount of milk in the carboplatin used in these survival studies (i.e., 120 mg/kg; continuous administration at 60 mg/kg/day for two days). One hour after the second dose of carboplatin in each round, PEGylated TPO compound 1 (l〇〇ug/kg) or vehicle (septic saline, SS, no preservative 0.9%) was injected at 1V (once). Sodium chloride) treatment, as described below. The doses administered are based on body weight (100 g/10 g body weight). 39 200808341 Study design: Gp N pre-treatment (ip), _1 &amp; 0 day 10 & 11 day trial project dose (iv) 2nd CBPL after each dose ~lh Collection of blood Eut 5 mice 1 25 vehicle (PBS) vehicle (SS) pseudo-agent, 0th, 11th, 7th, 10th, 18th, 21st, & 28th day 2 25 card uranium (70 mg/kg) vehicle (SS) pseudo-agent, Day 0, 11th, 7th, 10th, 18th, 21st, & 28th day 3 25 card start (70 mg/kg) PEGylated TPO compound 1 100ug/kg, 0th &amp; 11th day 7th, 10th , 18, 21, & 28 days, on days 7, 10, 18, 21, and 28, five mice from each of the experimental groups (25 small, / groups) were euthanized using C〇2_ asphyxiation and Bleeding through the heart ^. The blood sample was transferred to a separate EDTA (lavende M〇p) micro-valley for evaluation. The money of the control group was treated with vehicle = in the form of phase. The results are shown in Figure 15. During the period from 1G to 21 days, the death of the two ==f=f blood and the death of the second and the second death in the fourth (four) day occurred in the poor 1 2 = off 2, most of the anemia can be made Miscellaneous by high-volume chemotherapy; ^ display;; 'suppressed by the sleeve of 15 compound 1 can be increased by preventing the occurrence of anemia 200808341 Example 6 on the mechanism study, the mouse group as a vehicle or incremental card Platinum 9 (i.e., 60, 70 or 80 mg/kg) was continuously treated for 2 days (Day-1 &amp; Day 3). About one hour after the second dose of carboplatin, the mice were injected IV (-) with pegylated TPO compound 1 (l〇〇Ug/kg) or vehicle (sterile saline, SS, no Preservative 0.9% sodium chloride), as shown in Table 5. table 5

Gp --- Ν carboplatin (CBPL) on the -1 &amp; 0 day dose (ip) treatment (iv) on the 8th day (2ndCBPL after lh) collecting jk solution / analysis of euthanasia 3 mice 曰 1 —— — 3 Vehicle (PBS) Vehicle (*ss) Day 15 2 3 Card Turn (60mg/kg) Vehicle (*ss) Day 15 3 —-~ — 3 Card Turn (70mg/kg) Vehicle ( *ss) Day 15 4 --— 3 Card Start (80mg/kg) Vehicle (*ss) Day 15 5 3 Card (60mg/kg) Pegylated TPO Compound 1 (100ug/kg) 15th Day 6 —----- 3 Card start (70mg/kg) Pegylated TPO Compound 1 (100ug/kg) Day 15 7 3 Cardin (80mg/kg) Pegylated TPO Compound 1 Day 15 (100 ug/kg) On day 15, all treatment groups of mice were euthanized using C02-asphyxiation and bled via cardiac puncture. Blood samples were transferred to separate 10 200808341 EDTA (lavender-t〇P) microcontainers for blood supply. In addition, several organs (including the brain) of the small breast milk treated with 2×7 Gmg/kg (without or with PEGylated τρ〇 compound 1 synergy/oral therapy) were isolated and treated for the group 5

10 15 20 Weaving test. These tissue fractions were immunohistochemically processed for fibrinogen/fibrin detection. The small gas treated with the card surface of ~Li, caused a significant decrease in the number of platelets in 2x7〇mg/kg mice on the first day, and the small blood specific volume (HCT) treated at 60 and 70 mg/kg (separately). A dose-dependent decrease. These reductions in the number of platelets and RBCs caused by B can be completely inhibited by the treatment of PEGylated TPQ compounds. It should be noted that all 2x8〇mg/kg wei (single sputum study knot) 胄 dead money to euthanize (death). Interestingly 'all treated with 2x8Gmg/kg Wei and PEGylated TPO Compound 1 The mice survived to the end of the scheduled study and did not have platelet reduction or poorness at 15 days. Brain histological evaluation of the control mice showed normal small tubes. Many blood vessels contained red blood cells and had Dim fibrinogen contamination. Dim, intravascular fibrin eggs from pro-fibrin staining _ will - in the rhyme group mice, because fibrinogen is a normal a Li component. Treated by Cabot alone (2x70mg / The brain from mice of kg) contains small blood vessels that are completely blocked by the close contamination of fibrinogen/fibrin material. These are gray plugs often observed in the tissues of all mice in this dose group. The small blood vessels of the brain obtained from mice treated with carboplatin and PEGylated TPO Compound 1 showed normality, or had fibrinogen/fibrin staining that was only slightly darker than the control group. Only found in the group A microthrombotic.'', 42 200808341 These findings suggest that microthrombies are caused by chemotherapy, and because microthrombus is thought to be caused by mechanical dissociation of RBCs, it may be that these vascular events cause chemotherapy-induced anemia. In addition, the ability of the PEGylation compound i to prevent the development of these recorded events can be expected to be the element-like component, whereby the agent prevents the occurrence of anemia caused by chemotherapy. Finally, these thrombotic events may also cause high doses to be received. The mortality rate of chemotherapeutic animals. Therefore, the ability of PEGylated TPO compounds to prevent these gold plug events from occurring can be responsible for increasing the survival of these high-dose chemotherapy and pharmaceutical animals. Figure 18A and Figure 18B show PEGylation The effect of Tp〇 compound on platelet and hematocrit of cardiotherapy 1 + mouse [as described in Example 6] Figure 19 shows that administration of PEGylated τρ〇 compound i reduced carbotherapy Fibrinogen deposition and blood clots in the brain of mice, as shown in Example 6. Proposed effect 15 ® 16 series shows the PEGylated TPO compound from Saskatchewan! Anti-anemia effect Using the mechanism. As seen in Figure 16, 'chemotherapy causes small endothelial damage and inhibits hematopoiesis. In the absence of PEGylated τρ〇 compounds, when circulating platelets become activated due to altered endothelial cells and deposit on small vessel walls At the time, the reduction of small platelets was rapidly developed. This process was caused by the altered 2 〇 platelets produced by the compromised bone marrow. These activated platelets were deposited in damaged blood vessels due to fibrin deposition and developed into microvascular thrombi. These microvascular thrombosis mediate the mechanical destruction of red blood cells, causing the occurrence of anemia caused by chemotherapy. Since the synergistic treatment of beta-alcohol oxime compound 1 inhibited the damage caused by chemotherapy to the vascular endothelium, and / or improved the circulation to the plate Antithrombotic and anterior fibers 43 200808341 ΐ敫 / knife! ^ sex. Microvascular gold plugs cannot develop and maintain the structure of red blood cells. 4 B-alcoholized TP 〇 compound 丨 for the use of bone megakaryocyte precursors to promote the formation of normal platelets. Hemostasis is maintained and prevents anemia. The π-line shows the effect of certain lineages of the sputum on the hematopoietic cells of PEGylated τρ〇 compound 1. EXAMPLE 7 Binding Analysis The activity of a compound can be measured using standard relative luminescence unit analysis techniques. This assay is applied, for example, to murine engineered cells that stably express human receptors and to fluorescent receptor structures driven by the f0s promoter. This analysis can be carried out by exposing plasma to hTp〇r fbs/lux cells, C-mpl (hTPOr) and fluorescent receptor structures exhibiting human Tp〇 receptors in a concentration of 15 or more peptide compounds. 1δ hours. The cells were then cultured in medium containing fluorescein and the luminosity of the cells was measured using a luminometer. As shown in Figure 20, the polyethylated oxime 0 compound activated Baf/3 cells recombinantly expressing human TP0_R in a dose-dependent manner. As shown in Figure 21, when the cells were stimulated with PEGylated TPO Compound 1, a stronger TP0-R activation than the same concentration of TP0 was observed. The PEGylated τρο Compound 1 has an EC5 〇 of approximately 5 pM. While the above is a preferred embodiment of the invention, it is understood that the modifications and variations of the invention may be made without departing from the spirit and scope of the invention. 44 200808341 PEGylated TPO compound Ethylene glycolated TPO compound 1 pair [Simplified illustration] _ Figure 1 shows the therapeutic effect of hemoglobin amount as in Example 1. Figure 2 shows the example as in the example 1

10 15 Therapeutic effect of hemoglobin. Fig. 10 shows the therapeutic effect of the B-red ball number as described in Example 3. Figure 11 shows the therapeutic effect of B on hematocrit as described in Example 3. Figure 12 shows the PEGylated TPO compound as described in Example 3, B. The therapeutic effect of PEGylated TPO Compound 1 PEGylated TPO Compound 1 on the number of red blood cells is described. Figure 3 is a graph showing the therapeutic effect of the compound of Example 11 on hematocrit. Figure 4 shows the therapeutic effect on body weight as in Example 1. Figure 5 is a graph showing the therapeutic effect of the amount of hemoglobin as in Example 2. Figure 6 is a graph showing the therapeutic effect of the number of sputum &amp; red blood cells as in Example 2. Figure 7 shows the therapeutic effect of hematocrit as described in Example 2. Figure 8 is a graph showing the therapeutic effect of the body weight of the household as in Example 2. Figure 9 is a diagram showing the PEGylated TPO compound 1 as described in Example 3, pegylated TPO compound 1 to pegylated TPO compound 1 to pegylated TPO compound 1 to alcoholated TPO compound 1 alcoholized τρο compound 1 Alcoholization τρο Compound 1 20 200808341 Therapeutic effect on body weight. Pegylated TPO Compound 1 Pegylated TPO Compound 1 Figure 13 shows the therapeutic effect on hematocrit as described in Example 4. 5

10 15

20 Figure 14 shows the therapeutic effect on body weight as described in Example 4. The PEGylated TPO compound is described in Figure 15 which shows the therapeutic effect of body weight as in Example #1. Alcoholized TPO Compound 1 is anti-poor. Figure 16 shows the mechanism of action of the poly-B. Fig. 17 is a graph showing the effect of the sputum of the sputum on the hematopoietic cells of the PEGylated TP oxime compound 1. Figure 18A and Figure 18 show the effect of platelet and bismuth on the treatment of uranium-treated mice with PEGylated hydrazine compound 1 as described in Example 6. Figure 19 is a graph showing the effect of PEGylated bismuth compound 1 on fibrinogen and clot clots in the brain after treatment with a sputum as described in Example 6. Figure 20 is a graph showing the effect of PEGylated guanidine compound 1 on the activation of human TPO-R in Baf/3 cells as described in Example 7. Figure 21 is a graph showing that PEGylated TPO Compound 1 activates recombinant human TCKCr^Baf/3 cells in a dose-dependent manner as described in Example 7. 46

Claims (1)

.200808341 X. Patent application scope: ^.-(4) n pure anemia method, its package reduction - effective amount of TK) peptide compound to pay for this need. 2. If you apply for a patent scope! The method of the present invention, wherein the treatment is selected from the group consisting of: _, anti-" limb radiotherapy. 3. - a method for increasing erythrocyte production, which comprises administering an effective amount of τρ〇 peptide compound To a subject. _ 4, as in the method of claim 1, wherein the object is human. 5. The method of claim 2, wherein the τρ〇 peptide compound 10 is fortunate to one or more rhTP〇 and rhIL-ll ρ are low in immunity. 6. As in the method of patent (4) ljf, the τρ〇 peptide compound has the property of improving ρκ compared to one or more of rhTP〇 and rhIL_u. The method of claim 1, wherein the effective amount is from about 1 Ug to about 300 Ug/kg body weight per day. 15 8. A pharmaceutical composition for increasing red blood cell production, comprising a TPO peptide compound and a medicine The carrier is acceptable for mixing. 109. A method for treating anemia comprising the step of administering an effective amount of a TPO peptide compound to a subject in need thereof. ίο. A pharmaceutical composition for treating anemia, which comprises The τρ〇 peptide compound is mixed with 20 pharmaceutically acceptable carriers. 11. The method of claim 3, wherein the red blood cell line is selected from the group consisting of specific red spheres such as blast cells, reticular red blood cells, and red blood cells. 12. A method for preventing anemia after treatment, which comprises administering an effective amount of 47 200808341 TPO peptide compound to a subject in need thereof, and the τρ〇 peptide compound comprises the following structure: IEGPTLRQ (2- Nal) LAAR (Sar) (SEQ ID NO: 5). The method of claim 12, wherein the treatment is selected from the group consisting of toxic agents, anti-tumor agents, and radiation therapy. 14. The method of claim 12, wherein the effective amount is from about 1 ug to about 300 ug/kg body weight per day. 15. A method of increasing red blood cell production comprising administering an effective amount of a TPO peptide The compound is administered to a subject, and the TPO peptide compound comprises the following 10 columns of structure: IEGPTLRQ(2-Nal)LAAR(Sar) (SEQ ID NO: 5). 16 The method of claim 15, wherein The red blood cell system A lineage selected from the group consisting of a heteroerythrocyte precursor cell, a reticular red gold sphere, and a red gold sphere. 15 17· A pharmaceutical composition for increasing erythropoiesis, comprising a TPO peptide compound mixed with a pharmaceutically acceptable carrier And the TPO peptide synthesis system comprises the following structure: IEGPTLRQ (2-Nal) LALAAR (Sar) (SEQ ID NO: 5). 18. A method of treating anemia comprising the step of administering an effective amount of a τρο peptide to a subject in need thereof, and the TPO peptide compound comprises the following structure: IEGPTLRQ (2_Nal) LAAR ( Sar) (SEQ ID NO: 5). 19. A pharmaceutical composition for treating anemia comprising a τρο peptide compound mixed with a pharmaceutically acceptable carrier, and the TPO peptide compound kit 48 200808341 comprises the following structure: IEGPTLRQ (2-Nal) LAAR (Sar (SEQ ID NO: 5). 20. 5 21 ίο 22 • 23 15 A method for preventing anemia after treatment comprising administering an effective amount of a TP0 peptide compound to a subject in need thereof, and the TP0 peptide compound comprises the following structure: Where MPEG is a methoxy poly(ethylene glycol) having a molecular weight of about 20,000 Daltons. The method of claim 20, wherein the treatment is selected from the group consisting of a toxic agent, an antitumor agent, and radiation therapy. The method of claim 20, wherein the effective amount is from about 1 ug to about 300 ug/kg of body weight per day. A method of increasing red blood cell production, comprising administering a stimulating peptide compound to a subject, and the τρ〇 peptide compound comprises 49.200808341 wherein MPEG is a methoxy group having a molecular weight of about 20, _ Dalton (B. 24. The method of claim 23, wherein the red blood cell line is composed of specific red blood cell precursor cells, reticulocytes, and red blood cells A pharmaceutical composition for increasing erythropoiesis, comprising a mixture of a τρ〇 peptide compound and a pharmaceutically acceptable carrier, wherein the acesulfide compound comprises the following structure: Among them, MPEG is a methoxy poly(ethylene glycol) having a molecular weight of about 2 Å and a valence. 26. A method for treating anemia comprising the step of administering a ruthenium-containing peptide compound to a subject in need thereof, wherein the acesulfide compound comprises the following structure: 200808341 wherein MPEG has a molecular weight About 2 〇, 〇〇〇 Dalton's methoxy poly (ethylene glycol). 27. A pharmaceutical composition for treating anemia comprising a τρο peptide compound mixed with a pharmaceutically acceptable carrier, and the TPO peptide compound comprises the following five columns of structures: Among them, MPEG is a methoxypoly(ethylene glycol) having a molecular weight of about 2 Å, 〇〇〇 Dalton. Ίο 28 — A method for preventing the occurrence of a poor after treatment, which comprises administering an effective amount of a TPO peptide compound to a subject in need thereof, and the Τρ〇 peptide φ compound comprises the following structure: IEGPTLRQ ( 2-Nal) LAAR(Sar) 15 \ K(NH2) / IEGPTLRQ(2-Nal)LAAR(Sar). 2. The method of claim 28, wherein the treatment is selected from the group consisting of toxic agents, anti-tumor agents, and radiation. 51. The method of claim 28, wherein the effective amount is from about 1 ug to about 300 邶 /1 per day. 31. A method of increasing red blood cell production comprising administering an effective amount of a TPO peptide compound to a subject, and the TPO peptide compound comprises the following five structures: IEGPTLRQ(2-Nal)LAAR(Sar)\IK( NH2) • / IEGPTLRQ(2-Nal)LAAR(Sar). 32. The method of claim 31, wherein the red blood cell line is a lineage selected from the group consisting of a specific red blood cell, a reticulocyte, and a red blood cell. 33. A pharmaceutical composition for increasing erythrocyte production comprising a τρο peptide compound mixed with a pharmaceutically acceptable carrier, and the TPO peptide combination 10 comprises the following structure: 20 IEGPTLRQ(2-Nal)LAAR (Sar) \ K(NH2) IEGPTLRQ (2.Nal)LAAR(Sar)/〇34· A method for treating anemia comprising administering an effective amount of a τρ〇 peptide compound to a subject in need thereof Step, and the Τρο peptide synthesis 52 25 200808341 The system comprises the following structure: IEGPTLRQ(2-Nal)LAAR(Sar) \ K(NH2) / IEGPTLRQ(2-Nal)LAAR(Sar). 35. The next pharmaceutical composition for treating anemia comprising a TPO peptide compound mixed with a pharmaceutically acceptable carrier, and the TPO peptide compound is a scaffold structure: ^ IEGPTLRQ(2-Nal)LAAR(Sar)\ 15 K(NH2) / IEGPTLRQ(2-Nal)LAAR(Sar). 53