USOO6903068B1 (12) United States Patent (10) Patent No.: US 6,903,068 B1 Stanton et al. (45) Date of Patent: *Jun. 7, 2005
(54) USE OF COLOSTRININ, CONSTITUENT OTHER PUBLICATIONS PEPTIDES THEREOF, AND ANALOGS THEREOF FOR INDUCING CYTOKINES Kruzel et al. (Dec. 2001) “Towards an Understanding of Biological Role of Colonstrinin Peptides.” Journal of (75) Inventors: G. John Stanton, Texas City, TX (US); Molecular Neuroscience 17(3): 379–389.* Thomas K. Hughes, Jr., Galveston, TX Inglot, Junsz, and Lisowski Colostrinine: a Proline-Rich (US); Istvan Boldogh, Galveston, TX Polypeptide from Ovine Colostrum Is a Modest Cytokine (US); Jerzy A. Georgiades, Houston, Inducer in Human Leukocytes, 1996, Archivum Immuno TX (US) logiae et Therapiae Experimentalis (44) 215-224.* Elgert, "Immunology: Understanding the Immune System” (73) Assignee: Board of Regents, The University of Text (1996) Wiley-Liss 1st Ed. pp. 24–26 and 199-217.* Texas System, Austin, TX (US) Ngo et al. Computational Complexity, Protein Structure Prediction, and the Levinthal Paradox (1994) The Protein (*) Notice: Subject to any disclaimer, the term of this Folding Problem and Teriary Structure Prediction (#14), patent is extended or adjusted under 35 491-495. U.S.C. 154(b) by 488 days. Wells Addivity of Mutational Effects in Proteins (1990) Biochemistry (29): 37,8509-8517.* This patent is Subject to a terminal dis- Babbit, ed., The Vanderbilt Rubber Handbook, R.T. Vander claimer. bilt Company, Inc., Norwalk, CT, pp. 344-397 (1978). Bespalov et al., “Fabs Specific for 8-oxoguanine: control of (21) Appl. No.: 09/641,801 DNA binding.” J Mol Biol. Nov. 12, 1999:293(5):1085–95. (22) Filed: Aug. 17, 2000 Blach-Olszewska et al., “Stimulatory effect of ovine colos e -- is trinine (a proline-rich polypeptide) on interferons and tumor Related U.S. Application Data necrosis factor production by murine resident peritoneal cells.” Arch Immunol Ther Exp (Warsz). 1997; 45(1):43–7. (60) Continuation-in-part of application No. 10/281,652, filed on Buescher et al., “Clostral antioxidants: Separation and char Oct. 28, 2002, which is a division of application No. acterization of two activities in human colostrum, J Pediatr 09/641,803, filed on Aug. 17, 2000, now Pat. No. 6,500,798. Gastroenterol Nutr1. Jan. 1992; 14(1):47-56. (60) Provisional application No. 60/420,369, filed on Oct. 22, 2002, and provisional application No. 60/149,311, filed on Calingasan et al., "Protein-bound acrolein: a novel marker Aug. 17, 1999. of oxidative stress in Alzheimer's disease,” J. Neurochem. Feb. 1999;72(2):751–6. (51) Int. Cl." ...... A61K 38/00; A61K 38/02; Chao “Growth factor signaling: where is the specificity'?” A61K 38/08; A61K 38/18 Cell. Mar. 20, 1992; 68(6): 995–7. (52) U.S. Cl...... 514/2; 514/12; 514/13; Esterbauer et al., “Chemistry and biochemistry of 4-hydrox 514/14: 514/15; 514/16; 514/17; 530/300; ynonenal, malonaldehyde and related aldehydes, Free 530/324; 530/326; 530/327; 530/328; 530/329 Radic Biol Med. 1991;11(1):81–128. (58) Field of Search ...... 514/2, 12, 13, Fields et al., Synthetic Peptides. A User's Guide, W.M. 514/14, 15, 16, 17, 18; 530/300, 324, 326, Freeman & Company, New York, NY, pp. 77-183 (1992). 327, 328,329, 350, 334; 424/85.1,535 Fillmore et al., “Differentiation of PC12 cells with nerve growth factor is associated with induction of transin Syn (56) References Cited thesis and release,” J Neurosci Res. Apr. 1992;31(4):662-9. U.S. PATENT DOCUMENTS Gabbita et al., “Increased nuclear DNA oxidation in the brain in Alzheimer's disease,” J Neurochem. Nov. 4,938,949 A 7/1990 Borch et al. 1998;71(5):2034–40. 6,040,1805,595,887 A 3/20001/1997 JoheCoolidge et al. Gabbita et al., “Decrease66 in peptide methionine Sulfoxide 6,410,058 B2 * 6/2002 Gohlke et al...... 424/535 reductase in Alzheimer's disease brain, J Neurochem. Oct. 6,500,798 B1 * 12/2002 Stanton et al...... st 1999;73(4): 1660–6. 2003/009 1606 A1 5/2003 Stant t al. f f aO e a (Continued) FOREIGN PATENT DOCUMENTS Primary Examiner Elizabeth C. Kemmerer JP O6 041191 A 2/1994 Assistant Examiner-Christopher James Nichols WO WO95/OO155 1/1995 (74) Attorney, Agent, or Firm- Mueting, Raasch & WO WO95/30686 11/1995 Gebhardt, PA WO WO 98/14473 * 4/1998 s - a. al WO WO 99/65329 12/1999 (57) ABSTRACT WO WOOO/75173 12/2000 WO WO 01/11937 2/2001 The present invention discloses a use of coloStrinin, a W W 8. 2. 2: constituent peptide thereof, and/or an analog thereof as an immunological regulator and as a blood cell regulator in W W 8:45 32: animals including humans. WO WO O2/13851 2/2002 WO WO 03/33423 10/2003 37 Claims, 9 Drawing Sheets US 6,903,068 B1 Page 2
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(NAT) and glass-adherent (GAT) thymocytes in thymecto Zimecki et al., “Immunotropic properties of fractions iso mized mice,” Arch Immunol. Ther Exp, 1984:32: 191-196. lated from human milk,” Arch Immunol Ther Exp, 1984:32:203-209. * cited by examiner U.S. Patent Jun. 7, 2005 Sheet 1 of 9 US 6,903,068 B1 Fig. 1C s U.S. Patent Jun. 7, 2005 Sheet 2 of 9 US 6,903,068 B1 Fig. 2. A 5 | 150 120 90 60 30 DCF Fluorescence U.S. Patent Jun. 7, 2005 Sheet 3 of 9 US 6,903,068 B1 U.S. Patent Jun. 7, 2005 Sheet 4 of 9 US 6,903,068 B1 fig. 4 U.S. Patent Jun. 7, 2005 Sheet 5 of 9 US 6,903,068 B1 Fig. 5 B Mock-treated CN LAH U.S. Patent Jun. 7, 2005 Sheet 6 of 9 US 6,903,068 B1 U.S. Patent Jun. 7, 2005 Sheet 7 of 9 US 6,903,068 B1 Fig. 6B U.S. Patent Jun. 7, 2005 Sheet 8 of 9 US 6,903,068 B1 Annexi-PE Alexi-PE U.S. Patent Jun. 7, 2005 Sheet 9 of 9 US 6,903,068 B1 Tta. 9. 8 Apoptotic + Necrotic Cells Healthy Cells 100 80 60 40 20 s US 6,903,068 B1 1 2 USE OF COLOSTRININ, CONSTITUENT modulator Selected from the group of coloStrinin, a constitu PEPTIDES THEREOF, AND ANALOGS ent peptide thereof, an active analog thereof, and combina THEREOF FOR INDUCING CYTOKINES tions thereof, under conditions effective to accomplish at least one of the following: reduce 4.HNE-protein adduct This application claims priority to U.S. Provisional formation; inhibit 4HNE-mediated glutathione depletion; Application Ser. No. 60/420,369, filed Oct. 22, 2002, and is inhibit 4HNE-induced activation of p53 protein; or inhibit a Continuation-In-Part of U.S. patent application Ser. No. 4HNE-induced activation of c-Jun NH2-terminal kinases. 10/281,652, filed on Oct. 28, 2002, which is a Divisional of In one embodiment, the present invention provides a U.S. patent application Ser. No. 09/641,803, filed Aug. 17, method of down regulating 4.HNE-mediated lipid peroxida 2000 (issued on Dec. 31, 2002 as U.S. Pat. No. 6,500,798), tion in a cell. The method includes contacting the cell with which claims the benefit of U.S. Provisional Application Ser. a modulator Selected from the group of coloStrinin, a con No. 60/149,311, filed Aug. 17, 1999, all of which are Stituent peptide thereof, an active analog thereof, and com incorporated herein by reference. binations thereof, wherein: the active analog is an active analog of a constituent peptide of coloStrinin Selected from BACKGROUND OF THE INVENTION 15 the group of SEQ ID NO:1 through SEQ ID NO:34; the Colostrum is a component of the milk of mammals during active analog comprises a peptide having an amino acid the first few days after birth. Colostrum is a thick yellowish Sequence with at least about 15 percent proline and having fluid and is the first lacteal Secretion post parturition and at least about 70 percent Structural Similarity to one or more contains a high concentration of immunogloblins (IgG, IgM, constituent peptides of coloStrinin; and the active analog and IgA) and a variety of non-specific proteins. Colostrum does not interfere with cellular uptake of redox-Sensitive also contains various cells Such as granular and Stromal 2,7-dihydro-dichlorofluorescein-diacetate. cells, neutrophils, monocyte/macrophages, and lympho In one embodiment, the present invention provides a cytes. Colostrum also includes growth factors, hormones, method for inhibiting apoptosis in a cell (typically, due to and cytokines. Unlike mature breast milk, colostrum con DNA damage). The method includes contacting the cell with tains low Sugar, low iron, but is rich is lipids, proteins, 25 an effective amount of an apoptosis inhibitor Selected from mineral Salts, Vitamins, and immunoglobins. the group of coloStrinin, a constituent peptide thereof, an Colostrum also includes or contains a proline-rich active analog thereof, and combinations thereof. polypeptide aggregate or complex, which is referred to as In another embodiment of inhibiting apoptosis in a cell, a colostrinin (CLN). One peptide fragment of colostrinin is method is provided that includes contacting the cell with an Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro (SEQ ID NO:31), effective amount of an apoptosis inhibitor Selected from the which is disclosed in International Publication No. WO-A- group of colostrinin, a constituent peptide thereof, an active 98/14473. Colostrinin and this fragment have been identified analog thereof, and combinations thereof, wherein; the as useful in the treatment of disorders of the central nervous active analog is an active analog of a constituent peptide of System, neurological disorders, mental disorders, dementia, colostrinin selected from the group of SEQID NO:1 through neurodegenerative diseases, Alzheimer's disease, motor 35 SEQ ID NO:34; the active analog comprises a peptide neurone disease, psychosis, neurosis, chronic disorders of having an amino acid Sequence with at least about 15 percent the immune System, diseases with a bacterial and viral proline and having at least about 70 percent Structural aetiology, and acquired immunological deficiencies, as Set Similarity to one or more constituent peptides of coloStrinin; forth in International Publication No. WO-A-98/14473. and the active analog does not interfere with cellular uptake Although certain uses for coloStrinin have been identified, 40 of redox-sensitive 2,7-dihydro-dichlorofluoresce in it would represent an advancement in the art to discover and diacetate. disclose other uses for coloStrinin, or a component thereof, Other methods of the present invention include protecting that are not readily ascertainable from the information against DNA damage in a cell, and reducing the toxic effect currently known about coloStrinin or its constituents. 45 of B-amyloid or retinoic acid on a cell. These methods involve contacting the cell with an effective amount of a SUMMARY OF THE INVENTION compound Selected from the group of coloStrinin, a con The present invention relates to the use of coloStrinin, at Stituent peptide thereof, an active analog thereof, and com least one constituent (i.e., component) peptide thereof, at binations thereof. least one active analog thereof (e.g., peptide having an 50 The cell can be present in a cell culture, a tissue, an organ, N-terminal Sequence equivalent to an N-terminal Sequence or an organism. For certain embodiments, the cell is a of at least one of the coloStrinin constituent peptides), and mammalian cell. For certain embodiments, the cell is a combinations thereof, as modulators of intracellular Signal human cell. ing mechanisms. The Signaling molecules discovered to date For certain embodiments, the compound (e.g. modulator that are modulated include 4HNE adduct formation, GSH, 55 Such as an apoptosis inhibitor) is a constituent peptide of P53, and JNK. colostrinin. Preferably, the modulator is selected from the Furthermore, the present invention relates to the use of group of MQPPPLP (SEQ ID NO:1), LOTPQPLLOVM colostrinin, at least one constituent (i.e., component) peptide MEPQGD (SEQ ID NO:2), DQPPDVEKPDLQPFQVOS thereof, at least one active analog thereof (e.g., peptide (SEQ ID NO:3), LFFFLPVVNVLP (SEQ ID NO:4), having an N-terminal Sequence equivalent to an N-terminal 60 DLEMPVLPVEPFPFV (SEQ ID NO:5), MPQN Sequence of at least one of the coloStrinin constituent FYKLPQM (SEQ ID NO:6), VLEMKFPPPPQETVT (SEQ peptides), and combinations thereof, in the inhibition of ID NO:7), LKPFPKLKVEVFPFP (SEQ ID NO:8), apoptosis. Specifically, the apoptotic (cytotoxic) effect of B VVMEV (SEQ ID NO:9), SEQP (SEQ ID NO:10), DKE amyloid on SH-SY5Y neuronal cells and TNF-alpha. (SEQ ID NO:11), FPPPK (SEQ ID NO:12), DSQPPV (SEQ In one embodiment, the present invention provides a 65 ID NO:13), DPPPPQS (SEQ ID NO:14), SEEMP (SEQ ID method of modulating an intracellular signaling molecule in NO:15), KYKLQPE (SEQ ID NO:16), VLPPNVG (SEQ ID a cell. The method includes contacting the cell with a NO:17), VYPFTGPIPN (SEQ ID NO:18), SLPQNILPL US 6,903,068 B1 3 4 (SEQ ID NO:19), TOTPVVVPPF (SEQ ID NO:20), LOPE FIG. 1. Colostrinin inhibits formation of protein-HNE IMGVPKVKETMVPK (SEQ ID NO:21), HKEMPFP (i.e., 4-HNE protein) adducts. (A): 4HNE (25 nM); (B): KYPVEPFTESQ (SEQ ID NO:22), SLTLTDVEKLHL HO (100 uM); (C): CLN(10 ug/ml) pre-treatment followed PLPLVQ (SEQ ID NO:23), SWMHQPP (SEQ ID NO:24), by 4HNE (25 nM) exposure; (D): LAH (10 ug/ml) pre QPLPPTVMFP (SEQ ID NO:25), PQSVLS (SEQ ID treatment followed by 4HNE (25 nM) exposure; (E): HNE NO:26), LSQPKVLPVPQKAVPQRDMPIQ (SEQ ID protein adducts detected by Western blot analysis. Lane 1, NO:27), AFLLYQE (SEQ ID NO:28), RGPFPILV (SEQ ID 25 nM; lane 2, 12.5 nM; lane 3, 6.2 nM of 4HNE alone; NO:29), ATFNRYQDDHGEEILKSL (SEQ ID NO:30), lanes 4-6, CLN (10 ug/ml) plus 4HNE, 25, 12.5 and 6.2 nM, VESYVPLFP (SEQ ID NO:31), FLLYQEPVLGPVR (SEQ respectively. ID NO:32), LNF (SEQ ID NO:33), and MHQPPQ 1O FIG. 2. Colostrinin inhibits 4HNE-induced oxidative PLPPTVMFP (SEQ ID NO:34), and combinations thereof. stress. (A): 1, control; 2, colostrinin (10 ug/ml); 3, 4HNE (25 AS used herein, “a” or “an” means one or more (or at least uM); 4, 4HNE (25 nM) plus colostrinin (10 ug/ml); 5, one), Such that combinations of active agents (i.e., active lactalbumin hydrolysate (10 ug/ml); 6, lactalbumin hydroly oxidative stress regulators), for example, can be used in the sate (10 ug/ml) plus 4HNE (25 nM). (B): A representative compositions and methods of the invention. Thus, a com 15 position that includes “a” polypeptide refers to a composi FACS histogram of fluorescence of cells treated with 4HNE tion that includes one or more polypeptides. (25 nM) and CLN (10 ug/ml) plus 4HNE. “Amino acid” is used herein to refer to a chemical FIG. 3. Effect of CLN on 4HNE-induced loss of intrac compound with the general formula: NH-CRH-COOH, ellular GSH levels. Cells were mock-treated or treated with where R, the Side chain, is H or an organic group. Where R CLN (or LAH) and/or 4.HNE for 30 min, and is organic, R can vary and is either polar or nonpolar (i.e., o-phthalaldehyde-mediated fluorescence was determined as hydrophobic). The amino acids of this invention can be described in Materials and Methods. Open columns: 1, naturally occurring or synthetic (often referred to as mock-treated; 2, CLN (10 ug/ml)-;3, LAH (10 ug/ml)-; 4, nonproteinogenic). AS used herein, an organic group is a 4HNE (25 nM)-treated. Filled solid columns: 5, CLN (10 hydrocarbon group that is classified as an aliphatic group, a 25 ug/ml) pre- and 4HNE (25 nM)-treated for 30 min; 6, LAH cyclic group or combination of aliphatic and cyclic groups. (10 ug/ml) pre- and 4.HNE (25 nM)-treated for 30 min. The term “aliphatic group” means a Saturated or unsaturated FIG. 4. Inhibition of JNK induction by colostrinin. A linear or branched hydrocarbon group. This term is used to change in JNK's phosphotyrosine levels was monitored by encompass alkyl, alkenyl, and alkynyl groups, for example. SDS-PAGE analysis. Equal amounts of protein (50 ug) were The term “cyclic group” means a closed ring hydrocarbon fractionated, blotted, and probed with anti-phospho-(Thr group that is classified as an alicyclic group, aromatic group, 183/Tyr-185)-JNK antibody. Lanes 1 and 2, mock-treated or heterocyclic group. The term “alicyclic group” means a cells; lane 3, 8-(4-chlorophenylthio)-cAMP, an inhibitor of cyclic hydrocarbon group having properties resembling JNK activation; lanes 4 and 5, 25 nM 4HNE, lane 6, CLN those of aliphatic groups. The term “aromatic group' refers (10 ug/ml) alone; lane 7, 25 nM 4HNE plus 10 ug/ml CLN; to mono- or polycyclic aromatic hydrocarbon groups. AS 35 lane 8, 25 nM 4HNE plus 1 tug/ml CLN; lane 9, 25 nM used herein, an organic group can be Substituted or unsub 4HNE plus 0.1 lug/ml CLN. Stituted. FIG. 5. CLN reduces 4HNE-mediated activation of p53. The terms “polypeptide” and “peptide' are used inter PC12 cells were pre-treated with CLN or LAH and exposed changeably herein to refer to a polymer of amino acids. to 4.HNE. Three hours after treatment, cell lysates were These terms do not connote a specific length of a polymer of 40 analyzed by Western blot analysis. (B) p53; (A) correspond amino acids. Thus, for example, the terms oligopeptide, ing O-tubulin. 4HNE (25 nM), CLN (10 ug/ml), LAH (10 protein, and enzyme are included within the definition of lug/ml). polypeptide or peptide, whether produced using recombi FIGS. 6A-6D. (A) Normal morphology of SH-SY5Y nant techniques, chemical or enzymatic Synthesis, or natu control cells. Cells are mostly clumped, non-contact inhib rally occurring. This term also includes polypeptides that 45 ited (right arrow) with a few elongated cells present. Their have been modified or derivatized, Such as by glycosylation, refractability indicates they are healthy and growing nor acetylation, phosphorylation, and the like. mally. (B) Cells treated with Beta-amyloid (10 ug/ml added The following abbreviations are used throughout the on day 5) that show its toxicity. Note small round granulated application: cells with little refractability. (C) Differentiated SH-SY5Y 50 cells following treatment with CLN (0.1 lug/ml added on day 5 for 30 minutes). Touching cells are flat, contact inhibited A = Ala = Alanine T = Thir = Threonine (not clumped), left arrow, and more isolated cells are elon W = Wall = Waline C = Cys = Cysteine gated and neuronal in appearance, right arrow. (D) Cells L = Leu = Leucine Y = Tyr = Tyrosine protected from toxic (apoptotic effect) of Beta-amyloid by I = Ile = Isoleucine N = Asn = Asparagine 55 treating with CLN (Colostrinin 0.1 ug/ml added on day 5 for P = Pro = Proline Q = Glm = Glutamine 30 minutes+Beta-amyloid 10 ug/ml added on day 5). Cells F = Phe = Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu = Glutamic Acid are flat (upper arrow) or elongated (lower arrow) showing M = Met = Methionine K = Lys = Lysine typical morphology of differentiated cells (see FIG. 6C). (E) G = Gly = Glycine R = Arg = Arginine Inhibition of toxicity (apototic activity) of Beta-amyloid by S = Ser = Serine H = His = Histidine 60 CLN treatment (Colostrinin 3 ug/ml added on day 5 for 30 minutes+Beta-amyloid 10 ug/ml added on day 5). Note flattened (bottom arrow) and elongated (upper arrow) cells BRIEF DESCRIPTION OF THE DRAWINGS typical of SH-SY5Y differentiated cells. (F) Toxic The invention can be better understood with reference to (apoptotic) effect of retinoic acid (20 uMadded on day 1) on the following detailed description together with the 65 SH-SY5Y cells. The observed toxicity resembles cytopa appended illustrative drawings in which like elements are thology induced by viruses. Cytoplasmic bridging caused by numbered the same: Shrinking of cells once in contact with each other (upper US 6,903,068 B1 S 6 right arrows), shrunken granular cells (lower right arrow) totic (cytotoxic) effect of B-amyloid on SH-SY5Y neuronal and small round cells (lower left arrows). (G) Inhibition of cells and TNF-alpha or the apoptotic effect of retinoic acid. toxic effect of retinoic acid by treatment of SH-SY5Y with Such compounds (e.g. modulators Such as inhibitors) are CLN (20 uM retinoic acid added on day 1+1 ug/ml Colos referred to herein as “active agents.” Significantly, Such trinin added on day 5 for 30 minutes). Cells are well active agents can be administered alone or in various com organized showing typical morphology of differentiated binations to a patient (e.g., animals including humans) as a SH-SY5Y cells, elongation (lower arrow) and flattening medication or dietary (e.g., nutrient) Supplement in a dose (upper arrow). sufficient to produce the desired effect throughout the FIG. 7. Analysis of apoptosis by flow cytometry. (A) patient's body, in a specific tissue site, or in a collection of Induction of apoptosis by 4HNE (100 nM). UL, upper left; tissues (organs). UR, upper right: necrotic cells, LL, lower left: Viable cells, Colostrinin is composed of peptides, the aggregate of LR, lower right: apoptotic cells. (B) Absence of apoptosis in which has a molecular weight range between about 5.8 to mock-treated cells. UL, upper left, UR, upper right: necrotic about 26 kiloDaltons (kDa) determined by polyacrylamide cells, LL, lower left: viable cells, LR, lower right: apoptotic gel electrophoresis. It has a greater concentration of proline cells. 15 than any other amino acid. Ovine coloStrinin has been found FIG. 8. Inhibition of 4HNE-induced apoptosis by CLN. to have a molecular weight of about 18 kDa and includes PC12 cells were treated with CLN (1 lug per ml) for 15 min three non-covalently linked Subunits having a molecular and 4HNE (100 nM) was added. Twenty four hours later, weight of about 6 kDa and has about 22 wt-% proline. cells were harvested and stained with annexin V-PE and Colostrinin has been found to include a number of pep 7-AAD. 1, solvent alone; 2, 100 nM 4HNE, 3, TROLOX tides ranging from 3 amino acids to 22 amino acids or more. (vitamin E)4, col (internal control)+100 nM 4HNE; 5, CLN These can be obtained by various known techniques, includ alone (1 lug per ml); 6, CLN (1 lug per ml)+100 nM 4HNE. ing isolation and purification involving eletrophoresis and FIG. 9. Inhibition of UV-B-induced apoptosis by CLN. Synthetic techniques. The Specific method of obtaining Parallel cultures of PC12 cells were treated with CLN (1 lug colostrinin and SEQ ID NO:31 is described in International 25 Publication No. WO 98/14473. Using HPLC and Edelman per ml) or col (1 lug per ml) and exposed to LD50 of UV-B. Degradation, over 30 constituent peptides of coloStrinin Twenty four hours later, cells were harvested and stained have been identified, which can be classified into several with annexin V-PE and 7-AAD. 1, Mock-treated; 2, UV-B groups: (A) those of unknown precursor; (B) those having a (LD50); 3, col (internal control); 4, col+UV-B (LD50); 5, f-casein homologue precursor, (C) those having a f-casein CLN alone (1 lug per ml); 6, CLN (1 lug per ml)+UV-B precursor; and (D) those having an annexin precursor. These (LD50). peptides are described in International Patent Publication DETAILED DESCRIPTION OF ILLUSTRATIVE No. WO 00/75173, published Dec. 14, 2000, and can be EMBODIMENTS Synthesized according to well-known Synthetic methods. These peptides (i.e., constituent peptides of colostrinin), ColoStrinin, a complex of proline-rich polypeptides 35 which can be derived from colostrinin or chemically derived from Ovine colostrum, induces mitogenic Stimula synthesized, include: MQPPPLP (SEQ ID NO:1); LOTPQ tion and a variety of cytokines in human peripheral blood PLLOVMMEPQGD (SEQ ID NO:2); DQPPDVEKP leukocytes. It also possesses anti-oxidant activity in pheo DLOPFOVQS (SEQID NO:3); LFFFLPVVNVLP (SEQ ID chromocyltoma (PC12) cells. NO:4); DLEMPVLPVEPFPFV (SEQ ID NO:5); MPON It has been discovered that coloStrinin, at least one con 40 FYKLPQM (SEQ ID NO:6); VLEMKFPPPPQETVT (SEQ Stituent peptide thereof, and/or at least one active analog ID NO:7); LKPFPKLKVEVFPFP (SEQ ID NO:8); thereof (e.g., a peptide having an N-terminal Sequence VVMEV (SEQ ID NO:9); SEQP (SEQ ID NO:10); DKE equivalent to an N-terminal Sequence of at least one of the (SEQ ID NO:11); FPPPK (SEQID NO:12); DSQPPV (SEQ colostrinin constituent peptides) can be used as modulators ID NO:13); DPPPPQS (SEQ ID NO:14); SEEMP (SEQ ID of intracellular Signaling mechanisms. The Signaling mol 45 NO:15); KYKLQPE (SEQ ID NO:16); VLPPNVG (SEQ ID ecules discovered to date that are modulated include 4HNE NO:17); VYPFTGPIPN (SEQ ID NO:18); SLPQNILPL adduct formation, GSH, P53, and JNK. (SEQ ID NO:19); TOTPVVVPPF (SEQ ID NO:20); LQPE More Specifically, the present invention provides methods IMGVPKVKETMVPK (SEQ ID NO:21); HKEMPFP that involve: 1) reduction of the abundance of 4HNE-protein KYPVEPFTESQ (SEQ ID NO:22); SLTLTDVEKLHL adducts as shown by fluorescent microScopy and Western 50 PLPLVQ (SEQ ID NO:23); SWMHQPP (SEQ ID NO:24); blot analysis; 2) reduction of intracellular levels of ROS as QPLPPTVMFP (SEQ ID NO:25); PQSVLS (SEQ ID shown by a decrease in 2,7"dichlorodihydro-fluorescein NO:26); LSQPKVLPVPQKAVPQRDMPIQ (SEQ ID mediated fluorescence; 3) inhibition of 4HNE-mediated NO:27); AFLLYQE (SEQ ID NO:28); RGPFPILV (SEQ ID glutathione depletion as determined fluorimetrically; and 4) NO:29); ATFNRYQDDHGEEILKSL (SEQ ID NO:30); inhibition of 4HNE-induced activation of c-Jun NH2 55 VESYVPLFP (SEQ ID NO:31); FLLYQEPVLGPVR (SEQ terminal kinases. Furthermore, the present invention pro ID NO:32); LNF (SEQ ID NO:33); and MHQPPQ vides methods that down regulate the 4HNE-mediated lipid PLPPTVMFP (SEQ ID NO:34). These can be classified as peroxidation and its product-induced Signaling that other follows: (A) those of unknown precursor include SEQ ID wise may lead to pathological changes at the cellular and NOS:2, 6, 7, 8, 10, 11, 14, and 33; (B) those having a organ level. 60 B-casein homologue precursor include SEQ ID NOS:1, 3, 4, Also, the present invention relates to the use of 5, 9, 12, 13, 15, 16, 17, and 31; (C) those having a f-casein colostrinin, at least one constituent (i.e., component) peptide precursor include SEQ ID NOS:18 (casein amino acids thereof, at least one active analog thereof (e.g., peptide 74-83), 19 (casein amino acids 84-92), 20 (casein amino having an N-terminal Sequence equivalent to an N-terminal acids 93-102), 21 (casein amino acids 103-120), 22 (casein Sequence of at least one of the coloStrinin constituent 65 amino acids 121-138), 23 (casein amino acids 139-156), 24 peptides), and combinations thereof, in the inhibition of (casein amino acids 157-163), 25 (casein amino acids apoptosis, Specifically, the inhibition is related to the apop 164-173), 26 (casein amino acids 174–179), 27 (casein US 6,903,068 B1 7 8 amino acids 180–201), 28 (casein amino acids 202-208), 29 AS Stated above, active analogs of coloStrinin and its (casein amino acids 214-222), 32 (casein amino acids constituent peptides include polypeptides having Structural 203-214), and 34 (casein amino acids 159-173); and (D) Similarity. Structural Similarity is generally determined by those having an annexin precursor include SEQ ID NO:30 aligning the residues of the two amino acid Sequences to (annexin amino acids 203-220). optimize the number of identical amino acids along the A preferred group of Such peptides includes: MQPPPLP lengths of their Sequences, gaps in either or both Sequences (SEQ ID NO:1); LOTPQPLLOVMMEPQGD (SEQ ID are permitted in making the alignment in order to optimize NO:2); DQPPDVEKPDLQPFQVOS (SEQ ID NO:3); LFF the number of identical amino acids, although the amino FLPVVNVLP (SEQ ID NO:4); DLEMPVLPVEPFPFV acids in each Sequence must nonetheless remain in their (SEQ ID NO:5); MPQNFYKLPQM (SEQ ID NO:6); proper order. Preferably, two amino acid Sequences are VLEMKFPPPPQETVT (SEQ ID NO:7); LKPFP compared using the Blastp program, Version 2.0.9, of the KLKVEVFPFP (SEQ ID NO:8); and combinations thereof. BLAST 2 search algorithm, available at http:// The polypeptides of SEQ ID NOS:1-34 can be in their www.ncbi.nlm.nih.gov/gorf/b12.html. Preferably, the free acid form or they can be amidated at the C-terminal default values for all BLAST 2 search parameters are used, carboxylate group. The present invention also includes ana 15 including matrix=BLOSUM62; open gap penalty=11, exten logs of the polypeptides of SEQ ID NOS:1-34, which Sion gap penalty = 1, gapx dropoff=50, expect = 10, includes polypeptides having Structural Similarity with SEQ wordsize=3, and filter on. In the comparison of two amino ID NOS:1-34. These peptides can also form a part of a larger acid Sequences using the BLAST Search algorithm, Struc peptide. An “analog of a polypeptide includes at least a tural similarity is referred to as “identity.” Preferably, an portion of the polypeptide, wherein the portion contains active analog of coloStrinin or its constituent peptides has a deletions or additions of one or more contiguous or non Structural Similarity to colostrinin or one or more of its contiguous amino acids, or containing one or more amino constituent peptides (preferably, one of SEQ ID NOS:1-34) acid Substitutions. An “analog can thus include additional of at least about 70% identity, more preferably, at least about amino acids at one or both of the termini of the polypeptides 80% identity, and most preferably, at least about 90% listed above. Substitutes for an amino acid in the polypep 25 identity. tides of the invention are preferably conservative Colostrinin or any combination of its peptide components Substitutions, which are selected from other members of the or active analogs thereof can be derived (preferably, isolated class to which the amino acid belongs. For example, it is and purified) naturally Such as by extraction from colostrum well-known in the art of protein biochemistry that an amino or can be Synthetically constructed using known peptide acid belonging to a grouping of amino acids having a polymerization techniques. For example, the peptides of the particular size or characteristic (Such as charge, hydropho invention may be Synthesized by the Solid phase method bicity and hydrophilicity) can generally be substituted for using standard methods based on either t-butyloxycarbonyl another amino acid without Substantially altering the Struc (BOC) or 9-fluorenylmethoxy-carbonyl (FMOC) protecting ture of a polypeptide. groups. This methodology is described by G. B. Fields et al. For the purposes of this invention, conservative amino 35 in Synthetic Peptides: A User's Guide, W. M. Freeman & acid Substitutions are defined to result from exchange of Company, New York, N.Y., pp. 77-183 (1992). Moreover, amino acids residues from within one of the following gene Sequence encoding the colostrinin peptides or analogs classes of residues: Class I: Ala, Gly, Ser, Thr, and Pro thereof can be constructed by known techniqueS Such as (representing Small aliphatic side chains and hydroxyl group expression vectors or plasmids and transfected into Suitable Side chains); Class II: Cys, Ser, Thr and Tyr (representing 40 microorganisms that will express the DNA sequences thus side chains including an -OH or -SH group); Class III: preparing the peptide for later extraction from the medium Glu, Asp, ASn and Gln (carboxyl group containing Side in which the microorganism are grown. For example, U.S. chains): Class IV: His, Arg and Lys (representing basic side Pat. No. 5,595,887 describes methods of forming a variety chains); Class V: Ile, Val, Leu, Phe and Met (representing of relatively Small peptides through expression of a recom hydrophobic side chains); and Class VI: Phe, Trp, Tyr and 45 binant gene construct coding for a fusion protein which His (representing aromatic side chains). The classes also includes a binding protein and one or more copies of the include related amino acids Such as 3Hyp and 4Hyp in Class desired target peptide. After expression, the fusion protein is I, homocysteine in Class II, 2-aminoadipic acid, isolated and cleaved using chemical and/or enzymatic meth 2-aminopimelic acid, (Y-carboxyglutamic acid, ods to produce the desired target peptide. B-carboxyaspartic acid, and the corresponding amino acid 50 The peptides used in the methods of the present invention amides in Class III; omithine, homoarginine, N-methyl may be employed in a monovalent State (i.e., free peptide or lysine, dimethyl lysine, trimethyl lysine, 2,3- a single peptide fragment coupled to a carrier molecule). The diaminopropionic acid, 2,4-diaminobutyric acid, peptides may also be employed as conjugates having more homoarginine, Sarcosine and hydroxylysine in Class IV; than one (same or different) peptide fragment bound to a Substituted phenylalanines, nor leucine, norvaline, 55 Single carrier molecule. The carrier may be a biological 2-aminooctanoic acid, 2-aminoheptanoic acid, Statine and carrier molecule (e.g., a glycosaminoglycan, a proteoglycan, B-Valine in Class V, and naphthylalanines, Substituted albumin or the like) or a Synthetic polymer (e.g., a poly phenylalanines, tetrahydroisoquinoline-3-carboxylic acid, alkyleneglycol or a Synthetic chromatography Support). and halogenated tyrosines in Class VI. Typically, ovalbumin, human Serum albumin, other proteins, Preferably, the active analogs of coloStrinin and its con 60 polyethylene glycol, or the like are employed as the carrier. Stituent peptides include polypeptides having a relatively Such modifications may increase the apparent affinity and/or large number of proline residues. Because proline is not a change the Stability of a peptide. The number of peptide common amino acid, a “large number” preferably means fragments associated with or bound to each carrier can vary, that a polypeptide includes at least about 15% proline (by but from about 4 to 8 peptides per carrier molecule are number), and more preferably at least about 20% proline (by 65 typically obtained under Standard coupling conditions. number). Most preferably, active analogs include more For instance, peptide/carrier molecule conjugates may be proline residues than any other amino acid. prepared by treating a mixture of peptides and carrier US 6,903,068 B1 9 10 molecules with a coupling agent, Such as a carbodiimide. with a nontoxic Surfactant. Dispersions of the active agent The coupling agent may activate a carboxyl group on either can be prepared in water, ethanol, a polyol (Such as glycerol, the peptide or the carrier molecule So that the carboxyl group propylene glycol, liquid polyethylene glycols, and the like), can react with a nucleophile (e.g., an amino or hydroxyl vegetable oils, glycerol esters, and mixtures thereof. The group) on the other member of the peptide/carrier molecule, ultimate dosage form is Sterile, fluid, and Stable under the resulting in the covalent linkage of the peptide and the conditions of manufacture and Storage. The necessary flu carrier molecule. For example, conjugates of a peptide idity can be achieved, for example, by using liposomes, by coupled to Ovalbumin may be prepared by dissolving equal employing the appropriate particle size in the case of amounts of lyophilized peptide and ovalbumin in a Small dispersions, or by using Surfactants. Sterilization of a liquid volume of water. In a second tube, 1-ethyl-3-(3- preparation can be achieved by any convenient method that dimethylamino-propyl)-carboimide hydrochloride (EDC; preserves the bioactivity of the active agent, preferably by ten times the amount of peptide) is dissolved in a small filter sterilization. Preferred methods for preparing powders amount of water. The EDC solution was added to the include vacuum drying and freeze drying of the Sterile peptide/ovalbumin mixture and allowed to react for a num injectible Solutions. Subsequent microbial contamination ber of hours. The mixture may then dialyzed (e.g., into can be prevented using various antimicrobial agents, for phosphate buffered saline) to obtain a purified solution of 15 example, antibacterial, antiviral and antifungal agents peptide/ovalbumin conjugate. Peptide/carrier molecule con including parabens, chlorobutanol, phenol, Sorbic acid, jugates prepared by this method typically contain about 4 to thimerosal, and the like. Absorption of the active agents over 5 peptides per Ovalbumin molecule. a prolonged period can be achieved by including agents for The present invention also provides a composition that delaying, for example, aluminum monoStearate and gelatin. includes one or more active agents (i.e., colostrinin, at least Formulations of the present invention Suitable for oral one constituent peptide thereof, or active analog thereof) of administration may be presented as discrete units Such as the invention and one or more carriers, preferably a phar tablets, troches, capsules, lozenges, wafers, or cachets, each maceutically acceptable carrier. The methods of the inven containing a predetermined amount of the active agent as a tion include administering to, or applying to the skin of, a 25 powder or granules, as liposomes containing the active patient, preferably a mammal, and more preferably a human, agent, or as a Solution or Suspension in an aqueous liquor or a composition of the invention in an amount effective to non-aqueous liquid Such as a Syrup, an elixir, an emulsion, produce the desired effect. The active agents of the present or a draught. The amount of active agent is Such that the invention are formulated for enteral administration (oral, dosage level will be effective to produce the desired result in rectal, etc.) or parenteral administration (injection, internal the Subject. pump, etc.). The administration can be via direct injection Nasal spray formulations include purified aqueous Solu into tissue, interarterial injection, intervenous injection, or tions of the active agent with preservative agents and other internal administration procedures, Such as through the isotonic agents. Such formulations are preferably adjusted to use of an implanted pump, or via contacting the composition a pH and isotonic State compatible with the nasal mucous with a mucus membrane in a carrier designed to facilitate 35 membranes. Formulations for rectal or vaginal administra transmission of the composition acroSS the mucus membrane tion may be presented as a Suppository with a Suitable carrier Such as a Suppository, eye drops, inhaler, or other similar Such as cocoa butter, or hydrogenated fats or hydrogenated administration method or via oral administration in the form fatty carboxylic acids. Ophthalmic formulations are pre of a Syrup, a liquid, a pill, capsule, gel coated tablet, or other pared by a similar method to the nasal spray, except that the Similar oral administration method. The active agents can be 40 pH and isotonic factors are preferably adjusted to match that incorporated into an adhesive plaster, a patch, a gum, and the of the eye. Topical formulations include the active agent like, or it can be encapsulated or incorporated into a bio dissolved or Suspended in one or more media Such as erodible matrix for controlled release. mineral oil, DMSO, polyhydroxy alcohols, or other bases The carriers for internal administration can be any carriers used for topical pharmaceutical formulations. commonly used to facilitate the internal administration of 45 Useful dosages of the active agents can be determined by compositions Such as plasma, Sterile Saline Solution, IV comparing their in Vitro activity and the in Vivo activity in Solutions or the like. Carriers for administration through animal models. Methods for extrapolation of effective dos mucus membranes can be any well-known in the art. Car ages in mice, and other animals, to humans are known in the riers for administration oral can be any carrier well-known art; for example, see U.S. Pat. No. 4,938,949. in the art. 50 The tablets, troches, pills, capsules, and the like may also The formulations may be conveniently presented in unit contain one or more of the following: a binder Such as gum dosage form and may be prepared by any of the methods tragacanth, acacia, corn Starch or gelatin; an excipient Such well known in the art of pharmacy. All methods include the as dicalcium phosphate, a disintegrating agent Such as corn Step of bringing the active agent into association with a Starch, potato Starch, alginic acid and the like; a lubricant carrier which constitutes one or more accessory ingredients. 55 Such as magnesium Stearate; a Sweetening agent Such as In general, the formulations are prepared by uniformly and Sucrose, fructose, lactose or aspartame; and a natural or intimately bringing the active agent into association with a artificial flavoring agent. When the unit dosage form is a liquid carrier, a finely divided Solid carrier, or both, and then, capsule, it may further contain a liquid carrier, Such as a if necessary, Shaping the product into the desired formula vegetable oil or a polyethylene glycol. Various other mate tions. 60 rials may be present as coatings or to otherwise modify the Formulations Suitable for parenteral administration con physical form of the Solid unit dosage form. For instance, Veniently include a sterile aqueous preparation of the active tablets, pills, or capsules may be coated with gelatin, wax, agent, or dispersions of Sterile powders of the active agent, shellac, or Sugar and the like. A Syrup or elixir may contain which are preferably isotonic with the blood of the recipient. one or more of a Sweetening agent, a preservative Such as Isotonic agents that can be included in the liquid preparation 65 methyl- or propylparaben, an agent to retard crystallization include Sugars, buffers, and Sodium chloride. Solutions of of the Sugar, an agent to increase the Solubility of any other the active agent can be prepared in water, optionally mixed ingredient, Such as a polyhydric alcohol, for example glyc US 6,903,068 B1 11 12 erol or Sorbitol, a dye, and flavoring agent. The material used Flow cytometry: Relative changes in ROS levels were in preparing any unit dosage form is Substantially nontoxic determined as described previously (I. Boldogh et al., Psy in the amounts employed. The active agent may be incor chogeriatr Ann, 4:57-65 (2001)). Briefly, PC12 cells at 70% porated into Sustained-release preparations and devices. confluence were trypsinized and washed with EMEM con taining 10% FBS. Cells were re-suspended in EMEM (plus EXAMPLES 5% FBS) and loaded with 2,7'dichlorodihydro-fluorescein The invention will be further described by reference to the diacetate (HDCF-DA; Molecular Probes Inc.) (5 mM final following detailed examples. The examples are meant to concentration) for 15 min, at 37° C. then washed in growth provide illustration and should not be construed as limiting medium. Following centrifugation, the cell pellets were the Scope of the present invention. re-suspended in EMEM containing 10 mM HEPES (pH: 7.4). DCF-mediated fluorescence of treated and mock Examples 1-5 treated cells was determined by flow cytometry (Becton Materials and Methods Dickinson FACS Scan) using 488 nm and 525 nm excitation Cell cultures: Pheochromocytoma (PC12) cells were pro and emission Settings, respectively. Each data point repre vided by Dr. Regino Perez-Polo (University of Texas Medi 15 sents the mean fluorescence for 12,000 cells. cal Branch, Department of Human Biological Chemistry and Reagents: Colostrinin (CLN) was purified from ovine Genetics) and maintained in EMEM supplemented with colostrum, collected during the first milking (6-12 hours 10% fetal bovine serum, penicillin (100 IU/ml) and strep (hr) after lambing), according to the method developed by tomycin (100 micrograms per milliliter (ug/ml)). Exponen Janusz et al. (M. Janusz et al., FEBS Lett., 49:276-279 tially growing populations of PC12 cells were sub-cultured (1974)). A high content of proline (>23%) and lack of and used for all experiments. detectable alanine, arginine, histidine, tryptophan, methionine, and cysteine were confirmed by amino acid Western blot analysis: PC12 cells were plated at 7x10 analysis of CLN. A peptide control was prepared by trypsin cells/T75 flask. After exposure to 4HNE, colostrinin or their (Sigma-Aldrich) digestion of purified lactalbumin from combination, cells were collected and lysed in 50 millimolar 25 (mM) Tris, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 10% bovine milk (Sigma). The trypsin was then inhibited by glycerol and protease inhibitor cocktail (Supplemented with treatment with inhibitor (Invitrogen). SDS-PAGE con 1 mM NaVO, 2 mM EDTA, 1 mM phenylmethylsulfonyl formed digestion of lactalbumin into peptides, and the fluoride). Lysates were centrifuged at 14,000 g for 10 hydrolysate was referred to as LAH. minutes (min) (4 C.) and 40 ug of protein was fractionated Statistical analysis: The experiments were repeated at on a 10% SDS-polyacrylamide gel and transferred to least three times and Statistically analyzed for significant protein-optimized membranes (Amersham, Inc.). p53 was differences using ANOVA procedures and Student's t-tests. detected using specific antibody (DO1; Santa Cruz Data are expressed as meansitS.E. Biotechnology, Inc.) at a dilution of 1:300. Adducts were Examples 1-5 detected using an antibody to HNE-protein adducts 35 (Pharmingen, Inc.) at a dilution of 1:500. The anti-phospho Results JNK antibody (New England Biolabs, Inc., Beverly, Mass.) was raised against a Synthetic phosphopeptide Example 1 (SFMMT* PY* VVRYYR) corresponding to residues Colostrinin Reduces 4HNE-Protein Adduct 179-193 of INK. For visualization of primary antibody 40 binding, all blots were incubated with horseradish peroxi Formation in PC 12 Cells dase conjugated Secondary antibody (Amersham, Inc.) at a Fluorescent microScopy and Western blot analysis was dilution of 1:2000, followed by chemiluminescence detec undertaken to investigate the extent of 4HNE protein-adduct tion (Amersham, Inc.) and autoradiography. formation in cultures of PC12 cells in the presence of CLN. Immunocytochemistry: PC12 cells grown on cover-Slips 45 Cells were pretreated with CLN or LAH in the presence or were fixed overnight in PBS containing 2% paraformalde absence of 4HNE and then analyzed for the formation of hyde at 4° C. Cells were permeabilized by 0.3% Triton 4HNE-protein adducts. The results in FIGS. 1A and 1B X-100, washed in PBS then incubated with primary anti show that addition of 4HNE (25 nanomolar (nM)) or HO body in PBS containing 0.05% Tween 20 (PBS-T). After (100 micromolar (uM) resulted in a bright fluorescence, washing 3 times in PBS-T, FITC-labeled anti-rabbit IgG 50 localized to the cytoplasmic region of PC12 cells due to (Santa Cruz Biotechnology Inc.) was added. Cells were binding of antibody to 4HNE-protein adducts. When cells washed (5 times, for 10 min) with PBS-T and mounted on were pre-treated with CLN (10 microgram per milliliter microscope slides in anti-fade Solution (Dako, Inc.). Images (ug/ml)) for 15 minutes (min) and exposed to 4HNE (25 nM) of cellular immunofluorescence were acquired using a for 15 min (concentrations of CLN and time required for NIKON Eclipse TE300 scanning microscope. 55 effect were determined in preliminary Studies), the results Measurement of glutathione (GSH): In brief, PC12 cells indicated that CLN reduced fluorescence intensity (FIG. 1C) were mock- or pre-treated with CLN or lactalbumin to background level (data not shown). In the controls, hydrolysate (LAH), both in 10 ug/ml concentration and then pre-treatment of cells with an N-acetyl-L-cysteine (10 mM) exposed to 4HNE (25 nM). PBS-washed (twice) cells were and trolox (1 mM, a water-soluble-tocopherol) combination then extracted with 25% (w/v) metaphosphoric acid solution 60 significantly reduced 4HNE-mediated intracellular fluores containing 5 mM EDTA. After ultracentrifugation (105,000 CCCC. g for 30 min), 100 ul of 100 mM phosphate solution (pH 8.0) To determine whether the inhibitory effect of CLN was containing 5 mM EDTA and 10 ul of o-phthalaldehyde specific, CLN was substituted with digested lactalbumin OPA(OPD; Molecular Probes, Inc.) was added to the hydrolysate (LAH, Materials and Methods), which contains Supernatant, and the fluorescence intensity at 420 nm deter 65 a variety of peptides as does CLN. Results in FIG. 1D show mined with excitation set at 350 nm (A. P. Senft et al., Anal bright fluorescence in cells treated with LAH (10 ug/ml) Biochem., 280:80-86 (2000)). plus 4HNE (25 nM), which is similar to that seen with 4HNE US 6,903,068 B1 13 14 alone (FIG. 1A). These data indicate that CLN inhibits To determine whether the loss of intracellular GSH was adduct formation, and the effect is specific and could be the due to it's extrusion from the cells or oxidation, the level of result of a not yet-determined interaction between its con reduced GSH in the extra-cellular fluid was evaluated. Stituent peptides and cellular component(s). Relative to CLN- or mock-treated cells, 4HNE caused a To confirm the results generated by immunochemistry, 5 significant increase in OPA-GSH-mediated fluorescence Western blot analysis was used to investigate changes in when it was added to extracellular fluid (data not shown). 4HNE-protein adduct levels in cells treated with 4HNE LAH alone or LAH in 4HNE-exposed cells did not affect alone or CLN plus 4HNE, FIG. 1E (lanes 1, 2, and 3) shows GSH extrusion (OPA-GSH fluorescence). OPA did not show that 4.HNE alone induced a significant increase in levels of fluorescence when it was mixed with CLN, LAH or 4.HNE 4HNE-protein adducts, with molecular weights ranging alone. These results indicate that CLN mediates its effect on from 200 kD to 15 kD. CLN (10 ug/ml) abolished adduct GSH metabolism at the cell membrane level. formation, as shown in FIG. 1E lanes 4 to 6. Overall these data indicate that CLN can block the formation of 4HNE Example 4 adducts. From these results, it is believed that the inhibition of 4HNE-protein adduct formation by CLN is multi-factorial 15 4HNE-Induced Activation of JNK is Suppressed by and may involve mechanisms. Such as direct Scavenging CLN (binding) of 4HNE via cysteine, lysine, or histidine residues The effect of CLN on 4HNE-induced activation of JNK in in CLN, or by inhibition of 4HNE’s entry onto cells. PC12 cells was investigated. The activation of JNK was To determine whether CLN can protect mitochondria and monitored by Western blot analysis using a highly specific abolish the oxidative stress induced by 4HNE, PC12 cells anti-phospho-(Tbr-183/Tyr-185) JNK antibody (Materials were treated with CLN (with LAH as control) and/or 4.HNE and Methods). and the changes in ROS levels were monitored by the The data Summarized in FIG. 4 show that 4HNE alone is redox-sensitive 2,7'-dichlorofluorescein diacetate (HDCF a potent inducer of JNK phosphorylation (FIG. 4, lanes 4 DA) probe (1. Boldogh et al., Psychogeriatr Ann, 4:57-65 and 5). Pretreatment of PC12 cells with CLN (1 and 10 (2001); LeBel, Chem. Res. Toxicol., 5:227–231 (1992)). 25 ug/nM) or with an inhibitor of JNK activation 8-(4- Mock-as well as CLN (or LAH) pre-treated cells were chlorophenylthio)-cAMP prevented 4HNE-induced JNK loaded with HDCF-DA then exposed to 4HNE for 15 min. phosphorylation; 4.HNE-mediated phosphorylation was Changes in fluorescence intensities mediated by the oxidized reduced by 10 and 1.0 ug/ml CLN (FIG.4, lanes 7 and 8) to probe, DCF, were determined by flow cytometry. Prior to control levels (FIG. 4 lanes 1 and 2). CLN at 0.1 lug/ml data collection, propidium iodide was added to the Samples concentration did not significantly effect 4HNE-mediated for Sorting out nonviable cells. JNK phosphorylation (lane 9). The maximum level of phos phorylation of JNK in 4HNE-treated cells occurred between Example 2 15 and 30 min post-treatment as determined in preliminary Colostrinin Affects the Oxidative Metabolism in 35 studies (data not shown). These data indicate that CLN may PC12 Cells modulate oxidative metabolism (GSH levels, 4HNE-protein adduct formation) of cells potentially through JNK, a kinase A representative histogram showing the effects of the that is central to the cellular StreSS responses. treatments on ROS levels is shown in FIG. 2B. AS Summa rized in FIG. 2A, 4HNE (25 nM) induced a 4- to 5-fold Example 5 increase in DCF-mediated fluorescence, while CLN alone or 40 LAH showed no significant effect. Remarkably, CLN abol Colostrinin Inhibits 4HNE-Induced Activation of ished (while LAH had no significant effect on) HDCF p53 oxidation in 4HNE-treated PC 12 cells. Because constituent peptides in LAH did not alter 4.HNE-induced HDCF Whether CLN could modulate p53 levels after 4.HNE oxidation, it can be concluded that the effect of CLN is 45 exposure was also investigated. In FIG. 5, Western blot Specific, and may protect cells from ROS damage via its analysis shows that CLN reduces activation of p53 induced quantitatively unique and Specific peptide composition. by 4HNE when compared to cells treated with 4HNE alone. On the other hand, pre-treatment with the same concentra Example 3 tion (10 ug/ml) of LAH had no affect on 4HNE-mediated 50 p53 induction. These data Suggest that CLN, via its antioxi Effect of CLN on 4HNE-Induced LOSS of dant activity, can effect activation of p53, a key regulator of Intracellular GSH Levels cell proliferation, differentiation and apoptosis (G. Evan et To investigate whether the anti-oxidant effect of CLN was al., Science, 281:1317-1322 (1998)) and may explain mul due to protection of intracellular GSH levels, PC12 cells tiple biological effects (antioxidant, differentiation) of CLN. were per-treated (with CIN or LAH) and exposed to 4HNE, 55 as described above, and changes in GSH levels were deter Examples 1-5 mined fluorimetrically. The results summarized in FIG. 3 show that treatment with 4.HNE alone for 30 min (time Discussion determined in preliminary Studies) resulted in a significant It has been shown that CLN, a milk-derived peptide reduction of intracellular GSH levels as shown by a change 60 complex can modulate both cytokine production and cellular in OPA-GSH's fluorescence. OPA (o-phthalaldehyde or redox status. To study CLNs antioxidant effects, 4HNE was phthalic dicarboxaldehyde) is highly fluorescent when it is used for treatment of PC12 cells. 4.HNE is a 3-unsaturated conjugated to GSH (A. P. Senft et al., Anal Biochem, aldehyde generated endogenously during lipid peroxidation, 280:80-86 (2000)). Pre-treatment of cells with CLN (10 Specifically from the Oxidative degradation of arachidonic Aug/ml), however, significantly inhibited this change in OPA 65 and linoleic acids (H. Esterbauer et al., Free Raids. Biol. fluorescence (loss of GSH) while LAH had an insignificant Med., 11:81–128 (1991)). Further, 4.HNE is involved in both effect. normal and pathophysiological events in cells and tissues US 6,903,068 B1 15 16 that result in various chronic diseases. While micromolar rylates two sites within the activation domain. These factors concentrations of 4HNE is cytotoxic, at the nanomolar level are members of the basic leucine Zipper group that binds as it can be involved in activation of the Signal transduction homo- and heterodimeric complexes to AP-1 and AP-1-like pathways. For example, it has been shown that depending on Sites in the promoters of many genes and result in increased concentration, 4.HNE can affect proliferation and induce transcriptional activity. differentiation or apoptosis in cells. The present studies show that treatment of PC12 cells In the current studies, a concentration of 25 nM 4HNE with 4HNE causes JNK activation within 15 to 30 min. was used. This did not show toxic effects or induce apoptosis However, in CLN pretreated cells JNK activation was not but considerably increased the levels of 4HNE-protein only delayed or reduced, it was abolished. CLN was also as adducts (FIG. 1A) in PC12 cells. Remarkably, it was found potent as 8-(4-chlorophenylthio)-cAMP a specific inhibitor that CLN abolished 4.HNE-protein adduct formation, while of JNK activation. AP-1 phosphorylation, which is a later LAH (as control) at the same concentration had no effect event in the JNK Signaling pathway is presently under indicating CLN's Specificity. Due to the presence of a highly investigation. These findings are consistent with the idea that electrophilic carbon, 4.HNE is a potent alkylating agent able CLN has the ability to protect cells from oxidative stress and to react with histidine, lysine, Serine, cystein, and tyrosine 15 other consequences of 4.HNE exposure, including JNK Side chains in proteins, and thus modify their functions. activation, and its down-stream consequences. Although it was hypothesized that peptides of CLN, via its The Western blot analysis shown in FIG. 5, clearly component amino acid residues, were reacting with 4HNE demonstrated that p53 is normally present in a latent form and chemically reducing its concentration, no direct inter and that 4HNE induced its activation. It has been shown that action between CLN's peptide(s) and 4HNE were observed. p53 lies at the center of a network of complex redox 4HNE is known to modulate the activities of ATPases, interactions. In this network, p53 can control the timely phospholipase C, adenylate cyclase, GTP-binding proteins, production of ROS, but this activity is itself under the and protein kinase C. Furthermore, 4.HNE can react with the control of changes in cellular redox Status. Thus, p53 acti nucleophilic Sites in DNA, mitochondrial proteins and a vation in 4HNE-treated PC12 cells can occur in multiple variety of other nucleophiles, including GSH, resulting in 25 ways: it may be due to 4HNE-induced DNA damage, ROS, cellular StreSS responses and oxidative StreSS. In the present and/or activation of cell cycle regulatory kinases. Regardless studies, it was demonstrated that that CLN was able to of the mechanism of p53 activation, CLN showed a potent prevent a decrease in 4HNE-induced GSH levels. It is inhibitory effect. proposed that 4HNE-induced reduction in GSH levels may CLN induced differentiation in SH-SY5Y cells in a dose be due to glutathione-S-transferase (GST)-mediated conju dependant manner (Table 1A). The ability of CLN to induce gation of 4-HNE to GSH, or that GSH may be utilized in differentiation in these cells is shown in FIG. 6A, the control detoxification reactions of ROS. compared to FIG. 6C, a culture treated with 0.1 lug/ml CLN Taking into consideration that only 25 nM of 4HNE was (see figure legends). used, while intracellular concentrations of GSH are in the 35 It has been shown that colostrinin can inhibit the toxicity 0.5 to 10 mM range, reduction of GSH levels by GST or of Beta-amyloid in neural derived SH-SY5Y cells. Essen utilization by glutathione peroxidases may not explain the tially complete inhibition of the toxicity occurred at the 0.01 more than 50% loss of GSH. Therefore, the GSH levels in ug/ml level (Table 1B). FIGS. 6D and 6E shows the pro the extracellular fluid was evaluated. The large reduction in tective effect of 3.0 and 0.1 tug/ml of CLN on B-amyloid GSH levels (FIG. 3) may be due to extrusion of GSH from induced toxicity as shown in FIG. 6B. Since this toxicity is cells after 4.HNE exposure. Indeed, it has been discovered 40 the result of the apoptotic activity of Beta-amyloid that an increase in GSH in the extracellular milieu is in (3-amyloid), the data indicate that colostrinin is a potent response to 4HNE treatment. Most remarkably, CLN was inhibitor of apoptosis in neural-derived cells. This potent able to prevent this effect of 4HNE. activity indicated that even lower concentrations of colos Although some ROS production in 4HNE-treated cells 45 trinin would have to be tested to determine the potency and has been shown to be due to mitochondrial damage, it is anti-apoptotic dose response effect of coloStrinin in this believed that the 3- to 4-fold increase in ROS levels were System. However, a dose dependant development of differ due to GSH extrusion, which resulted in a perturbance of entiation did occur in the presence of Beta-amyloid in the cellular anti-oxidant defenses. Most importantly, CLN, but colostrinin treated cells (FIGS. 6D and 6E). not LAH, inhibited oxidation of HDCF strongly Suggesting 50 The results indicate that not only did colostrinin inhibit that CLN is involved in the activation of cellular antioxidant the toxicity of Beta-amyloid, but it also was able to induce defenses or possesses effective anti-oxidant activity via differentiation of the SH-SY5Y in a dose dependant manner regulating cellular GSH levels. in Beta-amyloid treated cells. This finding indicates that the 4HNE exposures have been reported to be linked with development of differentiation in Beta-amyloid treated cells c-Jun NH2-terminal kinases activation and c-Jun phospho 55 could be used as a biological assay for coloStrinin and one rylation. Three groups of mitogen-activated protein (MAP) of its important functions. kinases have been identified in mammals: the extracellular To determine whether this was reproducible and to deter signal-regulated kinase, the p38 MAP kinase, and JNKS mine the potency of CLN to inhibit retinoic acid toxicity, (also referred to SAPKs). JNKS are activated by a wide two concentration of retinoic acid were used to treat cells on variety of Stimuli, including ROS, DNA-damaging agents 60 day one of the experiment, 20 uM and 40 uM. * Control and inhibitors of protein Synthesis, and heat or OSmotic Wells were mock-treated for the duration of the experiment. Shock. These Stimuli appear to operate through Small G Retinoic Acid was left on the plate for the duration of the proteins of the Ras and epidermal growth factor (EGF) experiment or until washed off. Colostrinin was added to the family receptors and Sequential activation of various protein plate at the indicated doses. When added on Day 1, it was kinases. Targets of the JNK Signal transduction pathway 65 present during the entire experiment. When added on Day 5, include the transcription factors ATF2 and c-Jun. c-Jun binds it was incubated on the plate for 30 minutes at 37 C. and to the N-terminal region of ATF2 and c-Jun and phospho then removed. The wells were washed twice with PBS US 6,903,068 B1 17 18 before adding fB-Amyloid. Cultures were then observed under the microScope and graded. TABLE 1-continued The data reported herein shows the ability of CLN to Differentiating, Anti-Apoptotic and Protective Activity of Colostrinin block the activity of cells treated with 20 uM retinoic acid Against B-Amyloid and Retinoic Acid in Neuronal Derived SH-SY5Y (the toxicity developed too rapidly in cells treated with 40 Cells uM). Table 1C indicates that 1.0 ug/ml of CLN almost DOSE completely blocked the cytotoxicity of retinoic acid. The COLOSTRININ DIFFERENTI retinoic acid induced differentiation in these cells by day TREATMENT (MG/ML) TOXICITY ATION two, but the cells Started showing Signs of toxicity by day 6. CLN, 1.0 lug/ml, added on day one or for 30 minutes on day D. Control five of the experiment completely blocked the toxicity, and Mock - (Day 5 & 8) - (Day 5 & 8) Similar to the finding with Beta-amyloid, also induced the Treated (Day 1) cells to proliferate. FIGS. 6F and 6G further document the finding. FIG. 6F shows the toxicity induced by retinoic acid ++++ Approximately 100% of the cells 15 + Approximately 25% of the cells compared 10 FIG. 6G, which clearly shows differentiated Day of treatment cells (see legends). Day cells observed Colostrinin inhibited the eventual development of cyto toxicity by Retinoic Acid when added at the same time or 5 Example 7 days later. Colostrinin added at Day 1 also inhibited the development of toxicity by B-Amyloid added on Day 5 and Inhibition of 4HNE-Induced Apoptosis by CLN was dose dependent (data not shown). Apoptosis is a specific mode of cell death recognized by The ability of CLN to inhibit apoptotic effects of two a characteristic pattern of morphological, biochemical, and Substances, Beta-amyloid and retinoic acid, indicates it may molecular changes. Currently the hallmark of apoptosis in have potential biological use in many areas where apoptosis 25 Vitro is DNA fragmentation and changes in plasma mem plays a role, e.g., virus infections, chronic diseases and brane reorganization that allows for the Surface expression attempts to get Stem cells to grow and differentiate, among of phosphatidyl-D-Serine and result in increased membrane many others. permeability. The protection of cells against 4HNE by CLN Table 1. Differentiating, Anti-Apoptotic and Protective using increased permeability and cell membrane expression Activity of Colostrinin Against B-Amyloid and Retinoic of phosphatidyl-D-Serine was investigated. Acid in Neuronal Derived SH-SY5Y Cells Cells were simultaneously labeled with fluorochrome conjugated annexin V-PE (detecting PS asymmetry in the TABLE 1. plasma membrane, an early marker of apoptosis), Differentiating, Anti-Apoptotic and Protective Activity of Colostrinin 35 7-aminoactinomycin D (7-AAD) (detecting increased mem Against B-Amyloid and Retinoic Acid in Neuronal Derived SH-SY5Y brane permeability associated with both apoptosis and Cells necrosis). A dual laser flow cytometer (either a Becton DOSE Dickinson FACScan) was used for the simultaneous detec COLOSTRININ DIFFERENTI tion of the PE-conjugated annexin V (which is excited at 632 TREATMENT (MG/ML) TOXICITY ATION nm and emits at 660 nm), 7-AAD (excited at 488 nm and 40 emitting at 670 nm). Fluorochrome compatibility was A. Differentiating excellent, although careful intralaser compensation is Activity required for simultaneous use of PE and 7-AAD. 3.0 (Day 5) - (Day 8) ++++ (Day 8) The analysis of apoptosis by flow cytometry is shown in 1.0 (Day 5) - (Day 8) ++++ (Day 8) FIG. 7. The results of the inhibition of 4HNE-induced 0.1 (Day 5) - (Day 8) +++ (Day 8) 45 0.01 (Day 5) +/- (Day 8) ++ (Day 8) apoptosis by CLN are shown in FIG. 8. B. Anti-apoptotic Activity Example 8 B-Amyloid +++ (Day 8) - (Day 8) Inhibition of UV-Irradiation-Induced Apoptosis by 10 ug/ml (Day 5) 50 B-Amyloid 3.0 (Day 5) - (Day 8) +++ (Day 8) CLN 10 ug/ml (Day 5) B-Amyloid 1.0 (Day 5) +/- (Day 8) +++ (Day 8) Chronic repeated UV exposures are the primary cause of 10 ug/ml (Day 5) benign and malignant skin tumors, including malignant B-Amyloid 0.1 (Day 5) +/- (Day 8) ++ (Day 8) melanoma In experimental animal models, among types of 10 ug/ml (Day 5) B-Amyloid 0.01 (Day 5) + (Day 8) + (Day 8) 55 solar radiation, ultraviolet B (290–320 mm) radiation is 10 ug/ml (Day 5) highly mutagenic and carcinogenic compare to ultraViolet A C. Protection (320-400 nm) radiation. Based on current understanding of Against Retinoic DNA damage caused by direct UV radiation and by indirect Acid Activity StreSS via reactive oxygen Species and DNA repair mecha Retinoic Acid - (Day 2) ++++ (Day 2) 60 nisms are responsible for UV irradiation-induced skin tumor 20 uM (Day 1) development in human cells. Retinoic Acid +++ (Day 8) + (Day 8) UVB exposure leads to a time-dependent increase in the 20 uM (Day 1) production of intracellular peroxide and Superoxide anions Retinoic Acid 1.0 (Day 5) - (Day 8) ++++ (Day 8) 20 uM (Day 1) and may induce carcinogenic mutations and apoptosis. Retinoic Acid 1.0 (Day 1) + (Day 8) ++++ (Day 8) 65 Besides being a major cause of oxidative StreSS in the cells, 20 uM (Day 1) UVB-irradiation induces apoptosis by a large number of unrelated pathways Such as enhanced Fas transcription and/ US 6,903,068 B1 19 20 or mRNA stability, induction of transcriptional factors viz Sequence Listing Free Text c-fos, c-jun, SAP-1 and nuclear factor kB gene expression. A possible prevention of UV-induced skin cancer by feeding or topical use of antioxidants, Such as polyphenols, and Vitamins are observed. The following are all Synthetic peptide Sequences. The effect of CLN on ultraviolet B (UVB)-induced apo ptosis and DNA damage in cultured PC12 cells has been determined. The apoptosis was determined by flow cytom SEO ID NO:1 MOPPPLP SEO ID NO:2 LOTPOPLLOVMMEPOGD etry. The comet assay was employed to detect DNA damage SEO ID NO:3 DOPPDVEKPDLOPFOVOS in individual cell. The results shown in FIG. 9 indicate an SEO ID NO:4 LFFFLPVVNVLP inhibitory effect of CLN on UVB-induced apoptosis. CLN SEO ID NO:5 DLEMPVLPVEPFPFW treated cells also showed a significantly reduced DNA SEO ID NO:6 MPONFYKLPOM SEO ID NO:7 VLEMKFPPPPOETVT damage. Semi-confluent cells with >98% viability (tested SEO ID NO:8 LKPFPKLKVEVFPFP with trypan blue dye exclusion) were used in all experi SEO ID NO:9 VVMEV ments. PC12 cells were exposed to UV-B irradiation. Lethal 15 SEO ID NO:10 SEOP dose 50 (LD50) was determined in preliminary studies. SEO ID NO:11 DKE SEO ID NO:12 FPPPK Cells were irradiated by a dose result in 50% cell death. SEO ID NO:13 DSOPPV SEO ID NO:14 DPPPPOS CONCLUSION SEO ID NO:15 SEEMP SEO ID NO:16 KYKLOPE Taken together, these results show that CLN can be SEO ID NO:17 involved in regulation of the cellular redox status, GSH SEO ID NO:18 WYPFTGPIPN metabolism, and modulation of ROS-induced Signaling SEO ID NO:19 SLPONILPL SEO ID NO:20 TOTPVVVPPF mediated down-stream events (e.g., JNK, p53). These SEO ID NO:21 LOPEIMGVPKVKETMVPK results further Suggest a potential mechanism(s) by which 25 SEO ID NO:22 HKEMPFPKYPVEPFTESO CLN could modulate a network, resulting in cytokine, SEO ID NO:23 SLTLTDVEKLHLPLPLVO chemokine production, cell differentiation and may explain SEO ID NO:24 SWMHOPP it's beneficial effects on pathogenic processes involved in SEO ID NO:25 QPLPPTVMFP SEO ID NO:26 POSVLS AD and other chronic neuro-degenerative diseases. SEO ID NO:27 LSOPKVLPVPOKAVPORDMPIO Although the invention has been disclosed with reference SEO ID NO:28 AFLLYOE to its preferred embodiments, from reading this description SEO ID NO:29 RGPFPILV those of skill in the art may appreciate changes and modi SEQ ID NO:30 ATFNRYODDHGEEILKSL SEO ID NO:31 VESYVPLFP fication that may be made which do not depart from the SEO ID NO:32 FLLYOEPVLGPVR Scope and Spirit of the invention as described above and SEO ID NO:33 LNF claimed hereafter. All references, patents, and patent appli 35 SEO ID NO:34 MHQPPOPLPPTVMFP cations cited herein are incorporated herein by reference in their entirety as if individually incorporated. SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 34 <210> SEQ ID NO 1 &2 11s LENGTH 7 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &22O > FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 1 Met Glin Pro Pro Pro Leu Pro 1 5 <210> SEQ ID NO 2 &2 11s LENGTH 17 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &22O > FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 2 Leu Gln Thr Pro Gln Pro Leu Leu Glin Val Met Met Glu Pro Gln Gly 1 5 10 15 US 6,903,068 B1 21 22 -continued Asp SEQ ID NO 3 LENGTH 18 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 3 Asp Gln Pro Pro Asp Val Glu Lys Pro Asp Leu Gln Pro Phe Glin Val 1 5 10 15 Glin Ser SEQ ID NO 4 LENGTH 12 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 4 Leu Phe Phe Phe Leu Pro Wal Wall Asn. Wall Leu Pro 1 5 10 SEQ ID NO 5 LENGTH 15 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 5 Asp Leu Glu Met Pro Val Leu Pro Val Glu Pro Phe Pro Phe Val 1 5 10 15 SEQ ID NO 6 LENGTH 11 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 6 Met Pro Glin Asn Phe Tyr Lys Leu Pro Gln Met 1 5 10 SEQ ID NO 7 LENGTH 15 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 7 Val Leu Glu Met Lys Phe Pro Pro Pro Pro Gln Glu Thr Val Thr 1 5 10 15 SEQ ID NO 8 LENGTH 15 TYPE PRT ORGANISM: Artificial Sequence US 6,903,068 B1 23 24 -continued &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 8 Leu Lys Pro Phe Pro Lys Leu Lys Val Glu Val Phe Pro Phe Pro 1 5 10 15 <210 SEQ ID NO 9 &2 11s LENGTH 5 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 9 Wal Wal Met Glu Wall 1 5 <210> SEQ ID NO 10 <211& LENGTH 4 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 10 Ser Glu Glin Pro 1 <210> SEQ ID NO 11 &2 11s LENGTH 3 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 11 Asp Lys Glu 1 <210> SEQ ID NO 12 &2 11s LENGTH 5 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 12 Phe Pro Pro Pro Lys 1 5 <210> SEQ ID NO 13 &2 11s LENGTH 6 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 13 Asp Ser Glin Pro Pro Val 1 5 US 6,903,068 B1 25 26 -continued SEQ ID NO 14 LENGTH 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 14 Asp Pro Pro Pro Pro Glin Ser 1 5 SEQ ID NO 15 LENGTH 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 15 Ser Glu Glu Met Pro 1 5 SEQ ID NO 16 LENGTH 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 16 Lys Tyr Lys Lieu Glin Pro Glu 1 5 SEQ ID NO 17 LENGTH 7 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 17 Val Leu Pro Pro Asn Val Gly 1 5 SEQ ID NO 18 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 18 Val Tyr Pro Phe Thr Gly Pro Ile Pro Asn 1 5 10 SEQ ID NO 19 LENGTH 9 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide US 6,903,068 B1 27 28 -continued <400 SEQUENCE: 19 Ser Leu Pro Glin Asn. Ile Leu Pro Leu 1 5 <210> SEQ ID NO 20 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 20 Thr Glin Thr Pro Wal Wal Wall Pro Pro Phe 1 5 10 <210> SEQ ID NO 21 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 21 Leu Glin Pro Glu Ile Met Gly Val Pro Llys Val Lys Glu Thr Met Val 1 5 10 15 Pro Lys <210> SEQ ID NO 22 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 22 His Lys Glu Met Pro Phe Pro Llys Tyr Pro Val Glu Pro Phe Thr Glu 1 5 10 15 Ser Glin <210> SEQ ID NO 23 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 23 Ser Lieu. Thir Lieu. Thr Asp Val Glu Lys Lieu. His Leu Pro Leu Pro Leu 1 5 10 15 Wall Glin <210> SEQ ID NO 24 &2 11s LENGTH 7 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 24 Ser Trp Met His Gln Pro Pro US 6,903,068 B1 29 30 -continued <210> SEQ ID NO 25 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 25 Gln Pro Leu Pro Pro Thr Wal Met Phe Pro 1 5 10 <210> SEQ ID NO 26 &2 11s LENGTH 6 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 26 Pro Glin Ser Wall Leu Ser 1 5 <210 SEQ ID NO 27 <211& LENGTH 22 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 27 Leu Ser Glin Pro Lys Val Leu Pro Val Pro Gln Lys Ala Val Pro Glin 1 5 10 15 Arg Asp Met Pro Ile Glin 2O <210> SEQ ID NO 28 &2 11s LENGTH 7 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 28 Ala Phe Lieu Lleu Tyr Glin Glu 1 5 <210 SEQ ID NO 29 &2 11s LENGTH 8 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 29 Arg Gly Pro Phe Pro Ile Leu Val 1 5 <210 SEQ ID NO 30 &2 11s LENGTH 18 &212> TYPE PRT US 6,903,068 B1 31 32 -continued <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 30 Ala Thr Phe Asn Arg Tyr Glin Asp Asp His Gly Glu Glu Ile Lieu 1 5 10 15 Ser Lieu <210> SEQ ID NO 31 &2 11s LENGTH 9 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 31 Val Glu Ser Tyr Val Pro Leu Phe Pro 1 5 <210> SEQ ID NO 32 &2 11s LENGTH 13 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400> SEQUENCE: 32 Phe Leu Leu Tyr Glin Glu Pro Val Leu Gly Pro Val Arg 1 5 10 <210 SEQ ID NO 33 &2 11s LENGTH 3 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 33 Lieu. Asn. Phe 1 <210> SEQ ID NO 34 &2 11s LENGTH 15 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Description of Artificial Sequence: synthetic peptide <400 SEQUENCE: 34 Met His Glin Pro Pro Glin Pro Leu Pro Pro Thr Wal Met Phe Pro 1 5 10 15 What is claimed is: 60 NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), 1. A method for inducing a cytokine in a cell, the method DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF comprising contacting the cell with an immunological regu FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV lator under conditions effective to induce a cytokine, (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), wherein the immunological regulator is Selected from the VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK group consisting of a constituent peptide of coloStrinin, an VEVFPFP (SEQ ID NO:8), and MHQPPQ active analog thereof, and combinations thereof; 65 PLPPTVMFP (SEQ ID NO:34); and wherein the constituent peptide of coloStrinin is Selected wherein the active analog comprises a peptide having an from the group consisting of MQPPPLP (SEQ ID amino acid Sequence with at least about 15 percent US 6,903,068 B1 33 34 proline and having at least about 70 percent Sequence DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF identity to a constituent peptide of coloStrinin Selected FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV from the group consisting of MQPPPLP (SEQ ID (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF VEVFPFP (SEQ ID NO:8), and MHQPPQ FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV PLPPTVMFP (SEQ ID NO:34); and (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), wherein the active analog comprises a peptide having an VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK amino acid Sequence with at least about 15 percent VEVFPFP (SEQ ID NO:8), and MHQPPQ proline and having at least about 70 percent Sequence PLPPTVMFP (SEQID NO:34) and wherein said active identity to a constituent peptide of coloStrinin Selected analog induces a cytokine. from the group consisting of MQPPPLP (SEQ ID 2. The method of claim 1 wherein the cell is present in a NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), cell culture, a tissue, an organ, or an organism. DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF 3. The method of claim 1 wherein the cell is a mammalian FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV cell. 15 (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), 4. The method of claim 3 wherein the cell is a human cell. VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK 5. The method of claim 1 wherein the immunological VEVFPFP (SEQ ID NO:8), and MHQPPQ regulator is the colostrinin constituent peptide MQPPPLP PLPPTVMFP (SEQID NO:34) and wherein said active (SEQ ID NO: 1), an active analog thereof, or a combination analog modulates an immune response. thereof. 12. The method of claim 11 wherein the immunological 6. A method for modulating an immune response in a cell, regulator is administered as part of a dietary Supplement. the method comprising contacting the cell with an immu 13. The method of claim 11 wherein the immunological nological regulator under conditions effective to induce a regulator is administered topically. cytokine, wherein the immunological regulator is Selected from the group consisting of a constituent peptide of 14. The method of claim 11 wherein the patient is an 25 animal. colostrinin, an active analog thereof, and combinations 15. The method of claim 14 wherein the patient is a thereof; human. wherein the constituent peptide of coloStrinin is Selected 16. The method of claim 11 wherein the immune response from the group consisting of MQPPPLP (SEQ ID is a Specific immune response. NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), 17. The method of claim 11 wherein the immune response DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF is a nonspecific immune response. FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV 18. The method of claim 11 wherein the immune response (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), is the interferon response or antibody production. VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK 19. The method of claim 11 wherein the immunological VEVFPFP (SEQ ID NO:8), and MHQPPQ 35 regulator is the colostrinin constituent peptide MQPPPLP PLPPTVMFP (SEQ ID NO:34); and (SEQ ID NO: 1), an active analog thereof, or a combination wherein the active analog comprises a peptide having an thereof. amino acid Sequence with at least about 15 percent 20. A method for modulating leukocyte proliferation, the proline and having at least about 70 percent Sequence method comprising contacting leukocytes with a leukocyte identity to a constituent peptide of coloStrinin Selected 40 regulator Selected from the group consisting of coloStrinin, from the group consisting of MQPPPLP (SEQ ID a constituent peptide thereof, an active analog thereof, and NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), combinations thereof, under conditions effective to change DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF the number of leukocytes, FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV wherein the constituent peptide of coloStrinin is Selected (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), 45 from the group consisting of MQPPPLP (SEQ ID VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), VEVFPFP (SEQ ID NO:8), and MHQPPQ DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF PLPPTVMFP (SEQID NO:34) and wherein said active FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV analog modulates an immune response. (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), 7. The method of claim 6 wherein the cell is present in a 50 VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK cell culture, a tissue, an organ, or an organism. 8. The method of claim 6 wherein the cell is a mammalian VEVFPFP (SEQ ID NO:8), VESYVPLFP (SEQ ID cell. NO:31), and MHQPPQPLPPTVMFP (SEQ ID 9. The method of claim 8 wherein the cell is a human cell. NO:34); 10. The method of claim 6 wherein the immunological wherein the active analog comprises a peptide having an regulator is the colostrinin constituent peptide MQPPPLP 55 amino acid Sequence with at least about 15 percent (SEQ ID NO: 1), an active analog thereof, or a combination proline and having at least about 70 percent Sequence thereof. identity to a constituent peptide of coloStrinin Selected 11. A method for modulating an immune response in a from the group consisting of MQPPPLP (SEQ ID patient, the method comprising administering to the patient NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), an immunological regulator under conditions effective to 60 DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF induce a cytokine, wherein the immunological regulator is FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV Selected from the group consisting of a constituent peptide (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), of coloStrinin, an active analog thereof, and combinations VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK thereof; VEVFPFP (SEQ ID NO:8), VESYVPLFP (SEQ ID wherein the constituent peptide of coloStrinin is Selected 65 NO:31), and MHQPPQPLPPTVMFP (SEQ ID from the group consisting of MQPPPLP (SEQ ID NO:34); NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), and wherein the number of leukocytes is changed. US 6,903,068 B1 35 36 21. The method of claim 20 wherein the leukocytes are wherein the active analog comprises a peptide having an present in a cell culture or an organism. amino acid Sequence with at least about 15 percent 22. The method of claim 20 wherein the leukocytes are proline and having at least about 70 percent Sequence mammalian cells. identity to a constituent peptide of coloStrinin Selected 23. The method of claim 22 wherein the leukocytes are from the group consisting of MQPPPLP (SEQ ID human cells. NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), 24. The method of claim 20 wherein the leukocytes are DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF increased in number. FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV 25. The method of claim 24 wherein the leukocytes are (SEQ ID NO:5), MPQNFYKLPQM (SEQ ID NO:6), differentiated. 1O VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK 26. The method of claim 20 wherein the leukocyte regu VEVFPFP (SEQ ID NO:8), VESYVPLFP (SEQ ID lator is a constituent peptide of coloStrinin. NO:31), and MHQPPQPLPPTVMFP (SEQ ID 27. The method of claim 20 wherein the leukocyte regu NO:34), lator is the colostrinin constituent peptide VESYVPLFP and wherein the number of leukocytes is changed. (SEQ ID NO:31), an active analog thereof, or a combination 15 30. The method of claim 29 wherein the patient is a thereof. human. 28. The method of claim 20 wherein the leukocyte regu 31. The method of claim 29 wherein the leukocytes are lator is the colostrinin constituent peptide MQPPPLP (SEQ increased in number. ID NO:1), an active analog thereof, or a combination 32. The method of claim 31 wherein the leukocytes are thereof. differentiated. 29. A method for modulating leukocyte proliferation in a 33. The method of claim 29 wherein the leukocyte regu patient, the method comprising administering to the patient lator is a constituent peptide of coloStrinin. a leukocyte regulator Selected from the group consisting of 34. The method of claim 29 wherein the leukocyte regu colostrinin, a constituent peptide thereof, an active analog lator is the colostrinin constituent peptide VESYVPLFP thereof, and combinations thereof, under conditions effec 25 (SEQ ID NO:31), an active analog thereof, or a combination tive to change the number of leukocytes, thereof. wherein the constituent peptide of coloStrinin is Selected 35. The method of claim 29 wherein the leukocyte regu from the group consisting of MQPPPLP (SEQ ID lator is the colostrinin constituent peptide MQPPPLP (SEQ NO:1), LOTPQPLLOVMMEPQGD (SEQ ID NO:2), ID NO:1), an active analog thereof, or a combination DQPPDVEKPDLQPFQVOS (SEQ ID KO:3), LFF thereof. FLPVGVLP (SEQ ID NO:4), DLEMPVLPVEPFPFV 36. The method of claim 29 wherein the leukocyte regu (SEQ ID NO:5), MPONFYKLPQM (SEQ ID NO:6), lator is administered as part of a dietary Supplement. VLEMKFPPPPQETVT (SEQ ID NO:7), LKPFPCK 37. The method of claim 29 wherein the leukocyte regu VEVFPFP (SEQ ID NO:8), VESYVPLFP (SEQ ID lator is administered topically. NO:31), and MHQPPQPLPPTVMFP (SEQ ID 35 NO:34); k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 6,903,068 B1 Page 1 of 24 APPLICATIONNO. : 09/641801 DATED : June 7, 2005 INVENTOR(S) : G. John Stanton et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: Delete the title page and substitute therefore the attached title page. Delete Columns 1-36 and Substitute therefore the attached Columns 1-36. Signed and Sealed this Nineteenth Day of August, 2008 WDJ JON. W. DUDAS Director of the United States Patent and Trademark Office Page 2 of 24 (2 United States Patent (10) Patent No.; US 6,903,068 B Stanton et al. (45. Eate of Patent: Jun. 7, 2005 USE OF COEOSTRININ, CONSTITUENT SHER BLECATENS PEPTEES THE&EOF, ANE ANALOGS EREE FIR NEUNG CYONES Kruzel et al. (Dec. 2001) “Bewards an Understanding of Biological Rce of Colonstritir Peptides." Journal of investers: G.Eohn Stanton, Texas City, "EX {{S}; Molecular Neuroscience 17(3): 379–389, hostas K. Highes, Jr., Galveston, EX Englict, unsa, and Lisowski Colostinika Proline-Rich (3S); stwar Bodogh, Galveston, TX Polypeptide from vine Costura is a Modest Cytokine (JS), ery A. Georgiades, Houstors, Inducer in Human Eleukocytes. 996, Archivam immunolo TX (US) giae et: herapiae Experimentalis (44.215-238.* Eger, “lina:uaoiogy; inderstanting the iratase System Assignee: Board of Regents, The University of Eext (3996) Wisey-issist Exi. pp. 24-25 and 199-237. Texas System, Austin, TXIS Ngo et al. Computational Complexity, Protein Strixture Pre diction, and the Elevinta Paradox (3994 he Protein Foid Notice: Subject to asy discia:ster, the term of this ing Prob:ea and eriary Structure Prediction (#4), patcat is extended or adjusted under 35 491-95. J.S.C. 354(b) by 488 days. Wells Addivity of Mutationa: Effects in Proteins (1990) Bio chemistry (29): 37,8509-857," This patent is subject to a termiral dis Babbit, ed., the Ranieriil Ritar Hardiai, R.T. Wander caines. hift Company, inc., Norwalk, CT, p. 344-397 (978), Bespakov et ai. "Fabs specific for 8-oxogamine: control of Appi. No.: 09;64,881 DNA binding. Moi Biol. Now, 12, 1999:293(5): 1085-95. Blach-Olszewska et al., "Stimulatory effect of cwine coias Field: Aug. 17, 28.09 irinines proline-rich polypeptide cr: interferons and tuinar Related E.S. Applicationa Data aecrosis factor production by muriae resident peritoneal Sisional application. No. 68/149,3}, fied on Aug. 1, cel3s." 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Film (re et al., “Differentiation of PC12 celis with serve growth factor is associated with induction of transia synthe References Cited sis and release”. Neurosti Res. Apr. 1992;31 (4:562-9. Gabbit: et al. Increase inclear DNA oxidation in the E.S. PAENOCENTS brain is Alzheirer's disease.' . Netirician, Now, 4,938,949. A 1993 BCrch 33. 1998:71(5):2034-49. 5,593,88. A 2199; Coolidge et at, 604, 180 A. 3 J. Gabbita et al., “Eccrease in peptide methionize saifaxide 64,58 B2 862 Goike et al...... , 424,535 redictase in Alaheiner's disease brain, Neuracher. Oct. 6.5.93 Es: 23 S:to tal...... S42 1999;73(4): E 66-6. 23:09.5. A 5.33 Sosa, Cositinued FOREIGN PAFENT DOCUMENTS Priitary Eranzinier-Eizabeth C, Karrierer 649. A 99. Assistant Examiner...-Christopher 3ames Nichols WO95/E55 993 WO 95.3685 S95 (74. Attorney, agent, or Fiat-Muetiang. Raasch & 98.44 - 498 sharit, P.A. 99.6329 9. (57) ABSTRACT W 53 E. 1:33 CO The present invention discioses a use of colostrinii, a cor W 3f23S 201 WCFS stituent peptide thereof, and for an analog thereof as an WC 38.9 irritznological regulates and as a hlod ce: regulator its SC 383. animals including humans. {2:385 E.E. 333 23 37 Claims, No Drawings Page 3 of 24 US 6,903,068 BE Page 2 OHERELATONS Lovel et al., "increased DNA oxidation and decreased fevels of repair products in Azheimer's disease verticular CSF". Good et al., “Evideace of neutrona: oxidative damage in Neuracien. Fet. 1999;2(2):771-6, Alieheimer's disease Air Pathai, E. 996;149(3:2-8. ovellet al., “Secreased base excision repair and increased (iratara et al., "Flow cytometric qisantitation of inninof helicase activity is: Aizheiffer's disease 3rain, Brain Res. Faorescence intersity; proheins and perspectives. European Feb. 7, 2030,855(13:16-23. Working Group on Cirsical Ce: Arsalysis,” Cytoretery, Oct. 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E998; 33:27-37. are increased in Alzheimer's disease 3: Neira. Seg. Jagusa et al., "solation and characterization of a proli 1998:44(3):41.0-3, tie-rich polypeptide from owine colostria.' FEBS eit. Dec. Ostrea et al., “infidence of breast-feeding on the restoration: 15, 1934:49(2:278-9. of the ow scruti concentratio of vitamin E aid beta-caror Jansz et al., “Cherniea and physica: characterization of a terre it; the Bewborn infat." Ars J Obstet {ynecol. May proline-rich polypeptide fro?ii sheeps colostrifi, Biocherri J. 1986;354(5):104-7. Oct. 1, 1982; 99(1):9-5. Peurowa et al., "Nitri: oxide triggers a switch to growth JanxSz. et al., "Proline-rich polypeptide {{RP-aa immunic arrest during differentiation of 3e3ronal ceils, Nature, say mciliatory peptidie from owife constitur, 4rch innitol 4, 1995:375(6526:68-73. Tier Exp (Warsz. 1993:415-6):275-9. Piaseki et a:... “Coincidence between spontaneous release of Kim et al. “Simultaneous differetitiatin and quantitation of interferox and tumor recTasis factor by c.gistra: leukocytes erythrohiasts and white biood cells of a high throughput and the production of a colostrining by human risanitary clinical haematoiogy analyser Clint iah Haenatol Feb. glacid after normsi delivery," Arch innurial Ther. E.g. 1998:20(1):28-9. (Warsz), 399;45(1): 109-17. Kooy et al., “Oxidation of 2,3'-dichlorošiuorescin by perox Popik et al., “Coiostrinin, a polypeptide isolated from ear:y ynitric, Free Raic Res. Sep, 1997:27(3:245-54. milk, facilitates learing and Iretory in rats, harpsicol LeBel et ai, “Evaluation of the phe 2,7-dichiorofluores Biachern Behav Sep. 999;54): 183-9. cit as an indicator of reactive xygen species for nation and Prasad at al., “Regional riserabrase phospholipid alterations oxidative stress. Chen Ras Taxicot. 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Rothe et al., "Flow cytometric analysis of respiratory burst 1999:47(6):377.85. activity in phagocytes witt: hydroethidine and 2',3'-dichlor iwi et al., "he mode of action of nerve growth factor in rofurescin, J Leukoc Biol, May 1990; (5):440-8, PC12 celis, Mal Neurobial. 1988 Faii;2(3):23-26, Raya et ai, “Eva: sation of 2,7-dichlorifiutorescin aid 3 ovel et al., "Eievated thic harbituric acid-reactive stib d:ydrorhodamine 123 as fluorescert probes for irst racellular stances and assioxidiari enzyme activity in the brain in H2O2 in cultured entiethelial cels, Arch Biachann Biophys. Alzheimer's disease, Neurology Aug. 3995; May 1993:302(2}:348-55. $S(8)::593-501. Subbara et ai. 'Autopsy samples of Alzheimer's Cortex oveli et al., "Elevated 4-hydroxytoinema in wentricular show increased perixidation in vitrof Neurochen, ul. fluid in Alzheimer's disease," Neuroisioi Aging. Sep.-Oct. 3990:55(3):342-5, 1997; 8(5:457-6, Sciacier et ai. 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St. Jan. , 3938; 9}{2}:3? 8-4, Kiri et ai, "issu:i-ike Growth axia-i-mediatxi Negrite S8itti et 3. “Oxidative dataage in Atheirer's Nature, {Outgrowth it. Witra Requires Mitogen-stivated Protein Ju:... : 3, 1996, 382(6587); 20.3. Kiriase Activation." Journal of Riological Cheristix. Aag. SiSith et al., “Excess brain proteit oxidation and expyxe 22, 1997:272:34:21268-2: 273. 8ysfixietios is sofaai aging arti is: Aizheiaier's disease Kia & 3., iiiierential Regiation of Sasai: Recepts St. irac Nati cadSci & & 4, sec. i. 883; 8823: 354-3. strate-2 arii kiitogen-Activatei sotein Kina: Syrosite Swensρ; et si... Membrate lipids, selectively irriaished Phosphorylation by Pittsgatidy:iisasiio 3-Kinase inhibit in Aizheities braits, suggest synapse oss as a primary evers: in easy-ysisition is typics atti denyeitiation is late-rise: tors in SH-SY5Y urnar: Nearbiastosta Ce:s, Eriocsi. fissa (typic: ), if attracier, viar, 994; 8:23:833.87. hotog: 998;39(123: 4882-1889, i:kaiasisi & al., “Spixtaneous transionisation aii is titor ischyskar et ai, 'A Rose for Neclear i: its Neurs: fs:izatia fataxa enclothetist ceils, in Fitr Cei: ; Siof tifierentiation, journai of acroscience. Feb. 5, Siar, 399;263 Pit i:265.74. 28:8;204: 14t4-143. Tsuchiya et al., “in vivo visualization of oxygei: Eadies-de ley et al., Aditsios Maieuses in ynphyte frafficking peatient phottkinissi: -fetito?is 8:23atai (oxygen; itsi and Cotilis, astroenterolog}} {}ct. 2833;2}{4}:Editoria: cais is 8igiogical Systers. 3394233C:238-48, 3.38. 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Nexite Out giastis system ostrai of Endocriadag; 298; 3.85: growth its p 2 Ceis threagh Activation of the Rapi Patis :23.3. way, journai of Biologica; Cigristi: Sep, S, Saint et &, “artise Celi trafficking is liters and Early 2003:275{3): 29.53.288, ..ife is Dominated by the Muccisa; Addressin &adcAM-1 in Bagchi et al., “Comparative in vitro and its wive protein Fiastia:8," Gastroenteroiog: Oct. 2003; 12 it}: 853-864. kistase C activisio try selected pesticities &nd transition. Xia:S-sing at a “sating kinetics of potassivam chate: metasaits, ixicoiogy feters, 389?:33:3-37. assi effects of 388 growth factors is 8:2 ceis a raiyae: Bikiaiwi et ai. "Biologicai Roles of Firosiast Stew8: East with fracts tradei" icia Fierracai Six, £ets. 233:2262: tor-2." Endocrine Reviews, Feb. 1997;38(1):25-45, 103-3), Chiefs et al., "ithi:Eticease Tytosie Hydroxylase eve:s 2hea et ai. "...ithiite regulates protein tyrosiae ghosphatase bath is vivo and in vitro." 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Nature, 9:387: 35?-458. ki raia Res, 2000:85:53-3, Nakamura et al., “Redox regulation of celiuas activation.” Cheng et al., “Effects of itsCS: A4 transfection of: Ana Rey issurgi. 997; i3: 35-36s. 3-hydroxyaotea-triedisted apoptosis snd differentiatic Page et al. “4-Hydroxycotta: presetts Ni-kaggai axi at 8563 human erythrieukemia ceils, Arck Biochen. iio waiian as tumor necrosis factor expressiox by sixibiting phys, 1999;372:29-36. kappas phosphoryliatics and subsequent proteoiysis,” avies et al., “Photo-oxidatio of proteins and its sole is Big Cie, 9994:36:-ii.538, cataractigrisis, J. Patagiaat, hotoioi 8, 23i:33: Faria, e, 83. “HNE, interacts directiy with NK isoforts in 3.35. sistas: repatic stcisie criss, fis: 8es, Eavis et al., “Celuiar this and tective skyger species is 998:32:3942-3358. irug-induced apoptosis.” . 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Trends Biochen Sci, 289;25; 337-329, to buriangutathione S-transferase alpha 4-4 (higSTA-4) Sara et al., “A controld trial of selegitiae, alpha-tsic reveals preferential escalizatios is unaniiver mitocho piero, or both as treatinent for A3a.heister's disease, he &ia frci Biochex Biophy, 233:398: 9-33. 4:ieixei's 3isease Cooperaise Staix & Engi 8:ed isit et ai. “kedox &dalation of p53 catfortation: 33d 997:336:236-323. sequence-specisic iNA bindirig in vitre anteer Res, Sayre et a., '8-Hydroxyacterial-derived advanced tipid 993:53: 4,469.4473. peroxidation end products are increased in Alzheimer's dis Hari 8; 3. “itriplication of a saais (SiPase Raci in the acti esse." Nearciere, i98'i:68: 2092-397. satists of c-ix-N-teria kinases at heat sixk factoria Seat it is... “Exeterixiiiaggiastixione and giutatiote sisti response takeat shock. Bioi (here, 380: 2 is: 888-895. fide sing the firrescetise pruhe (-phthaiadehyde." 42x Singhes it al., “Mediation of serve growth factor-drive ce: Biochen, 238; 28:80-86, cycle arrest in PC3 cells by p53. Simcitateous differettia Sincair et al., “Altered piasina axioxicant status is subjects tion and protiferatiati suissequent to p53 fastionai it:activa with Axicitser's disease and was::is demesis,' it. Geri it." iigi Chen, 2.8-8275: 3829-3837. air Psygia is iggs;33: 383-845, Page 6 of 24 US 6,903,068 B Page 5 Jchida et al., “Modification of histidine resichares in proteins Zimecki et al., “he effect of a proline-rich polypeptide by reaction with 4-hydroxynonenal Proc Nat Aid Sci {FRP) on the humoral immune response, ii. PRF induces USA, 1992:39:4544-4548. differentiation of helper cells fryn glass-nonadherent thy Wagini et al., "Cytochrome P450 and parkinsotistin: protec trocytes (NAE) and suppressor celis from glass-adherent tive role of CYP2E. 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Ther Exp, 1384:32:203-209, * cited by examiner Page 7 of 24 US 6,903,068 B 2 t;SE OR COLESRENN, CONSENT it (WMMEPQGE: (SEQ (Es N8:23, EXPREVEKP PEPEEES HEREOS, AN ANACGS {{R}Fows SEQ to No.3; ifFEEYYXY.P SECREE TEREEE NCR CYNES NO:4); DEEMEWFWEPFPFW (SEQ O NO:3: 83CN FYKLPQ& SEC is Nc3:6; W Ex$KF2P{P}ETyi (SEQ CRESS-REFERENCE is 8ELAEE 3D NO;?); i.&ie8:K.KW Ew88P8 SEE {E} NC: S}; AS WWMEW (SEQ 33). NO:3: SEF (SEQ D NO:38); IKE SEC ENEB is FrpKSE: E3 NC:32, ESC:3W (SEQ 3 he present application cisins priority irgin j.S. paterit :E) Na3:23), 3F2PPQS (SEQEE NO; i3, SEEMF (SE 333 applicatist No. 60: $9,338, fied on Aug. 37. 999, which is NC;15, KYKce: SEQ DNC:16); W.PP:NWG (SEQ $23 ::porategic::its by reference. N8: 7; WYP{{{3PEPN (SE 3: NO:38; Si3N3.33. (SEQ fix}:s; TaiwVPs (SEQ 3) No.203; QPE. BAEON FENSENTS: 388VPKWKia:Wik (S80) it Ne:2); H KEMRFF KYWEREES SEQ is MO:22); S: 3:3:YEK: #3- Coiostrum is a component of the milk of mammals during PEPLW (SEQ 3 NO:23;SWMHEPR SEQ in Ne:24; the first few days after birth. Csiastix is a thick yetiswish p'WMF (SEQ E NO:25; QSwi.S SEQ 3) iiigi &isi is the first actical secretiris inst parturition and is N6:25; LSXPKY 8 pcKAVP{{k}&isi SEQ is contains & high conexistration of imiticagobins (igi, IgM, :NO:3: AFLLYQE SEQE) NO:28}; RGPFPlW (SEQ 3) axi ga &nd a yariety of non-specific proteins. Cokxtram N{3:29; ASR8RYQEE}}:GEEikS. S8C 383 NC:30}; ask contains vario.38 calis such as granuiar and stressia, FLY QEFWLSWR SEQ E. NO:32); LNF SEQ 33) celis, the strophis, racnocyte? acrophages, and symphor NC:33; asid MHQFFQPLPFTWSiFP (SEQ : XO:34: an cytes, oistux: aiso is cities growth factors, heroics, active analog tiereof, ssd castinations thereaf; with the 3rd cytokies, it like tisture breast rail, ostru: cox proviso that the irrinxmiogica: reguiatest is riot WESYv. tsias low sugar, Boy ros, bit is riciisipids, proteias, iii F (SE: BE3 &:3}}, i.e. ce: car the is a ceil citure, 8 exa 53.88, witä:::::s, assi in:iii:38giosis, tissue, as organ irrat grganisri, Hience, this metics can the {oic strun aise includes or contains a proline-rich atrict it in viva or in vitro, sciyetisie &ggregate, which is referred to as gistriii. its another bodigreat, there is pressities: a sixx is (Sae peptide fragment of colostriairs is Wai-Gil-Ser-3yr-Wai staduating at itsmits response it 3 ceii. She retiod: *re-a-Phe-Pix SEC 3: NO:3}, which is disciased its inclusies contacting the ceiwit at it:33:logicategiatx itesaaixia; tibicatios N. WO-A-98.3443. Coostrini Easier citiests effective testice at eas: the cytokite, Rind this fragment have treet identišeiss Seii in the treat whereir: the: immunological regulator is tisted above. The Rett of disorders £f the centra: Beryotis system, tietirologi ceii at he is a xi care, a tissue, at orgat, or isi organ cal distfiefs, inenta: disorders, demertia, retirodegenerative isi. Fiesce this tethox cat is carried oit is via or is -ia. diseases, Alzheimer's disease, actor neurone disease, is yet attather emigier, there is provided a mctiid for 38s chosis, serosis, chronic isolars of the is skie a disting an ia assie test: is 8 patient, she as:xx: systers, disesses with a bacterial and wirai acticiogy, and inciules adrixistering to iiie gatient it infarciogical agaired intological deficiencies 33 set forth in terra registor udes ccasificat: effective to induce at least of: tists?icaixatic. W-A-98:33, cytokite, whereit the irrinitiegies regitiator is listeri Altigh certairs asts for colostrinir have been identified, above. it wild segres: 333 advaceaeat is the art to discover ataxi the islating response ca: he specific of 3ispecific. isci:58 other 3ses for coostrinia, is a capotent theref, iypically, cine or more eyekines are directly induced using that are of radiy ascertainabie from the is formation cir tie poiypeptides described herein, which their resuits is an festy kaowa 3xxiciostriix of its coastituetts, regiators or a dowaegiation of xe or acre cather cytokines. Thus, sing various cornbinations of polypeptides SUMARY FE NEWENN. tiescribed herein, waxios cytokine prefies and it imite the presert insertion reistex to the use of colosiritsin, at respoises csi 88 prodiei, stic ray be specific crisis east of constitue: (i.e., cottponen} peptide theref, at specific. Examples of such insane respecises include the east (xx active analog thereaf (e.g., pepticia swing at: iatifex respecise as: antixxy groxission, is oag &s at N-terisinai Siguktice equiva:ent to st: N-territai sequence iseast one cytokine level ircresses, whether it he increased as 3f at least 88 of the coistricit constitiest peptiis, and a resuit of irst irsiucer: ent y otte of the peptides corrhisations thereof, as 8 cytokine-inducing agent, these describes hereira, or as a resis firsdirect it wicernet e.g., agents can be used as immLalogica: regulators to thodiulate through the interaction with another cytokine, a peptide is e.g. 8883;xce, iiitiit, inosis, asgxxxi, at therwise aiser, “active" as settereiss. &nd preferably groanate specific or nonspecific: Étransie its arosther eibodiment, thers is provided is asthod is i.esponses in patients, particiary animals iniuding nar assidulating 3ox cei: peciferation, he tethod incides it is sis: as husiasis. Shey car aise be used as b$ood cel: s contacting bisex is with a sixxx ce: registor $éected regulators to modiate (e.g., enhance, insibi, tradity, from the group of colostrinin, 8 constituent peptide thereof, >sea;, & Kiewise aster, preferabis, assigrate ceil3 as analog thereof, and continations thereof, iiids: candi, iat prošiferation or differentiation preferatiy, protrioting ticas effective to sharge the nitraber of biocid cesis. The proliferation aski differentiation) of bird ceilis, such as let b:cod cesis can be in 8 cci culture of as organisi. Sience. kgyes. this methixi cake carriei Exit in wise aridi: vitre. in ot: ein bodiment, the present investicit provides a in stili another ambitimes, there is gravitied a methai :3ethosi (fiducing a cytokine in a cell. The aethod includes for modulating bioad cel: proliferation in a patient coatasting the ceit with an intoiogica: regitiator Baier preferably, a higan patient. Eise nethodiackides aiminis enditions effective to indu: (i.e., induce the synthesis or tering to the patient a b$ood cel: regulator selected first the prod&io: £f 33 eas; 8: ytokie (either irectly or gross of coiostrinii, a cristituent epixie thereoi, an assadog indirectiy), whereit the iurnuokagici regulatar is selected thereof, and coixbinations thereof, sider coxiiiots ::fice from the group af MQPPFF (SECR D &O::: QPQ tive a thaige the nutriber of biod cesis, Page 8 of 24 US 6,903,068 Bi 3 4. The blood celis can be maintain bisod cells, such as These terris do not enrote a specific ength of a clymes if hiriars based citis. Freiershiy, the basices are increased arino acids. Shas, for ex&ingle, the texts otigopeptide, in sitsber, athough a decrease in number eat also be ties:- protein, and eazyge are iticided within the definitio: &f abi: in settsii sitations still as leukemia, myeokgatly etc., polypeptide or peptide, whether produceri using tecossibi &iore preserabiy, the biood ceis are increased its Eggber six : riarst seeixigsses, cietrica or exaysiatic systhesis, 3r fast iifferentiated. The bisex ceil regiator is preferably a cor ratiy occurring. This tetin is: it cities polypeptides that sittent systide of goistrain. save been modified orderivatized, such as by glycosylatio, its ether enhoirserts, the invertion provides the use of acetylation, 83sphsyatists, 338 theike, an immuotogical regulator or biood ceili regaiator in the She foilowing abbreviations are used throughout the Ex8nsistie if a restigases: for is: is le netois applicatios: 8&crihacier8i. The preseat it:a::it:3 as provides & ins:te-inducing Expositics: that incides a stractical carria &nd 3. & cease tastica. active agent sesected from the MPPPEP (SEC) RE NO:l}, Ysgax air s: {{PQ.i.C.VM&EPQ&I SEQ is 3 NO:23; QpD. . Seeta excite Ys 3 yrs." - Ese Koscieticise 8x sits sp3agik WEKip£ic:PFQWQS SEQ & XQ:3; FFFE.:"ww8WE.P. Fars & Frisis. is 3:1 r hai:iii. SEQE) NO:4); OLEMWLPWEPFFV (SEQD 83:5; F sex Phenylaxine E. : As 3: Asparticid Mix FYKiipchi (SEQ I ko:6); wi.EMKipe Ysg: 3ryptophar Es: 3 - istasic sci: PQE3v (SEQ SENC:7), LKPFPKLKWEVFEP (SEQ} 88ssessie is sity: No.8); vy MEV (SEQ 8 No:9; SEQF (SEQED NO:38); is Giy a sycae R & Agar Arginite ExE (SE: D&O::::::PPKSE{i} :38:12; BSQFPV Six Sex x. Stice :::is sistidise (SEQ it 80::3; 3PFPPQS (SEQ to No:4); SEEMP (SEQEENQ:5). KYKiPE SER NO:36: W.FeNVG Sec. 3) NO:37: WYegrget N SEQ in No:8; Sieg SAE XESCRO OF REFERRE NEi PL (SEQ to No:i9); QVVVPPF (SEQED NO:38; E3: EiSSES OF ENES& {}}EE&GKKEX:VFK SEQ Six NO:3:3; HKExt{F- The invertists have fi:3d that coiostriti:3, at east 38& KYWERTES. S.E. E. NO:23; SLE: YExit costities; i.e., gatponent pegside thereof, and or at $33st P: PEVE SEC 3D NO:33; SWMHQPF (SEQID NC3:24); cris B-tive acaog there of {e.g. a peptide having 3i. (pip's wife (SEQ is No.25): PQSwis (SEQ to --texasia sequence equivalent is an N-territa seq3:ce RO:26: ...SOPKWLWKAVP (8;&PER (SS: E of a least (as of the &oistini constituettegicies cat be NO:27); ALEYQE SEC ID NO3:28); GFFP:EW (SEQE) used to induce at east oxie cytokine (e.g., iiNifo, IFN-, {O:29: AIE&RYQEEGE: KSE SE: $; NC:3i: Eat, E-2, ii-4, 33-6, -i-, 3-33. Its cytoki: can be FLLYcEPW: GWR SEQ ID NO:32); LNF (SEQ D. either circctiy or indirectiy induced. This can resist is the 8.333; assii Mi3O3PQP.PFryx.FP SEQ 8.0:34; an modulation of an imarin: response or biood cels attifera 8:tive attaing theref 8d xibinatists thexi with the tion or dišerentiation greferabiy, the proaction of bleski toviso that the imminological regulator is not WESYW. cell praisferation, and more preferably, the prisotion if SEP SEQE3 NO:3:. tood ce: pro:iteration as: differentiation) in vitri arti in As seiherein, “a” or "an eas ose or more ratieast wiva, in arisals including marrisis such 3: his rais}. one, stach tist castiniaticsts of active ageists (i.e., active Stilis irr:Etalogicai teguistors and blood cell regislators initiagical regators of bogi ce: differe:tiatics: are resered to here: as “active agents. SigExificantly, Sch prooters), for exatispie, ca: be used in the compositions agents caribs administered alone or in various continations assi methods of the invention. Thus, a cisposition that to a patien (e.g. snisais including Ruasts) as a taedicatists inscr:d:s a psypeptide refers to 3 congasities that or sietary (e.g., statient; supplement is a sics: stificient to includes one or triore po:ypeptides, modulate one or store itainine responses throughout the Axis &id is {:sed serits to refer to a settsia cis paticist's body, it 3 specific tissue site, o in a siection: };f possi with the genera: Sorauia: 333i-CRH-COOH. tissi: sites. where R, the side chain, is H crat organic group. Where R is Mary its;&ific aid specific inaune responses 3r: {}rgaric, 8 cat wary axi is either polar is oppolar i.e., associated with exkocyte grifession as diffe'eatistics. is iropicstic), iiie amic acids of this invention canoe astu The overai in Enuological significate of the pesettisweis raily occurriag or synthetic (often referred to as tiots cat be 8: it of iaited to the flowing: IFN- is a tist-proteinegetic). As Rised hereins, at &rganic group is a potent initionaositiate that is is::start or the devéicy hydrocatios grasp that is classified as at aliphatic grip, is meat of the cytotoxic lymphocyte response (CTI). Ehis &ycii group to coatiatio: {f siphatic assi cycic grass, ia:use response is considered tess wery ins;xtent in pre ise tet "siphatic grug' meats a sistrated or prisaturatei is teeting histians and 8ximals for a variety of bacterial, wit33, linear or branched ayrocarbor group, This ten is used to parasitic, and fungal diseases. The fact that "NF-C is sists &compass asky, akety, sada:3cyayi groups, for exaspie. iridices is japatia: because Ni-c. is 3 isjor sciiwar if The term "cyclic group" meats a closed ring hydroxathon macrophages, among of her irrinxne ceilis, which ste in-go group that is classified 88 at 3icycii: group, aromatic group, tatt in host defense against infections. in additio, irc. {x :eterocyclic group, he traiycii group' seats is has see: shown to have 8ctivity agains: cance, directly cyclic hydrocarbox: group iraving properties resembling through its 3ytic activity and isdirectly through Baeroph those of aiipstic grips. The terra 'sromatic group refers ages. 3-3 is is:er it sportarst immute sitesiiator that con {{ {i}:30- or psycyclic artistic hydrocarisis groups. As trois both fi-yati 88-ci priction and action. its pro used hetein, at organic group can be sshstituted or unsubsti duction represent a negative feedback control for IFN- and tites. TNi-g todactics, Aagher cae of its halimaxk activities is ise tetras “polypeptide' 4tsi peptiis' are used inter the control of satisady production diring the huara: changeably hereit to teier is a polyner of anciso acids. insane responses, which is certainty is portari in any Page 9 of 24 S 6,903,0688 s s types of iaitections. In addition to 3-10's inimus activities, ing isotatics and airification involving tietroit-gress arti it 3 so has been shown to play a rose in the terrondiscribe synthetic techniques. This specific sched if shtaining systers by fixxiiating certisia stress responses said iratise xiigiitii; 333d SER SES N3:32 is described in itsternatists: responses. 3-3 has been shown to iridice the productis of Publication &c. W-A-88.443. Using Hei-C and Edel corticotropis for pititary cesis. Certickstropia works S mat: Degradation, oyer 33 castituent peptides of colastini. iwnstream in the hypociatic acireta axis indise gig isve been iderstified, which can be cssifiexi into seversi cocorticisteroids that ar: inherently inauticitodulatory. groups: {A} those of tanknown precursor; B those having a i.ike i3-3, the 8-4 is isg:tax: if the is eight:388 fB 8-casei actoogie precursor C) iii.33: 3Wing 3 -casein ceit responses, which 3re tie tiediators if the hittinoza precists; and (Estost havings annexit pixx:38or, These int::is response. Eisaiy, the i-3 is an inpatiaist F-- 8 peptides are described it international Pate: Publication isites, take: {{gether these findings suggest tita: exiosiri No, 88 (3:35, 3, fied in. 2, 288, :aising prixity its tii and is cripotent peptides have the ability to modulate just. , 399, and cist he synthesized according {{title geness via cytokie indigetic: ; variety of osi-deterse mechanisms as: 3d descrix is the 8x8asies Section, itese peptides existed by instrophages atti lysiphocytes at the ceilar (i.e. crisitset; peptides of costriai, which as be and his intrue sess we as the recedicine s derived from coiostrinin or chemicaily synthesized, include: systein. &Q'eig (SEC; i3, NO::}; it is Y&EPQGD Thus, the tethods and compositions of the present inves (SEg i Nox2); toge:VEK238°FQWCS {SE{ {{ tists 3 he 8titiexi to control tiraogical aid biosi ce: x):3); FFFLP wVNV.P SEQ to NO:4; DLE&PW differentiating activity, he 8:tive agits described hereir i.ew EPFPFS is: ENO:3: &pgr;YK33°CM SEQ: No. 6; WLEM KFPPPPQErv's (SEQ D No.:7), LKPF8. can be used isdiwiriusly, in various combinations, or cora kil.KWEVFPFe (SEE ENE:8); WWMEY (SELEN:9); baei with other reviously kiswa or newly invented ghar. SEP (Si3C E NO:30; K: {SEQ 3 NOx; }; *::K sacologicai agents, such as antiaxidants. They cat be used (SEQ to NO:32); DSQFV (SEQ II) NO:3); DPPPQs as adivasits for existing vacciastions as weli. (SEQ E. No:4); SEEME SEQ 33 3-8:5). KYK. QF8 in a prefered Triximes, site present inventics, provides (SEQ IE) No. 6; WLPPNWG (SEQ 3 NO:17); wY?:TG a 3-ethodikimositiating an inne response, Whetheritag PIPN (SEQ 13 NC}: 83. SL-QNR: P. (SEQ 23 No:19); is ti's grin wity, this petitoxinvolves inor:::::::g the eve: Twww:8 SEQ EE NO:20; ; c.98; M.GWPKV&ER. of at east one cytokite, which can be dote y known MWrk (SEQ NO:2); HKEM2FPKYPVEFFESQ frie:Kxis, such as tisciosed by got & al., Arch, insistanti. {SEC E N 3:22; S3. TVE&LEiii.; W Q (SEQ is figr. Exa, 4, 25-224 836, Biach-Oiseevska et ai. No:23): Swx Bey (SEQ is No.24; p.PPrvX Fa 3rch. in manai. Ther. Exp., 45, 43-47 (997); Piasecki et ai. (SEQ is NO:35; PQSVLS (SEQED NO:26, SKV. inch. pip;xioi. ?her Exp., 35, 39-27 (997); Bugises at iWE&KAWPQRE8:::{R (SEQ E3 NO:27; A.F.I.Y.E. ai. Fat. i. ix. Rapharpnacai, 7, 85-863 (995; aiki (SEED NO:28); RGPFFILW (SE. E. NC:29; A:FN Mishes & al., Selected fetixis in Ceiliar innan&iogy, RY: DEFEGEELKSE SEQ EE NC3): 'ESYWiF: Wii. Free:33,888. Specific in vitra aetiosis are iexcribed SEC is NO:3: Fix EPWLGPWR SEQ 3: 8:32); in the Ex8tapies Section. LXF (SEQEE NO:33; and MHQPPF; PFWMFF (SEQ in &otix preferred eibodiest, he present isvestion 83:33. These cars he ciassified as Šikows: A thise if provides a method far modulating hood cell protiferation tanknows preciscriticide SERE Nos:2, 5, i. 8, , , , {referably, proliferation and differentiatica). Whether it be 14, and 338 these tasting a 3-caseis. hyologit: precut is tist gris site, this refixiwassessionisting the ieve: sor icide SEQ is NRs: 3,3,3,5, 9, 2, 3.35. 8, , azi of increase or decrease in the Eumber of biotice is tearing 3; 8) those having a case: is precursor inciade SEQ if & speciegheotypig starker for differentistic, the types of Nis: 8 casei &mino acids 24-83, 33 caseis aimini; scids xis foragi air awaiiated, as disclosed by Siti et ai. Cliss. 88-92), 20 castir amic acids 93-382), 2: {casein artino iai. Hasanital. 2, 3-29 (1998; Grunwalekai. Methods acids 3:23-28, 22 casein annic acids 22-38), 23 34ai. Bioi. 9, 443-454 (399; 3:ss et ai. Ceii, is, 5. S {caseia amiso acids 339.356, 24 caseis an:to 3cids 56-63 i998; atti iratains et al., Cytaneir; 33, 68-8 357.:f3), 25 (casein artino acids 64-73), 26 casei :998). Specific in -itro methods are described it the attiao acids :28-379.2 caseitsatsino aciis 388-28, 28 Ex3x3& Sectica. Ecasein attias acids 282-288), 29 (casein antino acilis The peptides described herein may be used for the prolif. 213-222.32 (casein airixto acids 23-24, &nd 34 {casein station 88sior d8 creatistics: gf other types of geis 8s wei. 8xic aciis 59-33, atti (3 those saving 33 &nexits Criosirii) is coaposed of peptides, the aggregate of recursor incisis: SEQ is N:38 (at:texin artic acids which 38s & Etoiccalar weight rage between abyss, S.8 to 203-238. ab{1: 28 kiii)aitons (kia determixi by polyacryiasisie fit certain extinents, 3 prefered grog of 5:8: p33} ge electripboresis, it 3s a greater conceritration of proline tides does not include SEQ (E NO:31. A ritore preferred tars: y ::fier attico 3cici witt: {{siastiaiti has beet fissid group of such pepties incisies: MQFFLP (SEQ is to have a moleciar weight of about 8 kts arid irschscies 80::; Tapili &WMMEPQGE (SEE ENO:2); QP. three no-covalenty linked stibunits having a resexlsr pVEKPDLQFFVQS (SEQ E NO:33; LFFFLPWWN weight of abs: 6 kta and i38 about 22 wi-%grgiiite. Owine Wi.p {SEC D. NC:4); E EXPWLEVEFFFY (SEE E. cokstint has sists been show to contain the faiowing NO:5; M:NFYKLPQ&A (SEQ (ENO:6); WLEMKFPP. surner of residies per suburit: sysine-3; histidine-3: PQEW; SExit No.:7: KPFPKEKVEVFFFP (SEQE) arginine-3 aspartic acid-2; threatiric-4; serine-3; giutaraic NC:8; WYFFT(;IPN (SEQ iO NO3: 8, SLPQNIL?i. acid-6; prolin 3- l l ; giycine-2 alarine-3, waiine-5; SE: El No: 19, Torv WWPPF (SEQE) NC:28), :::thiasise-2, is citicise-2 eucire-6; tyrosie-i i:Ki::PFKYPEF:FESC SEC Ex NO:23, atti corr:3. isenyiaiarine-3; and systeine-6). nations thereof. Cytosisirii has bgc futsad to include a master of pep is the galypeptities of SEQ O Nes: 3-34 can bc in their tities ratgig iroa 3 axiac acids to 23 atairso acids or 38. free scist for or they can he airiidated at the C-:rinias, these cat be staired by various known techniques, includi carboxylate group. The present invention aiso incities ar3 Page 10 of 24 US 5,903,068 B} ogs of the polypeptides of SEQ is 8O8:3-34, which satrix:33S38862; open gas pe: yori, extensios: 882 includes possippicies hayig structura simiarity with SEQ pensity:-3, gap x.iropaifest, expecific. Ysarisiser3. 888 DXOs:-34. These peptides cassais forts as part of slager iter sm. in the corrigadisor of two arrines acid sequeates peptide. As “araig at 8 poiypeptide inckxits 8 east a using the BIAST search sigoriths, structurs similarity is portion of the polypeptide, wherein the portiors costains refered to as “ideatity” refersby, 3 active attaig if deletions of additions of oste or for: cuttigross or Bostcor colostriain of its eanstituent peptides has a strict Fai siri tigious grains sists, or Estaining ox: x skix 3Six : arity to cosiastinia or the 3rmare of its constituet: peptics substitutions. An'aniog" can this it kisse additions attaino preferably, one of SEiENQs:-34) of at east about 78% acids store orth of the terrairii of the polyg:gsides listed identity, nort: preferably, at east about 88% identity, and abers. Sist3titates far as aaiso acid in site pois petities of raost preferabiy, at least aixxt 90% identity, the invention are preferably conservative substitutions, Coistrini or any carnbination of its peptide congresserts which are selecteti festis isther tenbers of the class to which gractive anatags thereof case tierived preferably, isolated the arisines acid beiongs. For ex:pie, it is wei-k38wn is the sai gasified) satirrsity such as by extractio3 ion: 388ts: ast of protein bitchetistry that si smiric acid belonging to a or car be synthetically constructed using known peptide graig faxino acids saving a pattiasis size & c.3:a: polymerization tecisiques, for exarspie, it peptides of the teristic (such as charge, hysiropicbicity and hydrphicity} inyeatica tasy be synthesized by the solid phase tethod can genera?y be substituted for ancither amino acid without asing standard methods based on eithet t-butyloxycarbony substantially attering the structure of a polypeptide, BOC) or 3-Éiaorersy?aethoxy-carbonyi {8848C3 protecting for the purposes of this invention, Cisservative anino groups. This methodology is described by (i.B. fields et at acid stabstitutics are defitters to restle fox skhaagé of in Sinthetic Peptides. A Users Guide, W. M. *reaffias & arrino acids resistics froii, within case if the fosswing Caspary, New York, N.Y., pp. 7-383 (i992. Moreover, classes of residues; Class :: Alia, Giy, Set, he, asid Pra gene sequence escoding the cottistriain peptides or atlaiogs {representing six: aiphatic site chasis and hygroxy: grxis execsi can be constructed by knows isschrities Stich 8s side chains; Class E: Cys, Ser, TF and tyr (represetting expression vectors or piassids sid transfected intes suitabie side chains inciding at -23 car -Sii grog; Class li: tricroorganistris that will express the DNA sequences thus Six, Asp, Asia and it {eart-axy groxg ce::iis& side preparing the peptide for ater extractices frotriaenesii; in chains): Class V: iiis, Ag and Eys (represecting basic side whic: the microorganistri &re growth. For ex8:gies, S. at chains: Class W: i.e. Wai, i.e., Pie and kict representing No. 5,595.887 describes ineth:ds of for:ting a variety of 8:ydrophobic site chains; and Class Wi. She, p. Syr and testively strai peptides thraxagh expressioxx of 8 textti is represeating aromatic site chais, he classes aiso nast geae construct coding for a fusion proteia witich include related ania acids stich is 33iygari:typia Ciass includes 3 binding proteix. 83d one or more copies of the i; hoacsysteige in Class 3; 2-anii. 3-dipic acid, desired target peptide. Aftet expression, the fusicXi gotii is 2- a rain aging lic acid, Y-carboxygiitanic acid, isolated and leaved using cherica: aridor crx2y:Eatie métis is earsaxy3spartic acic, and tie corresponding sinc acid ods to pixitxike desired target peptide. amides in Cias: ft3; orithine, homoarginine, Nengthy : The peptides used in the Earthods of the present investion. y site, iisaithyi lysis, trian ethyl ys in , 2,3- aay be empioyed it is onvalett state {i.e., free peptide or diatria (propicsic 3cid, 2,4-dia::it:b::ty ris: 8&id, s sixgie peptide frages: cocaixi - 8 easier those:seh, fire hotoagiliae, sarcosine and hydroxylysine it. Class V: Ski peptides say also be engloyed as conjugates having 38t: stisked piety ai&isiaes, is tie site. { of visite, thar se (38ts: ct differext peride fragment bound to 8. 2-aminooctanc: acid, 2-amiacheptanoic acid, stains and singie carrier noiecaie, he carrier may be a bickgics car 3-yaiine is Cass w; and rapatihyakassines, substituted tier recae (e.g., a glycosamiracgiyesin, a proteoglycin, payiastines, tetrahydrisqixoise-3-carth:xylic acid, aibstasia &r the 8ke or a synthetic piyster {e.g., 3 poisaiky srid halogenated tyrosines in Class Wii, enggiyces or a synthetic chroatography skippeoEt3. preferably, active anxigs : costinia and its constit typicay, owattsiia. huia serut album, other proteins, ent peptides incisie polypeptides having a reiatively tag: polyet:yiese glycci, or tie Eike are esployed its the c3rtier. nitrater of proline resides, kecause groline is got a csi Such triodifications may increase the apparent affinity andfor ators againic acii, a “large tract' preferabiy Bea:38 that is age the stability as a peptide. The sizabes of peptide polypeptide includes at east atost 35% prairie by fragents associatei with xb3rici to each cars& car w8ty, number, and ratyre preferasiyat east astout 28% pretire (by bat from about to 8 peptides per carrier so?ecules are typi assabex, yiest Feierakiy, active attaiogs insistie are press caity chisined sixties startiard (xxxpisig conditions, tire residues than any other amino acid. For certain For instance, peptidefcarrie anciece caugates may be crisix discists, gteierred group (8 sacs active 88siogs ties pregisted by treating a sixture of peptities and carrier rol at include SEQEE 8:3. ecies with 8 coping agent, sisch as a cartrixiasisie, the As stated above, active scalogs of calestinia and its con casiping agent relay sctivate a carboxy: group o either the sitties: peptities inciatic poiypeptities having strictissa peptidecs react or with the carriera sticicophi: racecake {e.g., 80 that an the8xit extoxyl or hydroxy group similarity, Structura simiarity is gesseraiy deterisisted by group) on the other member of the peptidefeasier aircule, aligning the resiixes of the two amic acid sequences to restigix he covalent litikage of the sigtikie assi the tat optirize the turber of itiatics: attias: acids akxtsg the rier riotectie. For exagpie, conjugates of a peptide cispied engths of their sequences; gaps in either or both sequences to waibaais; asy be prepared by dissolving ciga arcuits are peritted it: Eaking site signett in order to captistice is of yogized pegside atti oyajitsis is a saai soiuzic (f the naster of icietitieai &mic acids, aiii!gh the artites water is a second tube, -ethyl-3-3-inseihyaatias acids is each seqxexca inst nonetheless reissain in their propyl-carboitride hydrichloride {EC; text times the proper ories, referaby, was agaic acid sequences sire exit axist of peptida} is disseived in a sis: arists: if wat. pared using the Bissip program, version 2,8.9, of the BiASf The EDC solution was added to the peptitie? ovalbain 'Fix 3 search algorithis, awaiiase x the worldwide wet at 5 sure 8adaiywed to react for a strather of hosrs. She aixture fictixis.nih.govigorit2.htm:. Preferatiy, tie isfai was say the dialyzed e.g. irio isosphsie afferex satise to wes for all BEAST 2 search parameters are issed, including obtain a purified scation of peptidefoyalistinia constigate. Page 11 of 24 t3S 6,383,068 B {egisscarrier incecise costigates preparisi by this sistics a pr:cageigeriod car 88 airieved by iscitxding 8gests is: ysicality contain alsoast 4 to Speptiis get oweswati nei delaying far example, astinian meets::$tarate 83rigeiati. &c. Ferrariations of the present avé:tica switate foe orai She press: i:we:tion aiso peovicies a sixtapositios: t:a: airsinistratist gay be reset:text as sistercie ::its 838 as includes one ox aste active agents (i.e., colostriair, at east tablets, troches, capsules, lozenges, waters, or cachets, esch age castica: peptic: thereof, or active itskog thereaf isf getting a predeterati:i5:3ourt of the actise ages: as 8 she insention assicae or store citrits, referatiya phasia: posities or granties, as iposomes contaising the Rative castica:y acceptaisie csaries, 33xe aethods of the investion: ages, or as a sciutitsia of suspeasion is 88 &KReos igier8 inside airfixisteringio, or agglying te site ski oš, a paties, nor-33arcas icii such as: Syriga: eixis, as 8ttsios. 8 prefersby a gates, 3rd Rixe preferabiya histian, a cost a draught, the anourt of active agentis stic that the 338g: isitio; cities investiot in as snout effective to produce ieve w; his effective to pr:duce the desired resist is the the desires sitect. Five active agents of the presentisyeatists $3ixieci. are forms?ased for esterat 3distratioE. cxxi, retai, &c. Kass spray fixemiations include 8sified ages: Ski {x gaxentata: aximissistasis tirecticx, .3883: g:lp, etc.). tions at is activg agent with geeservative age:Ets six isc is admissistatists can be via irec: ::sectii its tisskie, : toxic ageists. Six: foraviatists are preferabis assister to & interarterialisection, intervexas injection, or catherinternai pi and isotonic state compatibie swith the nasal trac{3}8 38missististicx gractises, ski as through the use is 38 3einbratics. Foxitiations for esta: or vagiai &isinistris inspianited prisp, or via contacting t:e compositios wit: & tion may be presetsteias & stagpository with & spitatsix carrier xsciss n&strate is scarrier desigExito facilitate trastis stics sixa butter, or hydrogenated $383 or sydroge:38tex sia: {xftive cosposition across 8:33c:3s sixate Sc3.85 ity carxxy}ic acids. Ophthaiac Éxistiatio:38 are 3e a suppository, eye irops, islater, or other sinistraininistes pared by a sittiarateriod to the nasai spray, except thist the tity: sexixxi or wis sai admissistatis is the forts of a syriag, E: aati isotanic factors are preferably &isix to match that is liquid, a pii, capsie, ge: cated abiet, or cxit sinist of the eye, topica: orauiation: 3:3ide the active agest ots: 3digitististica tiethox, he actise agents can be incre issoised Ex suspended it ore or Saor: sedia sixth 8s 33i: scratei into 83 Sidhesive gigste, a gasi, a gates, as: the ike, exa: aii, O&ESO, geoisiyskroxy see:33, or other bases used otik can be encapsulated stic corporate insic & bio-socii is for tagicalipharitacgitica fixinistic88. i:38trix for critissed release, scia dosages of the active agents can be deternisted by is carriers for intertai aiministration can be any said: cargaring 3xeir in six actisity and the is: $fig skiwits $8 consonly used to acilitate the interia: saysicistratics of aiiana: isodels, &exhis for extrapoison :ffffective six8 coxapasitions sat &sississa, sixtie satine soiatios, W sour ages in nice, assissier aniasis, kiwiasars are know8 its the tios ex the ike. Rarrier8 fir aciatinistratists thr33;gh risis art; for xattspie, sects. S.at. 8a, ,938,949. neirbranes ca: be any well-known in the art, Cartiers for the tablets, traches, pits, capsules, asid the like may assic siastration yracas 3e any carries se:3-kixxx is the att. xxxtatai; one consore of the Éxitywing: a bises stici as gi:33 Sh: štautations may be coiseticatiy preserted in it tragacanti, seasia, xxia stage or geistis: 8t Excipient sci: disage for atti aay be prepari by 33y 3f the sisticks 3 as dicaicia phosphate; 8 disintegrating agest such as cot33 waii kawr in the art of pharacy. $3 aetitis iscitade the stsici, potato skatch, aigiris: 8cici assi tise ice; S is six&t step of bringing the active agentists associatist with a cas such as isg3esign stearate; & sweekessing agent Stich as sier which coastities 3:38 {x tore accessory ingeietis, $8. six-rose frtise, lactase or aspasian: said 338tures or afii general, tie fortiukaikiik are: stepsised by aiiority is disti ficial isvoring ager. When the unit cosage forts is & aicy bringing is active gettists associatists with & it cspsule, it way further costsis a tigid c8:ries. 34:ci as 3. sii airies, a fixely isvities said carrier, or kit, 8xi fixa, is vegetable ai: or a poly:byiexegy: i.variors (xies 88teri necessary, siaping tie gradictinto the desirixiffinuatiosis, als gay to present ss (xistings or to 3therwise todity the $ornitiations suitable for parenterai airiaisitation co physical foxii if the solici wait des&ge fora. For instance, weriently include a sexie aqueous pr::sior fibe actise isie: g::is, or cagsties isy be safet with gesatist, wax. ageat, or disgressions of sexie: gowders of the active agitt. site:ac, or stigar and tie Bike. A syrup or exissy contaits which ax: prefixaby iscitoxic wi:is tie icod of the recipient. the or sacre (; a sweet:Eing ages, 3 x&ivative Stick & is:nic agents that can beixeisticei is 8x8 tigiiti preparatist aretty-crprepyigarsken, an age: to retard exys 8izatiot inclusic: sigists, stiffers, and scidium chloride. Statists of of this stgat, s: agent to increase the satubility of asy she the 3cive 3gen as be pregatxi is water, optics::sily sixts: Étgredient, such as 3 poiyiyiric aixirai, for exasagic: giya with a settoxic skifactaat, ispersions of its 8::ive age: S. arol or sortikoi, a dye. &ndiasting agent. The matégia usex tax is: registedia satex, cisatsi, 8 poise: (sixts as giycerit. its preparing axy 3: tosage form is substantiatiy 388xic pregyiese gisai, iiqxiii poiyetisiese glycos, anxi is ise, is the 3racists epiosei, he 3&tise age:st say ts: $3xxx -3- vegetatie is, glycero esters, and mixtures thereof, he rated into sustained-release preparatics arc devices. uititiate isage foets is skiie, iii, 33x3 statie undes tie cositiots catangiactixe 33d storage, ixiecessary faii. 3S EXESS ity can be achieved for exatispie, by 33 gigaxies, by Teixei:tion wi: x&iatiher iscribed by refresce to the capitying the sppropriate partice six 8 in the case of isiowing detaiei &aggies, 38e existspies se 83&art is dispersicas, or by sing surfactants. Stetiigation of a liquid provide itsstration 3:33 shoxiaotix: {x}:series: 88 initing reparation a bic achieved y &ny casseriest aethod that the scope of the present inventiar, Ali peptides were dis preserves the bioactivity of the sitive ages, preferskiy ty soiyed is a taiataxi sai: Scission &xidiaris St. i8ter sterizatia. referexigeticks for piepairig powders Freparation of Peptities: inc::ie vastars drying as freeze citying of the sterie 3. Wasis pre-338 tied resin it El itectisie solutions. Subsequent microbia: castianitiation {diasethylfitaaraisie, ties itsin xxageteiy. can be prevented sing variots attiniercial ageists, fee 2. Add 3rt of 20% piperidineff MF to resia, Shake for 5 &Xangie, antitact::ia 3:{ivisi and &stifiaga &geists tiraxes, ties: itain. inclusiiig garabes-s, chirobtaiot, 883roi, sethic acii. 3, Aisi sotsier 38 is of 28% piperidits::83. Stake for ixitxserossi, 333i:heike, Association of the actise agents ove: 33 hittites. Page 12 of 24 jS 6,903,0688: ... pair reactix vess: a was es: with 88 fossif regitive has defense bastier against diseases associatei taxes, it is was stice with Chi (diciarostatio}}. sii the textata: sist. Check theasis sing the sihysici; test a tie beads shad 8& biu. ABE 5, 8 caping step was carried out as itiows: a. repaire the fixing solutist: itainee Finoc (i.e., itgreyiasetity:oxycitrixxy} axiao said.: iris: 8.43 X: 388 iiii:38: {: trao: 2-3H-8ergistriaxi-3-y}}- & icrosses: 3 cists i,.3,3-tetratisethyistorisia hexafluorop8:sphat:8. eptide :St. 3. ses: hydroxytagstasizo?e-sig3348 airfi:EA anot S388:8 8 --- S. {diisopropylethyletine; aid 8: . &838 h. Add it8 soiliitii: to tie exits assistake it a gaiti: 8 . a823 of 33 it:tes. 3 '8s, 33.33. 883 :) 8. *x- 33 is rais reactic: vesse: sad wash the resis again with &: 3:38 ::f festifies arts with SC. gigs. --- S8 F. Farfor the xia:yirin test: if itsitive {ric coiour - SS SEQXC 0 3S proceed & step 2 axi costinae synthesis; if negative site 8:8 8 $8. coks: a reixth is steg Saari recap:e the satise Fiaccating 8 3. --- S3 8:iii. 8. -: 8. After the synthesis was eotypiste, tie pigstick was 8&: 8 &EE s s N 8. cie&ved from tie resis with 5% ii., 5% passic, 3% 3. S- 333 Thionisote, 3% E1 (etianedichio), 3% riisopropylsilane 3. 8 8: 83%. A for hors. &EEE3 s 9. After 2 hor:rs, fiter its to cold Mi3E triety; thirty NB: 8 : 388 ether the precipitats: peptide was it wastic: twice wit: 3,3 s -- 4&S 8, - 3S38 coid MEBE axi dried usicier nitroger: gas, SE: 8 s it. The triotectia weight if he systhesised was checked Ex: E8 8 , 89.3 by 88trix-Assisted i38er Besogion. Six8-&f Fight hiass SS 8. SE 23 Spectroscopy (ixtS, 3:3 the purity was checked by &ECEE E8 NE 82 it using a C-38, 38 Angstros, 3 x &ciara. it as: : 338 it distics. 3888&d. Ce3 riferatio: 3. N 838 the giantity of periphera: i:iod exkocyte (F8E stiriss . R S3 SEQEE Es &tiots w88 deterinities: by measuring the ataouat of ; NE 38: B-thystidine (1.8 to 2.8:{iyraidinexciture incorporated 83x 3's inta tripitate cultures (8x323; sist:esiassisted with 3.3 33 soilstei:ii. 888 its constit: pegicies (CC} for 3 hours. SEEE 38: 2 --- 3. Fi-thyridine was the added and aliased to incorporate for S:3: E. 4. ES -- S8. 24 h;33rs. Staphy?ococca enterotoxin A (SEA, also referred , 26 8:S to 88'ssipes assigea, a specief ce:33itage, was sex as 88: 33 s : & {{sitive 88troi and for coxsparativs purposes, Coistritis 3.3 S. six ios 38d high iren containing baisy &ralasiated :s 3. 2. SS C:2:3: --- 8: 33d .38 were also usei in so:e experine:ks to determiae : 8. 28 the elative simulatexy &ctivity if these products. Rasiyas 8.8 8 3:38 tivity wa: seasured in 8 Maisix 388 Birect Bits Counter , C : Six replicas of 8&xiua treated critutes were asses to deter SE: s N wa--- &SS3 isite the 1888 backgrotti ixiptasks courts. Eise data is g 38 expresses as the near 8-thyroidine equats per airste Excise {C& sixws ackgrox:33d. R&xits of one cat of sists of six ti-i-x statisgadisetia flying::c882: SciFrascytes experimeats are show below it. Tatia: , - w is exists isdiction syrp.83&asts apioriticacy: sects assistics of lying:6&ts sixtix teacocytes it case sessitat exiostrait aid its castigext sexisis is ; State isdictio: fiyippaxiaasaic iscaya are exce::t. ixitxcers of 38. proliferation. Active cascists. -ass: excit: testions targeti frt: 88 grai to 3.2 xiii. Nixe peptides 8883 &tivity x 388xt coincias &epsiae: By 24-&x 8tti colosirisis and coiostratts were tested. Eertairs resides i-ixexicia&iaerestatix. Eggpeated to have greater activity is others with teaaxi Cytokine stadies: iguat increase is presiserative activity being roughly {} aiostricit is reviously been show is the iteratire to titles above backgrox3d. taggars that sit: Raag of tise advice is:- said Nics, as 33 s-3-Ser-is-Sai-x- regides, the &tive range of poisier8ion: initias was i.e.-:-irk (SEX33) {:33, which is isclosed its inter Fe3e3st sists: coscertaios as igw as Q.: gia: skii: 8: :ati:38: *:8:a::io; c. Q-A-88.4473. Sixs, sixties potext activity. Seine of the gestides said store activity th;as were diate t isvestigate th: individuai peptides, satistrini alie. As other interesting firsting is that coios Cytokine concentratists were also tietetired fox cais tries agge:388 is beiver:gby 80 exquisaientation of actis foiewing 72 scars of incubatics with concentrations ci ity as $35ttixia, SEA has the greatest activity and this is costii and its castixie: peptics: {{C} tanging first: sists as: 388xxx;eitise ic: its classificatios: 3s a super anti 3888, isgui, 33d&s:c-strum and high- a low-iron baby Sea, iii. petiferatian is at impxtsat art of the tracte for a £ristiii) st variots distix, Stigeraaza:3t fittiis t&sp83: both for ge::creating antigea reactive ceis a: wete the subjected to enzyme-inked irrangiosorbes; assay induction of axterosis modulating cytokiries. is the new... 6 (ESA) for the fix:ssig gamerciaisy availabic: cytok. 388 these grexexses are essentiai is a siding bicyck for ises: $8texier83-garta (*-i- is: 8xxosis factor siphs &xeiop:8ext }f 38 gigai trixie resportsc and provides iii-x}, interistikix i-, 3-3. i-30, and ii-32. Page 13 of 24 US 6,903,068 Bi 3. i airies represt::ts the resis of approximately 238 singis assays, viox: specificals, in these stidies it was find tha: iAi E2-cottiyesi imary of the petities its dig colostritin induced if and that the data correspotsis with hi-tlyricise incorers takitesiadiced is titats eakocyte cultures statisted with Boas Talies 3 &nd 3), sterestigy the triaxiakira yaikite C. capistria is cresis faiik rais3. isitating activity of Easy of the peptiis was not iiiuted cist SEC is 8 3. until the 3.0 or i. i girl concentratics of peptide wers NB: s E.283 SEK. :) 88 3. 88: 4:34 33rd Shaded numbers in Taois 2), or in the Eise of IFN-y 3:S 3. 83. and NF-cy induction by SEQED NO:31 atti SEQi NC: 8 85 $3.3 gigai rather 82: righer saceatressioxs. Shis fading s 32 as: 3S axy be consistent with a phasic response ::ce those if her. Ciss: 88. K. SS 88 a8. s Etoses or of toxicity preseat in higher concertistians, SS &S The ability to induce EF8-y by some of the pegfides ar s sik decayed over six. For gaitugie, SEQ E NO:3 st 3, Classers: S. as: a. 3. S3. s 63 agai at the begitivitigs; the sixties insduced 324 g {F}-y 8. s: 838 3. sti ared in tie as: experiments initex is secciasic Beweis. 3. S. Although the eptides iest the F8-y inducing activity awer a SE, s 85. Resicci of four months when stored in solatioxi, stine of the ERE 2 peptities were stili aibie to induce :NF-ct, Il-6, and IL-12, S8:3 but the Sevcis produced were sorcwitat swer that is tics E3:38 easiet studies, in contrast, inductics of TNF-ct, and ::-38 by SE: 33 83 coistiain arid coloss E was stili wery high 3t this tiac. E3:33 SER is E. 135, the couplexed peptides making up coistrinin ird s: coicistraat may be mere stabigaidiot coatinations of pep SE: I tides is coastinia and coiestrum may retroxe patent. Addis :::: tier8 factors that may accessi for the wasistic8s efthe pep tides in these st3dies include: 3 stara waiiatixis in the Timurie state of the individuals donating the eukocytes, 2. SER3. the possibility that aggregation occurred in sainpies stored it NB: BS, thus teracing in effective aximber of 8xolecuies she to Eact, 3rd 3 the possibiity that the is dividzai peptides tray SEQ 8:3 be stitject to oxidative siamage or satise estae inactivating s: grocess, fire fact that the peptide, SEQ is NC;8, which sti SEC 3. induced fN-Y is the last experiment (Example 3 was stored it 33% iMSO suggests 83 oxidative process or aggregation 338cities issy he resportsiisie iriss, reactics of itsic Eas its Sctivity it peglide sanges stirred in phosphate suffered 3: Eart: 38:18 (PBS), iowever, the sainpies in PBS appeared to be in with Sisution, at the titsis of the indiction experiments, Coci SE 88: Cyckies sitxsed liais: eukocytectures signitiates with SE& EE (C. cocarus or conserviai trilk &rass. g sererssssssssssessesssssssssssssssssssssssessee EEE SEE {NCE. E. s 8. N. SEK : (8x. igi: iggin gas se 8 ratasesseewggregagawasawaaaaaaaaaasses Eagle. SEES 8. & SEQia . SEQ: 8:8 : 88:3 N: . SEC is SEQ E. N: . O: St E 0. s: Ei as Eros: SERE East: N it lix SER: is: 35 SS Page 14 of 24 S 3.903,068 81 s s A83.8 2-continued AB8E 2-coastixaxi Cyakass isdictiishisai askeye casses sixtiae: its xixes induced is ktaxa le:kocyte cultures stalaxi wish CP, cocktail afxisteria is firgi's, C3 coststra is exsicciairs. -Ex3s. Sssage SEg: E. 3. 3S 88: S.9 SESEs 8. S. s: S, SEE East : t E: irst R a 3 3&riki: Essai : 3. with Ers {istral Raw SS3 : SEA Sess: sangs es 2S, SEQi. SE 88: : SE)}} SS s: NCEN. SE 33 s RAN 2: s E-3 38 Exp. ii. gia: aft (g gi SERE 8 NS Eggs. Rs: ck.8ttish SS3 SEGi. X N: - N: Extes. s as: SEK. :) r N K: SS E3 3.8 SEE ):38 2. N:8 R. N SEE E. 8: 48. SERE s: - SEQ Eart 8.2 Gs is E. . SS3 3ria SEED Costes: 3 3:4 SEA 8.8 85.3 S&A E. s: SEERINOsci-ani: Si, Raw Colaster, and Costrinitiere reefs:s. SE six sis. Saise day, 88 *SSENs:38, :3, 33, and 32 were recastitutes: 3; disasteria, SEC is 3:3: The reiative abilities of the various peptides to itsilict: g cytskires are siiswa in Eats: 3. She peptities were risikexi according to their sibilities to induce tie indicated cytokitle ticistria by first xing sting the raw authers at the 8, ign: x-see tration, flowed by ligfini coacetrations and the higher 83 teetitiatikas, i.e., 3 and 38 gix it cast be taxed 8t Cilitan SEC is: , 8,3,3, at 33 were thers overal inducers in airtost aii cytokine atti kood ce: proliferation &xperi 'it': S.: meats. Peptides SERENCs:7, 4, anti 5 were generally less E. 2 effective as inducers, Coistrinits 3rd coisstrain ranked get E88:8: eratiy is the aidie, however, any 3:5 isd 3: it distics of SERE celestin were sed, t:us actual cornparison are not accu :: rate since specific proteiis presert aid their coticestratists SE}}}} were not determixed for criostrain. it is important to rote, 3. however, that casiostrain contaited seibstatzes that cosid 88: indice cytokiries in a similar fashion to coistriniti and its SEE) corportent peptides. s: When the cokstriaxis sonstituerii peptides kawirig a -casei: precisor (SEQ (ENCs; 8, 9, 2, and 23 weste SES cosapared to the initiaisy testeriSExx 8 NRs.38 and 3: the s: Batter were better inducers, SEQ SINC:33 was generally the best inducer of those peptides having a -casein precursor, it SERE) was also fossix is at Erfaith; ioxx. irc.: tasy fixtaxia is3duced x83: higher levels of cytokites that the Enfiti high isos fog initia, Page 17 of 24 US 6,903,068 Bi • exiii.ac.3 klis S. i. 3 is a 3x3: .. ::.2. E. K:33: CSESS: Artistia: Sega eks Ex E8. 6.a3a SES 8:33; esci: Articis. Secrete : systis...it gag tidis &:e. Sees: is ss & S. Sek 8:3 at ::: S.C. s. Six Ex& 88 sea Se Wa: . s <33 - SSE : E 33 8 xis ES: 3, ses, it is w23 R32S 3...if:::ia. ::ieck as SR K3232 (TEEE 8:FR3:3CX: Sea existin a refeiai. Sexserties : syrtist: case.ie six 3SSE& 8 88: 83 : 3.x is a Eyre 's Exx. iris Sir st: s & SE I: ; ES S. TE: ; (ES:8: Artic:3& Segates $3.3 ox: {escrip:{x >f Aleksix is 3. sexes:ce: s::itats. 83. ... gk set Lys as F: xx is as 33s S3; tex is: it 5. S 432 SE E R Ka Es S. sex is is (.33: CSK83.SS: &lrtificial Sergence 8:- Exces v223 788.8 &FQSMATICE: Electrigtiari at Artificial set sents: 33.8 stile at is 33 332 x 8& is is i.e. ys is sit is is is 3:8, c. S case, S. K3: . SE if FEG i. s: is: st 383.38%; AskiSicia: Sexssessee as: &EEER. 3888&E:::::: S-seristiar; af 8:tilisia: sagarages sy: tetic setti is Page 22 of 24 JS 693,0688. 3. w-cc.ief x:33: "Es Esri:SAE2: Eesax$gotiss E &xisticssi Sega-ra: S::::::sec: stice £3. 3s SSSES: 33 a te: Sex: Ex 3E SEE is a sex city to We A's &23. 383 & 8: 3 sc set 3 asks EE: 23 ASS; assifs: sessie as Se:33: &:23x E5 E8883,383 he scist of 8::::::::... 3x3xxxixe: syrtiet: eas: s& S&ESCE. S. as s.s S& E is 33 is 3: . 3 : ag33s (RGASiSE: Artificia. Sixtecs s: sis: K233s (3:::R 83Rs: i: excitia;; g :rtiš:3a. Seggage sy::::::tic Existice seax SEE.E; 3. se: ; 3. tax ra. is is to is as a x s s e cair: 3, he method of claim where; the iaisogica: i. A met:acifix inducing a cytokine is a cei, tiss tre:hod 33 tegristor is the coistinia constituent peptide &QPFPEi 8ptising: sistacting the ceili with at intrigogical reg- {SE: NO; , at active alaig thereof, or a combinati: at Q: 3:3der signitiasis festive to induce a cytiziae, thereof. whereis is: itsunokagics regiator is seected for the f, A. Eggs: for acculatig as it:::::::::spotse it 8 ceii, gross consisting (if a coastitue: peptide of colostriain, as the tethod compising contacting the cei with an inautic active attaig thereof, 33d&bitatists tiereof; 38 logica: regulatios under ce:ditions effective to iridace a whereis the constitueat peptide of golestrinia is selected cytokine, w8erein the 88m:Kogical regulater is selected iroa the group consisting of hig::Pip (SEO from the group consisting of a costitiest peptide f N: , ASFQFLQVMXEQGIX SEQ Ex NO:23, co-strisis, 3:3 actise analog theref, and combinations pop;DVRK-f: Qakgways SEC in Ra:3, LFs. het f: x a F.WGWE 3 SEC E3 NC:4, EMFyLPygeRRY were 3e constituent peptide of cokstriain is selected fran the group consisting of &f{{ {SE} is NO: , ; RTPQP:LQVMMEPQGD, SEQ ID NO:2), EQ:POWEKESQPF&WS (SE& O Ko:3), E8. FEW:WLF (SEQE) NO:4), EAE:WWEPFSPW SEC is NO:53, SFXFYKLivi SEQEE) 8:6), wherein the active assaics comprises a peptide having at: WLEMKFFPPPQETVT (SEQE) to:7. KFPCK. aEiso scid sequence with & cast about 5 percent pre W8 WFFFP SEC D. KO: 83, sad MHPPQ. iiae 33d having at leas: six:30 percent sequence ide P:L-PTyMEF (SEQE3 NC3:33; and tity to 8 constituent peptide of coiositirin selected frarr; wherein the active analog of prises 3 peptiis having at the grog coasistiag of 8:PsP3 SEQ X xxi, &ntino acids>ersc: with stest aixit 85 percent gro EGEPQP.E.VSMEFQ3 (SEQ ii) No.2), ice. ite and having at east about its percent seqience ide:- ESWEX81.88 &WS (SE : B K3:3), LFS sity to 3 crostituent peptide if cokostinia sixt: first: F: FWGVEP SEC II XO34, OLExivilip WEEPEREv the grasp consisting of MCFPE i (SEQ F3 O: }, (SEQEE NO:5), MgSFYK is: SEQ 3) NO:s, 3 TPR I.QWh(88.833 (SEQ 33 NC:2), QP. WLEMKFPP{PQETYr (SEQ to No.:7, LKpfrck. EV EXI: QPFWS (SEQ : I XO;3, LFF V : VFPFF (SEQ is 8C.;8, and & Hoppg. Fi PWGVE (SEG E NO:43, ELE&EW3PWEPFe: PLPix F (SEQERNC:34 and whereia said active (SEQ O Ng:53, MPQNFYKLFQM (SEQ O NOx6, 838ogisinces a cytokine, WE.8&KFre:QEW; Sai : N:7), Kapc&- 2, the tiethod of cisira whereiz. the ce: is present is a WEWFFP (SEQ X3:8, aad MHQPPQ cell cufur, a tissue, as organ, r &n organistri. Fi?PFWMFi SE ENO:34) and wherein said active 3. The etics: six wherei se e: is 3 a.asiaatar 333iog Radiates an issure response. ce. 7, the method of claim 6 wherein the cit is present in a ... hexlethcs.ioficiaim 3 sheet it: i.e. eigahuaat celi, ce: culture, a tisstie, 38 xgar, at as organist. Page 23 of 24 JS 6,903,058 B. 33 3. 8. The method of cisin 6 wherein the cell is a tamatian vivipp (SEQ RD NO:8), WESYY: SP (SEC. ii) ce: xOx3, and M.H.QPPse FFS v MFP Seq I 9, he had of skii. 8 wherei, the cei is a funnar ceil. NO:34); 8. She seriod of ciaiti 6 sixtein: tie it:::::::siogicai whereia the active &taskg coagrises & peptise having &is regaixar is the coastinia constituent peptide M.PFP. atrixo acid sequence with at east abst is patcent pro {SE83 ER NO:3), an actise assaig thereof, or 3 stabistics its and having at Best aixit 8 percent sequence ider &rf. ity to a coastituent peptide of colostinia selected freign it. A rathi for Boitatixg 83 imarities resposse it a this greap consisting of MQ???i: {S83 to 8C3), patient, 8xe ethird coa prising sixiristing to the patient iTPFCY SAMESQG} {SE : N3, IQPP an innunciogical regulator usier conditions effective to SVEKOLOPFQWQS (SEQ is KO:3), i.FF ixitxe is cytskirse, wire: the it::::::cirgical regulator is is away; a SER its No:4), 3: Ext:W.WEFF8FV selected fixin the grip consisting of 3 constitiest piptide (SEQEDxo:5), MPQNFYKLPQ&i (SEQ (E) NC:6), of coistria:s, an active attaliog tiereof, asid (X53 biza:35 we&KFFePEQEW; SE: ; 8:2, ...Kiriak vEvrpF (SEQ NC:8), WESYWPLFF (SEQ 33 wherein the constitxeat peptide of {x}ossii is seieckexi iroa, the group consisting of MQPPPiP {SEQ: E No:3), and MHRFQFPPEVMF? SEQ is No: , LQ'qet-Low MMEP}{SE} (SE {E} NC:2), :NO:34: Roge:WEKP: PERWS (SER E. KC:3, 38. and wherein the sumber of Fiskocytes is changed. FrvoV (SEQ No:4), DEXPVLPWEPFFEW 2. Ehe freshasis of giaits 38 whereis tie egocytes are {SEQ is 8:3, MONEYS&M SEQ 38 NC:6), present in a ce: cuttre or its organis E. W.EXAXEggpporv'T (SEQI) No.:7, LKPFPCK 22, Etic ixecisi of ciaits 38 wherein the eukocytes are WEv}{F} {S}:{ i N:8, and Mihi i :33:333i:a:: cells, P8:WSA8 (SEQE) NO:34); asid 23, is right faii. 22 w8te: this eitkyites site whereins the activitirakog comprises 8 peptide having at sist3333 ceilis, aixiac acid sequence witi at east about is percent pro 24, he metaci of claim 2 whereii: the leukocytes ste Éireatsiaawing at east about 78 percent.8quiesce idea increase it taxes. ity to a coastitucnt peptide of cascaisinis selected for 25, he method of claia 24 w8ereirs that leukocytes are the group consisting of MQPPIF (SEQ is NO: ), ifetisted. {}}''QPL. QY&sific G3 SEQ 3:3 N33), 39°p 26. The prietisod of clairex 2E wherein his Ekocyte regis RWEK2OLOFFQYQS (SEQ is KO:3). LFF. $or is a constitxeit peptide of casiostinia, *PWGyi (SEQ NC:4. OLExEVIEWEPFPF 2, The trie:xxi of caii:28 wherein its eikocyte regis SEQ 3E 80:S), MPQNFYKiilya SEQ 33:30:6), tot is the crossinin constituent peptide WSSYWP:...F (SEX: VLEMKFagPPQErvi (SE& E No.:7, LKPFCK 333 NO:33, at actixe 8:aikg thereof, or a corebination WEWFPF; SE8 : I NC 8, and MiqPPQ eEf. {i Piaf'WNiFF (SEQ is NO:34) and whereirs said active 28. The tethod of cairn 28 wercise is kocyte 8giar aRai: 8xxitiates 38 it:::Res excise, taris the coastrinin constituent peptideMQPFPL (SEQ fi 3. The arthod of claiti 1 wherein the im:::to:ogica: N838, as active analog thereof, or a corbitatics; theref regulator is administered as park is a dietary siggienent. 29. A method for modulisting leukseyte prliferatiot in a 3. The method of ciam ... wherit the ixit togica: patient, the racisodcomprising admissistering to the patiest is reggiator is administered typically. Seskayte regulator selected from the groug catsisting of 4. The Exxi of ciaisi 1 wherei Eise patest is an colostrinix, a constitiest peptide theref, Sri active anaig animal. :ereof, axi cabitatioxxs thereof, wid: ::3diti; effec 5. it the:hod of ciaiti is where the pst:ext is a tive to charge the number of eukocytes; hunkar, whereit be constituent pride of koks&inis is selected 6. The Esekhod of giai ii whereirs theixxistics response first tie grough sisting of MQFP} SEC I) is a specific iristine response. NO::), TPP i{WXixi:QGI (SEQE) NC:2), . The tiethod of giaix it whereii: thei:nsuite respase XQP}WEKFDLQRFQWQS SEQ 3) KO:33, LF8 is a ranspecific is airie res.333e. FLEWGVP (SEQ is NO:A, DEMF'vi PWEPFPFY i8. The rivethod ofciaix E wherein the ixttaine response (S8 its XC:5}, XiPQ8:FYKLFQM SEQ is NO:6), is tie interferon response of antibody productiox. W.E.M.KFIPSPQE"W SEQE) NO:7), KPFPCK. 9. The method of claim it witherein the iristinoicgica: VEwsFrf: SEQ E. Nix8, WESYW2:...F8 SE} { registor is the coiostrikin constitxea: peptide 8:QF8: No:33, and MHQ.F.P.Pi"WMEP (SEQ ED (SE ENO: ), an active analog thereoi, or a cabination N834); erecyf. where is active 88&log stiprises a Reptide having 38 28. A nethod for nodulating eukocyte proliferation, the sing acid sequence with at eas: a-cut Spes cent pror Elethod (SEprising contactitgeikorytes with 8 eukocyte iiie at having at east 83.8 exce:3t sequesseeida regulatir selected fox the group consistiag of Exxiostricii. 8. tity to a constittent peptide of coiostrinin selected from constituent peptide thereof, an: &ctive sysiog theref, and the group consisting of 38:FPI. (S8 is NR3:32, combiaates thereof, inder conditions effective to kage Qipcil. V&MERCGL (SEQ E NO:2), QFE. the number of eukocytes, saw:Kpic foWS (SEQ E. KO:3), LFF wiercia: tire coastitueix peptide of cesistinia is selectix Frygvir (SEQR Nex4), DEEMFVE:WEPFPFW frog the grip consisting of MP?PLF (SEQ iD (SEQ it xo:5), MPQXFYXLegyi (SEQ 3) NO:63, No: i , Li2O'L{{W&MEFQ&E (SEQE) NC:2), Y: Ex KEgppgEry (SEQ to 80:7, Ke$8CK Expr:WEKii-Q:PFCWCS (S80} {} KQ3, LFF. WEWFPF (SEQ No.83, WESYV3 Fir SEQ ID FLPWG WLF (SEQED NO:43, i.EMEWLFVEFFPFW bic:33, and x}{ee: i.e. wh: Fe (SEQ is SER BE NCS, SFQ:YKirch: SEQED &:6. NO:34. V EM&FPPPEW (SEs. If No.73, KPRPK and w:iesei: the sumiher of keiskocytes is cisaged. Page 24 of 24 US 6,903,068 B1 35 36 30, he raethod of claim 29 wherein the patient is a ID NO:3i), an active analog thereof, or a combination histian, thereof. 3. The method of claim 29 wherein the leukocytes are 35. The method of claim 29 wherein the leukocyte reguia increased in timbe, toris the colostrini constituent peptidevi(PPi'ili (SEQ is 32. The method of claim 31 wherein the leukocytes are "“” thereof, or a combination thereof. differentiated, 36. The method of claim 29 wherein the leukocyte regula tor is administered as part of a dietary suppiement. 33. The timethod if cast 29 wherein the ekocyte regula- 3i, he method of clair; 29 wherein Ele regula or is a constituent peptide of coistinin. tor is administered topically. 34, he aethod of claim 29 wheris the eukocyte regular tor is the coiostrinin constituent peptide WESYWPilf? (SEQ UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 6,903,068 B1 Page 1 of 1 APPLICATIONNO. : 09/641801 DATED : June 7, 2005 INVENTOR(S) : Stanton et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: Column 32, line 61, Claim 1, delete “SEQID KO:3' and insert --SEQID NO:3-; Column 33, line 5, Claim 1, delete “SEQID KO:3 and insert --SEQID NO:3-; Column 33, line 31, Claim 6, delete “SEQID KO:3' and insert --SEQID NO:3-; Column 33, line 42, Claim 6, delete “SEQID KO:3' and insert --SEQID NO:3-; Column 34, line 1, Claim 11, delete “SEQID KO:3' and insert --SEQID NO:3-; Column 34, line 13, Claim 11, delete “SEQID KO:3' and insert --SEQ ID NO:3--: Column 34, line 47, Claim 20, delete “SEQID KO:3' and insert --SEQ ID NO:3--: Column 34, line 60, Claim 20, delete “SEQID KO:3' and insert --SEQ ID NO:3--: Column 35, line 30, Claim 29, delete “SEQ ID KO:3 and insert --SEQ ID NO:3-; Column 36, line 7, Claim 29, delete “SEQID KO:3' and insert --SEQID NO:3-; Signed and Sealed this Thirty-first Day of March, 2009 4 (O-e- JOHN DOLL Acting Director of the United States Patent and Trademark Office