Title of Invention	PEPTIDES WITH PHYSIOLOGICAL ACTIVITY
Abstract	A substantially purified peptide having wound healing activity, the peptide comprising a sequence of 44 amino acids, that is : K-K-L-K-K-E-E-N-P-L-E-L-K-E-K-L-K-E-K-K-N-P-L-P-S-K-E-E-E-K-A-S- P-F-D-K.-I-T-E-T-P-D-M-S (SEQ ID NO: 8), the peptide being linear.

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FORM 2 THE PATENTS ACT 1970 [39 OF 1970] COMPLETE SPECIFICATION [See Section 10] "PEPTIDES WITH WOUND HEEALING ACTIVITY" CHANDA ZAVERI, a US citizen of 6740 Los Verdes Drive, Suite 8, Rancho Palos Verdes, California 92075, United States of America, GRANTED 10-11-2006 The'following specification particularly describes the nature of the invention and the manner in which it is to be performed :- WO 01/96527 PCT/US01/18830 BACKGROUND OF THE INVENTION This invention is directed to peptides with various physiological activities, 5 particularly wound healing. Wound healing is a complex biological process that differs according to the wound type: acute or chronic. The principal elements of wound repair are the immediate events of hemostatis and stimulus for inflammation, then inflammation and cell proliferation 10 and migration, then followed by molecular synthesis, collagen polymerization and cross-linking, remodeling, and wound contraction. Inflammation is characterized by vasodilation, increased vascular permeability, leukocyte infiltration, bacterial killing, and macrophage-based stimulation of cellular proliferation and protein synthesis. 15 In cell proliferation and migration, fibroblasts appear within 2-3 days and dominate wound cell population during the first week. For the initial 2-3 days, their activity is confined to fibroblast replication and migration. At days 4-5, fibroblasts begin to synthesize and secrete extracellular collagen. Fibroblasts produce GAG and collagen. 20 Angiogenesis is essential to wound repair and scar formation. Capillary proliferation is required to support fibroblast migration into wound and fibroblast metabolic requirements, in the absence of angiogenesis, such as in ischemic ulcers or arteriosclerosis obliterans, fibroblast migration arrests and wound healing fails to proceed.' 25 Angiogenesis has the stages of cell attachment, basement membrane degradation and migration, proliferation, and differentiation, and is associated with epithelial cell migration. Molecular synthesis includes collagen synthesis and proteoglycan-synthesis, 30 Collagen synthesis begins with the intracellular phase of monomer synthesis. Secretion into WO 01/96527 PCT/US01/18830 the extracellular space then occurs, followed by polymerization into collagen fibers and cross-linking to increase tensile strength. Remodeling typically begins 3 weeks after injury. Equilibrium between 5 collagen synthesis and degradation is achieved. Wound remodeling begins and will continue for 2 years. There is a progressive increase in tensile strength as Collagen EL is replaced by Collagen I. Epithelialization is the hallmark of successful wound repair and occurs in four phases: mobilization, migration, mitosis, and cellular differentiation. 10 Granulation tissue contains numerous capillaries and has a support matrix rich in fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and periocytes. If vascular endothelial growth factor (VEGF) is removed, there is an absence of granulation tissue, and wound angiogenesis and the wound healing process cease. 15 In chronic wound healing, there is typically an absence of epithelial migration, excessive granulation tissue, and fibrosis, with scarring and impaired function possibly being present. Although many advances have been made in the understanding of wount 20 healing, the healing of wounds still presents a considerable challenge to the clinician. This i; particularly true in patients who are diabetic, who have impaired circulation of the skin, o who are susceptible to infection, such as the result of being in an irnmunocompromise condition. Additionally, when such wounds do heal, they frequently heal with cosmeticall undesirable consequences such as scars or discoloration. 25 Accordingly, there is a need for an improved method of wound healing that; particularly suitable for application in patients with diabetes, who have poor circulation in the skin, or who are immunocompromised. There is a further need for treatments and method that can reduce or eliminate the consequences that can occur from wound healing, such 30 . scars and discoloration. There is an additional need for factors that are Well-tolerated and be used with other treatments in such patients. SUMMARY One aspect of the present invention is a substantially purified- peptide having a physiological activity, the peptide pomprising a sequence from 28 to 44 amino acids, including therein a sequence of L-K-E-K-K (SEQ ID NO: 1), the peptide having a physiological activity selected from the grouP consisting of wound healing activity, immunostimulant activity, and growth factor activity, wherein the peptide is in linear or cyclic form, and wherein the peptide is in unear form, the amino terminus is optionally acetylated. The sequence within thi? peptide can be L-K-E-K-K-E (SEQ ID NO:2) or L- K-E-K-K-E-V-V-E (SPQ ID NO: 3). The invention incluldes the following peptides: (1) a substantially purified peptide having wound healing activity, the peptide comprising a sequence of 28 amino acids, that is Ac-S-D-A-A-V-D-T-S-S-E-I-T-T-K-D-L-K-E-K-K-E-V-V-E-E-A-E-N (SEQ ID NO: 4), where "Ac" indicates that the amino terminus is acetylated, the peptide being linear; (2) a substantially purified peptide having wound healing activity, the peptide comprising a sequence of 35 amino acids, thatis: K-L-K-K-T-E-T-E-Q-K-N-P-L-E-V-L-K-E-K-K-E-V-V-E-L-K-E-K-K-V-V-I-E-N-P (SEQ ID NO: 5), the peptide being linear; (3) a substantially purified peptide having immunostimulant activity, the peptide comprising a sequence of 38 amino acids, that is: Ac-A-D-K-P-M-G-E-L-A-S-F-D-K-A- G-L-K-E-K-K-E-T-L-P-T-K-E-T-I-E-E-E-K-R-S-E -I-S (SEQ ID NO: 6), the peptide being linear; (4) a substantially purified peptide having wound healing activity, the peptide comprising a sequence of 44 amino acids, that is: Ac-A-N-K-G-Q-A-P-G-E-A-M-K-P-S-F-L-K-E- K-K-E-V-V-E-R-S-K-E-E-E-G-P-A-K-M- N-L-V-I-E-M-P-K-D (SEQ ID NO: 7), the peptide being linear; and , (5) a substantially purified peptide leaving wound healing activity the peptide comprising a sequence of:44 amino acids, that is: K-K-L-K-K-E-E-N-P-L-E-L-JC-E-K-L-K-E-K-K-N-P-L-P-S-^-B-E-E-K-A-S-P-F -D'K-J-T-E-T-P-D-M-S (SEQ ID NO: 8), the peptide being linear,. WO 01/96527 PCT/US01/18830 Another aspect of the present invention is an isolated nucleic acid molecule encoding a peptide of the present invention. Typically, this nucleic acid molecule is DNA. Yet another aspect of the present invention is a vector comprising DNA 5 according to the present invention operably linked to at least one control element that influences the expression of the DNA Still another aspect of the present invention is a host cell transfected with a vector according to the present invention such that the cell expresses the peptide encoded by 10 the vector according to the present invention. Another related aspect of the present invention is a method of producing a substantially purified peptide having a physiological activity comprising the steps of: (1) culturing a host cell according, to the present invention; and 15 (2) isolating the peptide produced by the host cell to produce the substantially purified peptide. Yet another aspect of the present invention is a pharmaceutical compositior comprising: 20 (1) a peptide according to the present, invention in a physiologically effective quantity;,and (2) a pharmaceutically. acceptable carrier. A particularly preferred pharmaceuticaUy acceptable carrier comprises watei 25 Seamollient, a -polymer selected from the group consisting of carboxymethylcellulose an Still another aspect of the present invention is a method of use of a peptid .60 . . Recording to the present invention for promoting wound healing, stimulating the immun system or stimulating pituitary/hypothalamus activity in a mammal in need therec 'cpmpi!sing administering an effective quantity of a peptide according to the present inventio ' having the appropriate activity to the mammal. PCT/US01/18830 WO 01/96527 These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where: Figure 1 is a graph showing the effects of the peptides TB4 and TAi on endothelial cell migration in a Boyden chamber migration assay; Figure 2 is a graph showing the effects of the peptides TB4 and TAi on proliferation of endothelial cells at 4 hours and 24 hours after stimulation as measured in a MTT (tetrazolium) assay; Figure 3 is a photograph of an ulcer on the shin of a first patient (A.T.) before treatment; Figure 4 is a photograph of the ulcer on the shin of A.T. after one week of treatment with TA1, showing improvement; Figure 5 is a photograph of the ulcer after an additional week of treatment; Figure 6 is a photograph of the ulcer after further treatment with TB4; Figure 7 is a photograph of the ulcer after additional treatment with TB4, showing continued improvement; Figure 8 is a photograph of the ulcer after treatment with TB4 had been temporarily suspended for one week; Figure 9 is a photograph of the ulcer after treatment with TB4 had been restarted; Figure 10 is a photograph of the ulcer after further treatment; Figure 11 is a photograph of the ulcer after nine days of additional treatment; Figure 12 is a photograph of the ulcer after an additional three weeks, showing additional improvement; Figure 13 is a photograph of an ulcer on the finger of a second patient (P.M.) with diabetes mellitus before treatment; Figure 14 is a photograph of the ulcer after one week of treatment with TA5, showing considerable improvement; ' WO 01/96527 PCT/USO1/18830 Figure 15(a) is a photograph of a new ulceration that subsequently developed on the finger of P.M.; Figure 15(b) is a photograph of a second ulceration that subsequently developed on the finger of P.M: Figure 16 is a photograph of the ulcerations after one week of treatment with TB4, showing considerable improvement; Figure 17 is a photograph of the ulcerations after another five days of treatment with TB4, showing considerable further improvement; Figure 18 is a graph showing the effect of varying concentrations of TA] on keratinocyte migration as measured in the Boyden chamber in an in vivo experiment in rats; Figure 19 is a graph showing the effect of the topical TAi and normal saline (NS) on gap closure in the wounds in vivo for 4 and 7 days post-injury; Figure 20 is a graph showing the effect of the intraperitoneal TA1 and normal saline (NS) on gap closure in the wounds in vivo for 4 and 7 days post-injury; Figure 21 is a graph showing the effect of the topical TA1 and normal saline (NS) on wound width in the wounds in vivo for 4 and 7 .days post-injury; Figure 22 is a graph showing the effect of the intraperitoneal TA1 and normal saline (NS) on wound width in the wounds in vivo for 4 and 7 days post-injury; Figure 23 is a chart showing a model of wound healing that depicts possible points of action of peptides according to the present invention; and Figure 24 is a graph showing the effect of peptides TA1 and TA5 on the stimulation of collagen synthesis in chronic wounds in humans as compared with tretinoin and vitamin C. DETAILED DESCRIPTION I. PEPTIDES HAVING PHYSIOLOGICAL ACTIVITY A. General Description One aspect of the invention is substantially purified peptides having a physiological activity. The physiological activity can be one or more of wound healing WO 01/96527 PCT/US01/18830 activity, immunostimulant activity, and growth factor activity, particularly growth factor activity directed toward the pituitary or hypothalamus. Peptides according to the present invention typically have a spectrum of activities that includes wound healing activity and can include one or more of the other activities recited above. The peptide comprises a sequence 5 of from 28 to 44 amino acids and includes therein a sequence of L-K-E-K-K (SEQ ID NO:l). This sequence can be extended so that it is L-K-E-K-K-E (SEQ ID NO:2) or L-K-E-K-K-E-' V-V-E (SEQ ID NO:3). As indicated below with respect to the specific peptides that are ' aspects of this invention, this sequence is substantially invariant. 10 Peptides according to the present invention can be linear or circular. If the. peptide is linear, the amino terminus is optionally acetylated. In the context of this specification, the term "substantially purified" refers to a state of purity that is at least 50%, preferably at least 70%, more preferably at least 85%, and 15 still more preferably at least 95%, and in which the peptide having physiological activity is present in the substantial absence of other peptides or proteins having physiological activity. B. Specific Peptides of the Invention 20 1. Peptide TA1: One of the peptides of the present invention is a 28-amino acid peptide having wound healing activity, designated TAj. The peptide has the sequence SEQ ID NO:l: Ac-S-D-A-A-V-D-T-S-S-E-I-T-T-K-D-L-K-E-K-K-E-V-V-E-E-A-E-N. In this peptide and other 25 peptides of the present invention, the notation "Ac:' at the amino terminus of the peptide indicates that the amino terminus is acerylated. Generally, this acetyl group can be cleaved without impairing the function of the peptide. This peptide is linear. This peptide has an acetylated amino terminus and has a molecular weight of 3071 Daltons. The isoelectric point of this peptide is 4.1, indicating a predominance of acidic amino acids. This peptide has 30 wound healing activity and may be derived by cleavage of thymosin. 2. Peptide TA.sub.2 Peptide TA.sub.2 is a 35-amino-acid-peptide also having wound healing activity which has a sequence of SEQ ID NO:5: K-L-K-K-T-E-T-E-Q-K-N-P-L-E-V-L-K-E-K-K-E-V-V-E-L-K-E-K-K-V-V-I-E-N-P. This peptide is linear and has a free, unblocked amino terminus. This peptide incorporates the conserved sequence of L-K-E-K-K-E-V-V-E (SEQ ID NO:3) 3. Peptide TA.sub.3 Another peptide that is an aspect of the present invention is a peptide designated TA.sub.3. This is a peptide of 38 amino acids that has immunostimulant activity. This peptide has the sequence SEQ ID NO:6: Ac-A-D-K-P-M-G-E-I-A-S-F-D-K-A-G-L-K-E-K-K-E-T-L-P-T-K-E-T-I-E-E-E-K-R-S-E -I-S. This peptide is linear, and has an acetylated amino terminus. This peptide incorporates the conserved sequence of L-K-E-K-K-E (SEQ ID NO:2). 4. Peptide TB.sub.4 Peptide TB.sub.4 is a peptide of 44 amino acids having wound healing activity. This peptide has the sequence SEQ ID NO:7: Ac-A-N-K-G-Q-A-P-G-E-A-M-K-P-S-F-L-K-E-K-K-E-V-V-E-R-S-K-E-E-E-G-P-A-K-M-N -L-V-I-E-M-P-K-D. This peptide is linear. The amino terminus of this peptide is acetylated. This peptide contains the conserved sequence L-K-E-K-K-E-V-V-E (SEQ ID NO:3). 5. Peptide TA.sub.5 Another aspect of the invention is a peptide designated TA5, which has 44 amino acids. This peptide has wound healing activity. This peptide has a sequence of 44 amino acids, which is SEQ ID NO: 8: K-K-L-K-K-E-E-N-P-L-E-L-K-E-K-L-K-E-K-K-N-P-L-P-S-K-E-E-E-K-A-S-P-F-D-K-I -T-E-T-P-D-M-S. This peptide is linear and has a free, unblocked amino terminus. This peptide includes the highly conserved region L-K-E-K-K (SEQ ID NO: 1). WO 01/96527 PCT/US01/18830 In addition, peptides having conservative amino acid substitutions except in the highly conserved sequences of L-K-E-K-K (SEQ ID NO:l), L-K-E-K-K-E (SEQ .ID NO:2), or L-K-E-K-K-E-V-V-E (SEQ ID NO:3) are within the scope of the present 5 invention. It is a well-established principle of protein and peptide chemistry that certain amino acid substitutions, entitled "conservative" amino acid substitutions, can frequently be made in a protein or a peptide without altering either the conformation or the function of the protein or peptide. Such changes including substituting any of isoleucine (I), valine, and leucine (L) for any other of these amino acids; aspartic acid (D) for glutamic acid (E) and 10 vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine for threonine (T) and vice versa. The above-mentioned substitutions are not the only, amino acid substitutions that can be considered "conservative." Other substitutions can also be considered conservative, depending on the environment of the particular amino acid. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can be alanine and valine. Methionine (M), 15 which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Cysteine (C) can frequently be replaced by serine when cysteine's capacity to form disulfide bond is either 20 undesirable or unneeded. Still other changes can be considered "conservative" in particular environments. As indicated above, the peptides of the present invention can be either circular . or linear. The specific peptides described above, however, are linear. Some of the peptides 25 of the present invention have their amino termini blocked, typically by acetylation. However, ' ' these acetyl groups can be cleaved by hydrolysis without interfering with the function of the peptides. 50 II. NUCLEIC ACIDS ENCODING THE PEPTIDES Another aspect of the present invention is isolated nucleic acids encoding a peptide according to the' present; invention, particularly, the peptides of SEQ ID NO:4, SEQ WO 01/96527 PCT/USO1/18830 ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. As used herein, the term "nucleic acid" includes both DNA and RNA and both single-stranded and double-stranded forms; if double-stranded, DNA-RNA hybrids are also included. Recitation of a single-stranded nucleic acid sequence also includes its complement according to the generally . 5 accepted Watson-Crick rules for base pairing. Nucleic acids encoding these peptides can either be DNA or RNA; however, in many applications, DNA is preferred. The term "isolated" is used herein to indicate that the nucleic acids are present in substantial isolation from nucleic acid molecules that do not encode a peptide according to 10 the present invention. In the context of this specification, the term "isolated" refers to a state of purity that is at least 50%, preferably at least 70%, more preferably at least 85%, and still more preferably at least 95%. However, nucleic acids according to the present invention can be incorporated 15 into larger nucleic acid molecules such as vectors for transfection of appropriate host cells and production of a peptide according to the present invention, and the term "isolated" is nol to be interpreted to preclude this incorporation into larger, genetically-engineered molecules not occurring in nature. 20 The sequence of the nucleic acids is chosen according to the conventiona triplet genetic code to encode the amino acid sequence of the particular peptides. Because the genetic code, which specifies amino acids by triplet codons in the nucleic acid sequence, i degenerate, and many amino acids are specified by more than one codon, all possibl alternatives of codons can be used. However, in some cases, the efficiency of transcriptioi 25 and/or translation of the nucleic acid sequences can be affected by the codon selection. 1 such cases, it is preferred to use codons that provide increased efficiency of transcriptio: and/or translation of the nucleic acid sequences. 30 III. VECTORS AND HOST CELLS Another aspect of the present invention is a vector comprising a DN according to the present invention operably linked to at least one control element the 10 WO 01/96527 PCT/USO1/18830 influences the expression of the DNA. These control elements can be promoters, operators, enhancers, or other nucleic acid sequences that affect the expression of the DNA. The vector can be derived from either prokaryotic or eukaryotic sources. The vector can comprise sequences of chromosomal, non-chromosomal, or synthetic DNA sequences. Typically, these vectors include one or more cloning sites that contain restriction endonuclease sequences that are readily cleavable by specific restriction endonucleases. It is generally preferred that these restriction endonucleases yield cohesive or "sticky" ends for more efficient cloning in of the desired sequence. Some suitable prokaryotic cloning vectors include plasmids from Escherichia coli, such as colEl, pCRl, pBR322, pMB9, pUC, pKSM, or RP4. Prokaryotic vectors also include derivatives of bacteriophage DNA such as Ml 3 and other filamentous single-stranded DNA phages. Other vectors, such as baculovirus vectors, can be used. * Examples of useful expression controlled sequences are the lac system, the trp > system, the tac system, the trc system, major operator and promoter regions of bacteriophage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters of SV40 and other .0 sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof. Vectors useful in yeast are available. A suitable example is the 2μ plasmid. Vectors for use in animal cells are also known. These vectors include derivatives of SV40, adenovirus, retrovirus-derived DNA sequences, and shuttle vectors derived from combinations of functional mammalian vectors, such as those described 25 above, and functional plasmids and phage DNA. Another suitable vector is the baculovirus vector. In general, however, it is preferred to use a vector that is suitable for 30 expression in E. coli. Vectors are inserted into a host cell for expression. Typically, these vectors are inserted info a host cell by methods well-known in the art, such as transfection, 11 WO 01/96527 PCT7US01/18830 transformation, electroporation, direct injection of the DNA, Iipofection, and other well-understood methods. The method to be used can be chosen according to the host cells selected and the size and conformation of the DNA. Some useful expression host cells include well-known prokaryotic and eukaryotic cells. Some suitable prokaryotic hosts 5 include, for example, E coli, such as E. coli SG-936, E, coli HBlOl, E coli W3110, E coli x1776, E. coli x2282, R coli DHL, and E coli MRCI. Other bacterial and fungal host cells could be used, such as Pseudomonas, Bacillus species, such as Bacillus subtilis, and Streptomyces. Other host cells that can be used are eukaryotic cells such as yeast and other fungi, insect cells, animal cells, such as COS cells and CHO cells, human cells, and plant 10 cells in tissue culture. TV. METHODS OF PREPARATION OF PEPTIDES 15 A. Solid-State Peptide Synthesis Peptides according to the present invention can be synthesized by standard solid-state peptide synthesis methods, such as those described in M. Bodanszky, "Principles of Peptide Synthesis" (Springer-Verlag, Berlin, 2d ed., 1993). This involves synthesis on an 20 insoluble polymer such as a styrene-divrnylbenzene copolymer that is derivatized. The sequence of reactions used is standard. B. Genetic Engineering 25 Peptides according to the present invention can be prepared by genetic engineering. In general, a method of producing a substantially purified peptide according to the present invention having a physiological activity comprises the steps of: (1) culturing a host cell transfected with a vector comprising DNA encoding the peptide operably linked to' at least one control element that influences the expression of the DNA; and (2) isolating the 30 peptide produced by the host cell to produce the substantially purified peptide. WO 01/96527 PCT/US01/18830 Expression methods are described in, e.g., D.V.. Goeddel, "Gene Expression Technology" (Academic Press, San Diego, 1991). In general, such methods are. well known in the art. 5 Once expressed, the'peptides of the present invention can be isolated by standard protein isolation techniques including ion-exchange chromatography on resins such as diemylaminoethylcellulose or carboxymethylcellulose, chromatography on size exclusion media (gel filtration), isoelectric focusing, chromatofocusing, and other standard methods, such as those described in R.K. Scopes, "Protein Purification: Principlesand Practice'-(3d 10 Ed., Springer-Verlag, New York, 1994). tf polyclonal or monoclonal antibodies are prepared to these peptides, these antibodies can be used in affinity chromatography by standard methods such as those described in the above-identified Scopes book. Such methods for the preparation of 15 polyclonal antibodies or monoclonal antibodies are well known in the.art and need not be described in further detail here. In general, polyclonal antibodies are produced by injecting the peptides of the present invention, with or without a suitable adjuvant such as Freund's complete adjuvant, into an antibody-producing mammal such as a rat, a rabbit, a sheep, or a goat. The peptide can be coupled to a carrier protein such as keyhole limpet hemocyanin. 20 Once polyclonal antibodies are produced, cells producing such polyclonal antibodies can be fused with appropriate fusion partners by standard techniques to yield hybridomas producing monoclonal antibodies of defined specificity. V. METHODS OF USE Peptides according to the present invention can be administered by a number . of routes. When used for wound healing,, they are typically administered topically to the skin " or other mucous membranes. However, when they are administered as immunostimulants or as growth factors to stimulate pituitary or hypothalamus function, they can be administered by other routes, such as intramuscularly, intravenously, intradennally, or by other routes. A preferred dose is 0.5 ml of 100 μg/ml solution of the peptide or a dose of 50 ug of the peptide. WO 01/96527 PCT/USO1/18830 The dosages to be administered can be determined by one of ordinary skill in the art depending on the clinical severity of the disease, the age and weight of the patient, the exposure of the patient to conditions that may affect the course of wound healing, the 5 existence or nonexistence of underlying systemic problems such .as diabetes, impaired circulation, and immunocompromised status, and other pharmacokinetic factors generally understood in the art, such as liver and kidney metabolism. The interrelationship of dosages for animals of various sizes and species and humans based on mg/m of surface area is described by E.J. Freireich et al., "Quantitative Comparison of Toxicity of Anticancer Agents 10 in Mouse, Rat, Hamster, Dog, Monkey and Man," Cancer Chemother. Rep. 50: 219-244 (1966). Adjustments in the dosage regimen can be made to optimize the therapeutic response. Doses can be divided and administered on a daily basis or the dose can be reduced proportionally depending on the therapeutic situation. 15 Methods according to the present invention can be used to treat humans or socially or economically important animal species such as dogs, cats, horses, sheep, cows, goats, or pigs. Methods according to the present invention are not limited to use in humans. The peptides of the present invention that promote wound healing are suitable 20 for use in the following situations in which wound healing is required: (1) diabetic foot and leg ulcerations, including neuropathic ulcerations, decubitus lesions, and necrobiosis lipoidica diabeticorum'; (2) vascular ulcerations, including venous stasis ulceration, arterial ulcerations, varicose vein ulcerations, post-thrombotic ulcerations, atrophie blanche ulcerations, congenital absence of veins/ulcerations, congenital or traumatic arteriovenous anastomosis, 25 temporal arteritis, atherosclerosis, hypertension (Martorell's ulcerations), thrombosis, emhalism, platelet agulation, ankle. blow-Qut ayndtome^ or hemangiomas; (3\ decubitus, ulcers or pressure source (e.g., with bed rest); (4) traumatic ulcerations, such as those caused by external injuries, burns, scalds, chemical injuries, post-surgical injuries, self-inflicted injuries, lesions at an injection site, neonatal or perinatal trauma, or sucking blisters; (5) 30 infestations and bites, such as those caused by spiders, scorpions, snakes, or fly larvae (mydriasis); (6) cold injury, such as pemiosis (erythrocyanosis frigida), or cryoglobulinemic ulcerations; (7) neoplastic ulceration, such as those caused by basal cell carcinomas, WO 01/96527 pcmrsoi/18830 squamous cell carcinomas, malignant melanomas, lymphoma,, leukemia,"Kaposi's sarcoma, tumor erosion, midline lethal granuloma, or Wegener's granulomatosis; (8) blood diseases with ulcerations, such as polycythemia, spherocytosis, or sickle cell anemia; (9) skin diseases with ulcerations, such as tinea, psoriasis, pemphigoid, pemphigus, neurotic excoriations, 5 trichotillomania, erosive lichen planus, or chronic bullous dermatosis of childhood; (10) metabolic disease ulcerations, such, as those associated with diabetes mellitus or gout (hyperuricemia); (11) neuropathic ulcerations, such as those associated with diabetes mellitus, tabes dorsalis, or syringomyelia; (12) ischemic ulcerations, such as those associated with scars, fibrosis, or radiation dermatitis; (13) vasculitis ulcerations, such as those 10 associated with lupus erythematosus, rheumatoid arthritis, scleroderma, immune complex disease, pyoderma gangrenosum, or ulceration associated with lipodermatosclerosis; (14) infectious ulcerations, such as: (a) viral ulcerations, e.g. those associated with Herpes simplex or Herpes zoster in an immunocompromised or normal individual; (b) bacterial infections with ulcerations, such as those associated with tuberculosis, leprosy, swimming pool 15 granuloma, ulceration over osteomyelitis, Buruli ulcer, gas gangrene, Meleny's ulcer, bacterial gangrene associated with other bacterial infection (e.g., streptococcal infection), scalded skin syndrome, ecthyma gangrenosum (such as can occur in children infected with Pseudomonas aeruginosa), and toxic epidermal necrolysis; (c) mycotic ulcerations, such as those associated with superficial fungal infection or deep fungal infection; .(d) spirochetal 20 ulcerations, such as those associated with syphilis or yaws: (e) leishmaniasis; (f) mydriasis; or (g) cellulitis; (15) surgical ulcerations, such as those associated with closed incisions or excisions, open incisions or excisions, stab wounds, necrotic incisions or excisions, skin grafts, or donor sites; or (16) other ulcerations, such as those associated with skin tears (traumatic ulcerations), fistula, peristomal ulcerations, ulcerations associated with aplasia 25 cutis congenita, ulcerations associated with epidermolysis bullosa, ulcerations associated with ectodermal dysplasias, ulcerations associated with congenital protein C or S deficiency, ulcerations associated with congenital erosive and vesicular dermatosis, ulcerations associated with acrodermatitis enteropathica, and amputation stump ulcerations. 30. The peptides of the present invention can also be used to promote wound healing in other conditions. WO 01/96527 PCT/tTSOl/18830 VI. PHARMACEUTICAL COMPOSITIONS • Another.aspect of the present invention is pharmaceutical compositions that include peptides according to the present invention. In general, a pharmaceutical 5 composition of the present invention comprises: (1) a peptide according to the present invention in a physiologically effective quantity; and (2) a pharmaceutically acceptable carrier. The physiologically effective quantity can be determined by one of ordinary 10 skin in the art with reference to the dosages described above. " Conventional pharmaceutically acceptable carriers known in the art can include alcohols, e.g., ethyl alcohol, serum proteins, cholesterol, human serum albumin, liposomes, buffers such as phosphates, water, sterile saline or other salts, electrolytes, 15 glycerol, hydroxymethylceUulose, propylene 'glycol, • polyethylene glycol, polyoxyethylenesorbitan, other surface active agents, vegetable oils, and conventional anti¬bacterial or .anti-fungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. A pharmaceutically-acceptable carrier within the scope of the present invention meets industry standards for sterility, isotonicity, stability, and non-20 pyrogenicity. A preferred pharmaceutically acceptable carrier comprises Seamollient, water, a polymer selected from the group consisting of carboxymethylcellulose and hydroxyethylcellulose, and at least one preservative selected from the group consisting of 25 methylparaben and propylparaben. Preferably, the pharmaceutically acceptable carrier comprises both methylparaben and propylparaben. A particularly preferred carrier has 78.5% by weight of water, 20% by weight of Seamollient, 0.5% by weight of methylparaben, 0.5% by weight of propylparaben, and 0.1% by weight of carboxymethylcellulose. Seamollient is available from Philip Rockley, Ltd. (East Setauket, NJ) and is an extract of Hawaiian sea 30 plants that acts as an emollient and moisturizer. It contains chlorphenesin, phenoxyethanol, propylene glycol, and sodium dehydroacetate as preservatives. Another particularly preferred carrier has 78.5% by weight of water, 20% by weight of Seamollient, 0.5% by weight of 5 WO 01/96527 PCT/US01/18830 hydroxyethylcellulose, 0.5% by weight of methylparaben, .and 0.5% fay weight of propylparaben, Other carriers can be used and are known in the art The invention is illustrated by the following Examples. These Examples are for illustrative purposes only and are not intended to limit the invention. 10 EXAMPLES Example 1 Effects of TB4 and TA1. on Endothelial Cell Migration 15 The effects of TB4 and TAi on endothelial cell migration were determined. To investigate the possibility that these peptides effect the migration of normal human vascular endothelial cells (NHVEC) in vitro, a Boyden chamber migration assay was performed. 20 The results of these are shown in Table 1 and in Figure 1. In the experiments of Table 1 and Figure 1, normal human microvesicular endothelial cells are used at 5 x 104 cells per well. The wells are coated with 100 μ.g/ml bovine serum albumin Type TV. These results demonstrate that both TB4 and TA1 act as chemoattractants for 25 endothelial cells, stimulating the migration of NHVECs in Boyden chambers. At 'concentrations of 100 ng/ml, both TB4 and TA1 significantly enhanced cell migration over migration in the presence of media alone. Notably, TB4 and TA1 also revealed heightened responses at this- concentration when compared to three positive controls: vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and fibroblast growth factor 30 (FGF). In addition, endothelial cell migration revealed a dose response to both peptides, as both TB4 and TA1 reached a maximal effect on migration at concentrations of .100 ng/ml. WO 01/96527 PCT/US01/18830 TABLE 1 EFFECT OF PEPTIDES TB4 AND TA1 ON CELL MIGRATION Treatment Concentration Cell Number per HPF Mean SD Chamber 1 Chamber 2 Chamber 3 Blank Medium only 0 0 0 0 0 Negative Control Medium only 12 30 13 18 10 Positive Control VEGF 10 ng/ml 88 95 91 93 5 TB4 10 μg/ml 70 77 66 71 6 TB4 5 μg/ml 55 65 57 59 5 TB4. 1 μg/ml 45 47 63 52 10 TB4 0.5 μg/ml 33 39 50 41 9 TB4 0.1 μg/ml 18 12 26 19 7 TA1 10 μg/ml 51 55 66 57 8 TA1 5 μg/ml 35 64 72 57 19 TA1 1 μg/ml 64 69 43 59 14 TA1 0.5 μg/ml 52 53 57 54 3 TA1 0.1 μg/ml 38 45 33 39 6 WO 01/96527 PCT/TJS01/18830 Example 2 Effects of TB4 and TA1 on Endothelial Cell Proliferation 5 The effects of TB4 and TA1 on endothelial cell proliferation were determined using a MTT (tetrazolium) assay. The results are shown in Figure 2 for 4 hours and 24 hours and for three concentrations of these peptides, 10 ng/ml, 100 ng/ml, and 1000 ng/ml. These results show that these peptides stimulate endothelial cell proliferation. At a concentration of 10 10 ng/ml, TB4 and TA1 maximally stimulated cell proliferation approximately 2-fold and 3-fold over that of the control, respectively. Example 3 15 Effects of TB4 and TA1 on Endothelial Cell Proliferation To examine the effects of TB4 and TA1 (10 ng/ml) on NHVEC migration, a scratch would closure assay was used. When a confluent monolayer was scratched, migration of cells into the wounded area was significantly increased in the presence of TB4 20 and TAj compared to migration with media alone. Thus, these peptides significantly enhance endothelial cell migration, • proliferation, and angiogenesis, and may play an important role in the wound healing process. 25 '_ Example4 Clinical Effects of TB4 ; TB4 used in vivo revealed several clinical effects. One was the effect on 30 reducing the erythema of the healed wound (e.g., on the shin). There was not post¬inflammatory erythema and hyperpigmentation. Frequently, the skin color was norma] and not the expected deep red-purple with brown coloration that would take months to resolve on the leg. The wounds in other areas also did not leave residual erythema or hyperpigmentation WO 01/96527 PCT/US01/18830 that is typically seen in wound healing. To extend this concept, TB4 was tested on poikiloderma vasculare of the neck secondary to chronic sun exposure, with indications that the erythema was reducing, although not enough of the peptide was available to continue the evaluation. Another effect seen is the reduction of melanin pigmentation on the areas of treatment with TB4. There is a definite reduction in the clinical intensity of brown melasma, lentigines, post-inflammatory hyperpigmentation and ephelides after treatment with the TB4 peptide. Example 5 Pharmaceutical Composition According to the Present Invention 15 A pharmaceutical composition according to the present invention comprises 78.49999 g of water, 20 g of Seamollient; 0.5 g of hydxoxyethylcellulose, 0.5 g of methylparaben, 0.5 g of propylparaben, and 10 u.g of the active peptide. 20 Example 6 Clinical Course of Patients Undergoing Treatment with Peptides According to the Present Invention 25. Patient 1 (A.T.) A.T. was seen with a history of an injury on the left lower anterior leg (shin) which had occurred approximately five months previously. The injury started with a bruise on the left shin; but approximately seven weeks later the area started to get erythematous. 30 The patient was sent by the general internist to the local hospital wound healing center, where the wound was debrided and Silvadene cream was used. However, the patient was not progressing well and grafting was considered. The patient did not want to undergo surgery. The underlying problem was venous stasis with the development of ulceration secondary to WO 01/96527 PCT7US01/18830 minor trauma. The ulcer was about 3.0 cm by 1.0 cm insize with a. 1-cm rim of mild erythema. There was a small superior 4 mm deeper area of the ulcer. The wound at this stage is shown in Figure 3. The patient was given a preparation containing TA[ to be used twice daily to the small 4 mm area. After 1 week, improvement was noted as shown in Figure 4. The treatment with TAi was continued for another week twice daily. After another week, the deeper 4 mm area of the ulceration was now.level with the rest of the ulcer as shown in Figure 5. At this point, treatment was changed to a preparation containing TB4 to be applied twice daily to the entire ulcer. After the entire ulcer had been treated for seven days with TB4 twice daily, it was noted that the size of the ulcer was starting to reduce. It was now 2.3 cm x 0.8 cm with more reepithelialization and slightly more erythema surrounding the ulcer as shown in Figure 6. The wound center physician was concerned about infection (cellulitis around the ulcer). The patient was placed on oral antibiotics for 10 days. However, when the surrounding erythema was palpated, there was no tenderness. The wound appeared to be repairing and increasing the vascular flow to the ulcer base and surrounding skin. The patient felt that after treating the entire for seven days she improved by 40%. The patient was given more preparation containing TB4 for the next week. The internist felt that the patient should not undergo surgery for grafting due to cardiac problems in view of the improvement in the wound. After another seven days, the status of the ulcer was as shown in Figure 7. The ulcer continued to reepithelialize from the bottom of the ulcer. The depth of the ulcer was very shallow. Although the size of the ulcer was the same, there were peripheral islands of healing occurring making the entire ulcer smaller. The patient had not taken any antibiotics. At this, point, the administration of TB4 was temporarily suspended and the patient used Silvadene cream and Vigilon. WO 01/96527 PCT/US01/18830 After another seven, days, the status of the: ulcer was as shown, in Figure 8. After the patient had used Silvadene and Vigilon, the ulcer continued, to improve. The administration of TB4 was restarted and Silvadene was stopped. 5 After another eight days, the status of the ulcer was as shown in Figure 9. The ulcer had reduced to 2.0 cm x 0.5 cm with the upper pole almost closed. The patient was still using the Vigilon which may have absorbed the TB4 preparation. The patient was told to stop the Vigilon and continue twice daily with the TB4 preparation. 10 After another six days, the status of the ulcer was as shown in Figure 10. The ulcer was closing in and was 1.9 cm x 0.4 cm. The upper pole had just a 2-3 mm zone to close. After another nine days, the status of the ulcer was as shown in Figure 11. The 15 ulcer was reducing very well. The ulcer size was now 0.8 cm x 0.3 cm with the TB4 preparation continued. After another seven days, the ulcer had almost completely closed with a dry scab formation occurring. The TB4 preparation was continued, once daily, treating the 20 scabbed area. After another two weeks, the amount of scab had reduced with no ulceration, as shown in Figure 12. The TB4 preparation was continued once daily to the scab. 25 In summary, considerable improvement was shown with administration of peptides according to the present invention in this venous stasis ulceration. Once the wound healing preparation stimulated reepitheKalization there seemed to be some continuation of the wound repair even during the week the patient was not using the peptide. The erythema surrounding the ulcer may related to the repair process in the initial stages. The erythema 30 reduced and disappeared once the major portion of wound repair occurred. WO 01/96527 PCT/US01/18830 Patient 2 (P.M.) The patient had a history of diabetes mellirus for greater than 10 years and had developed ulcerations of her fingers secondary to bums since she developed diabetic 5 neuropathy and lost sensation in her fingers. The glucose levels of the patient were not kept under control. The patient developed heat burns with blistering and erosions on the patient's fingers. The ulcers are cleansed and Silvadene cream is used for wound healing, which usually takes 2-4 weeks. Occasionally, systematic antibiotics are given. 10 The patient was seen after having burned the distal phalanx of the left middle finger with a hairdryer 10 days before. The patient had taken some leftover Ceftin 250 mg twice daily and has used Silvadene cream twice daily for the 10 days, but the ulcer would not heal. The status of the ulcer at this time was as shown in Figure 13. The ulcer was defended and the patient was given a preparation containing TA5 to use on the ulcer base twice daily. 15 This ulcer was on the lateral thenar side of the left middle finger. One week later the status of the ulcer was as shown in Figure 14. There was a dramatic improvement of the ulcer base. Reepithelialization was occurring well at the base of the ulcer. The patient had developed another ulceration on the same finger, left middle 20 finger, on the palmar side two days prior to the visit. The patient was requested to use the preparation containing TA5 to both ulcerations. One week later after using the preparation containing TA5 twice daily to the •, new shallow ulceration there was marked improvement, with the ulcer being approximately 25 '• 70% resolved. The older, deeper ulceration was also healing more rapidly than previously. . Approximately six weeks later, the patient had developed new ulcerations of the right index finger on the palmar-ventral side (5 days), and two ulcerations on the right middle finger on the distal digit lateral thenar side (5 days) and the second digit on the dorsal 30 side (2 weeks history). These ulcerations had developed after the patient had handled hot items. These ulcerations are shown in Figure 15(a) and Figure 15(b). Dicloxacillin (500 mg twice daily) was given for 10 days. The patient was given TB4 wound healing preparation to be used twice daily to the ulcerations. WO 01/96527 PCT/US01/18830 After another week, the patient had experienced considerable (70-80% improvement) in the ulcerations after using TB4 wound healing preparation. This is shown in Figure 16. After another five days, the ulcers were approximately 90% cleared as shown in Figure 17, despite the fact that the patient's diabetes was still out of control. Example 7 Effect of TA1_[ on Wound Healing In Vivo To study the effect of TA1 on wound healing in vivo, full thickness punch wounds were made in rat skin and treated with TA1 topically or by intraperitoneal injection with a seven-day study interval. For topical treatment, 5 p.g of TA1 was applied in 50 pi saline on injury date and 48 hours post-injury. For intraperitoneal treatment, 60 pg of TA1 was administered in 300 μl saline on injury date and then every other day. The effect of varying concentrations of TA1 on keratinocyte migration as measured in the Boy den chamber is shown in Figure 18. The effect of the topical TAi on gap closure in the wounds in vivo is shown in Figure 19 for 4 and 7 days post-injury. There was an 18% ± 12% decrease in gap size versus a normal saline (NS) control, which is significant for the seven-day results, (p 0.04). For intraperitoneal administration as shown in Figure 20 for 4 and 7 days post-injury, there was a 42% ± 9% decrease in gap size versus a normal saline (NS) control, which was highly significant (p 0.0007) for both the four-day and seven-day results. The effect of the topical TA1 on wound width in the wounds in vivo is shown in Figure 21 for 4 and 7 days post-injury. There was a 15% decrease in wound width, versus a normal saline (NS) control, which is highly significant for the four and seven-day results, (p 0.0001). For intraperitoneal administration as shown in Figure 22 for 4 and 7 days post- WO 01/96527 PCT/US01/18830 injury, there was a 42% ± 9% decrease in wound width versus a normal saline control, which was highly significant (p 0.0001) for the four-day and marginally, significant (p 0.08) for the seven-day results. In summary, the 4-day post-injury findings indicated accelerated reepithelialization, wound contraction, and keratinocyte migration, as well as increased capillary ingrowth and collagen deposition. Although Applicant does not intend to be bound by this model, a possible model of the actions of TA1 is shown in Figure 23. In this figure, "ACT 1" refers to the peptide TA1 Similar results were seen with TA5. In conclusion, these peptides appear to interact with G-actin, mediate cell proliferation, migration, and differentiation, and stimulate angiogenesis. These peptides are also involved in differentiation of vascular endothelial cells and accelerate the migration of vascular endothelial cells into the scratch wounded area, the peptides also accelerate the production of matrix metalloproteinases that may degrade basement membranes during angiogenesis. They show efficacy with topical application and intraperitoneal injection. The potency of these intraperitoneal effects support high diffusibility in tissue. Example 8 Pilot Study in Humans of TA_^ and TA^ in Comparison to Vitamin C and Tretinoin A pilot study of TA1 and TA5 was performed in humans with 20 volunteers with chronic wounds. Skin biopsies were taken at the baseline and at two months, subjected to histological analysis, irnmunobistological analysis, and objective and subjective obse'rvation. Immunohistochemistry endpoints were epidermal differentiation, epidermal proliferation, and markers of inflammation and were assayed by using anti-CD3 monoclonal antibody. Preliminary results from this study in terms of collagen synthesis are shown in Figure 24. These results indicate that the peptides TA1 and TA5 were more effective in stimulating collagen synthesis than tretinoin or vitamin C. WO 01/96527 PCT/USO1/18830 ADVANTAGES OF THE INVENTION Although the present invention has been described in considerable detail, with reference to certain preferred versions thereof, other versions and embodiments are possible. Therefore, the scope of the invention is determined by the following claims. I claim: 1. A substantially purified peptide having wound healing activity, the peptide comprising a sequence of 44 amino acids, that is : K-K-L-K-K-E-E-N-P-L-E-L-K-E-K-L-K-E-K-K-N-P-L-P-S-K-E-E-E-K-A-S- P-F-D-K.-I-T-E-T-P-D-M-S (SEQ ID NO: 8), the peptide being linear. 2. A pharmaceutical composition comprising: (a) the peptide of claim 1 in a physiologically effective quantity; and (b) a pharmaceutically acceptable carrier. 3. The pharmaceutical composition of claim 2 wherein the pharmaceutically acceptable carrier comprises an extract of Hawaiian sea plants that acts as an emollient and moisturizer and includes chlorphenesin, phenoxyethanol, propylene glycol, and sodium dehydroacetate as preservative, water, a polymer selected from the group consisting of carboxymethylcellulose and hydroxyethylcellulose, and at least one preservative selected from the group consisting of methylparaben and propylparaben. Dated 23rd December, 2002

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IN-PCT-2002-01868-MUM-WO INTERNATIONAL PUBLICATION REPORT(23-12-2002).pdf