WO2009010968A2 - Traitement de maladie par des peptides antimicrobien ou leurs inhibiteurs - Google Patents

Traitement de maladie par des peptides antimicrobien ou leurs inhibiteurs Download PDF

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WO2009010968A2
WO2009010968A2 PCT/IL2008/000977 IL2008000977W WO2009010968A2 WO 2009010968 A2 WO2009010968 A2 WO 2009010968A2 IL 2008000977 W IL2008000977 W IL 2008000977W WO 2009010968 A2 WO2009010968 A2 WO 2009010968A2
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Prior art keywords
disease
cathelicidin
inflammatory
autoimmune
cell
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PCT/IL2008/000977
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English (en)
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WO2009010968A3 (fr
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Yitzchak Hillman
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Yitzchak Hillman
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Priority claimed from IL184611A external-priority patent/IL184611A0/en
Priority claimed from IL187627A external-priority patent/IL187627A0/en
Application filed by Yitzchak Hillman filed Critical Yitzchak Hillman
Priority to EP08776610A priority Critical patent/EP2200628A2/fr
Priority to JP2010516648A priority patent/JP2010533705A/ja
Priority to CA2696833A priority patent/CA2696833A1/fr
Priority to AU2008277257A priority patent/AU2008277257B2/en
Publication of WO2009010968A2 publication Critical patent/WO2009010968A2/fr
Publication of WO2009010968A3 publication Critical patent/WO2009010968A3/fr

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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1729—Cationic antimicrobial peptides, e.g. defensins
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Definitions

  • the present invention relates to methods of treating diseases using anti- antimicrobial peptide (AMP) and/or AMP-like molecule (AML) and in particular cathelicidin type AMPs, and to methods of identifying compounds capable of regulating, decreasing or increasing activities/levels of AMPs/AMLs so as to enable treatment of diseases.
  • AMP anti- antimicrobial peptide
  • AML AMP-like molecule
  • cathelicidin type AMPs in particular cathelicidin type AMPs
  • the present invention relates to methods of treating diseases by using cathelicidin or cathelicidin fragments or cathelicidin analogs or compounds capable of regulating the levels/activity of cathelicidin, such diseases including dysregulated cell proliferation/differentiation leading to bone loss or degradation, osteoporosis osteoarthritis, or to other autoimmune diseases such as multiple sclerosis, arthritis, psoriasis, and to malignancies such as carcinomas, which are associated with inflammation, to metabolic diseases, obesity, insulin resistance, diabetes type 2, diabetes type 1 and related diseases.
  • diseases including dysregulated cell proliferation/differentiation leading to bone loss or degradation, osteoporosis osteoarthritis, or to other autoimmune diseases such as multiple sclerosis, arthritis, psoriasis, and to malignancies such as carcinomas, which are associated with inflammation, to metabolic diseases, obesity, insulin resistance, diabetes type 2, diabetes type 1 and related diseases.
  • the present invention relates to methods of identifying compounds capable of regulating levels of cathelicidin or other AMPs or to increasing or to decreasing activity/levels of AMPs so as to enable treatment of diseases including autoimmune and inflammatory diseases such as, multiple sclerosis, arthritis, metabolic disorders such as diabetes, obesity and malignant diseases such as carcinomas, which are associated with inflammation, dysregulated cell proliferation/differentiation, angiogenesis and/or metastasis.
  • diseases including autoimmune and inflammatory diseases such as, multiple sclerosis, arthritis, metabolic disorders such as diabetes, obesity and malignant diseases such as carcinomas, which are associated with inflammation, dysregulated cell proliferation/differentiation, angiogenesis and/or metastasis.
  • cathelicidins are immune regulators and are over expressed locally in autoimmune diseases. They are also expressed systemically through bone marrow such that normal plasma concentrations average around 1.2 ug/ml to 1.5ug/ml (Journal of Immunological Methods 206_ 1997.53-59). Regulation of their expression is essential for homeostasis. AMPs are involved is skewing dendritic cell activation between ThI and Th2 inflammatory processes via Toll-like receptors and therefore are involved in homeostasis (J Immunol. 2004 Jan 15; 172(2): 1 146-56). Controlling or maintaining such homeostasis is performed by either increasing or decreasing of level/activity between the various AMPs.
  • Cathelicidins are mainly expressed by Vitamin D3 (calcitriol), via vitamin D3 receptor elements (VDRE) and Vitamin D3 itself has a modulating influence on cathelicidin expression both as an agonist via calcitriol/VDRE and by a negative feedback mechanisms (Marshall T BioEssays 30: 173-182, 2008).
  • This VDRE/cathelicidin pathway is unique to humans and furry/haired animals such as rodents for example whose skin is less exposed to sunlight do not possess this pathway.
  • cathelicidin forms a major immune regulator for diseases which are known to be also regulated by vitamin D3.
  • autoimmune diseases represent diseases of major clinical and economic impact. These include major diseases such as psoriasis, rheumatoid arthritis, type I diabetes, inflammatory bowel diseases, and multiple sclerosis for which no satisfactory treatment methods are available. Similarly, malignant diseases, such as skin carcinoma, breast carcinoma, colon carcinoma, head and neck carcinoma, hepatic carcinoma, lung carcinoma, renal cell carcinoma, urinary bladder carcinoma, and the like, represent numerous lethal diseases for which no satisfactory treatment methods are available.
  • AMPs antimicrobial peptides
  • AMPs are cationic peptides which display antimicrobial activity at physiological concentrations under conditions prevailing in the tissues of origin. AMP synthesis and release is regulated by microbial signals, developmental and differentiation signals, cytokines and in some cases neuroendocrine signals in a tissue-specific manner.
  • AMPs are classified into two major groups in humans; cathelicidins and defensins. AMPs appear to have common characteristics that enable them to affect mammalian cells in a way that does not necessarily function through a ligand-receptor pathway, and that, being small, and highly ionic or hydrophobic or structurally amphiphilic, AMPs can bind mammalian cell membranes. They are able to penetrate through the cell membrane to the cytoplasm. For example, it was shown that granulysin penetrates and damages human cell membranes dependent upon negative charge (J.
  • Cathelicidins contain a conserved "cathelin" precursor domain. Their organization includes an N-terminal signal peptide, a highly conserved prosequence, and a structurally variable cationic peptide at the C-terminus. The prosequence resembles cathelin, a protein originally isolated from porcine neutrophils as an inhibitor of cathepsin L (hence, the name cathelin).
  • the 37 amino acid-long human cathelicidin, LL-37/hCAP18 has a hydrophobic N-terminal domain in an ⁇ -helical conformation, particularly in the presence of negatively charged lipids.
  • LL-37 is enzymatically cleaved from the C-terminus of hCAP18 precursor via enzymes such as neutrophil elastase and proteinase 3.
  • LL-37 functions in synergy with other AMPs, and can directly activate host cells. Inappropriate cleavage of the cathelicidin hCAPl 8 pro-peptide by endogenous proteases can produce pro-inflammatory fragments of the cathelicidin (Nat Med. 2007 Aug;13(8):975-80).
  • cathelicidins such as LL-37 to both kill bacteria and regulate immune responses is a characteristic of numerous AMPs.
  • the peptide can influence host immune responses via a variety of cellular interactions, for example, it has been suggest to possibly function as a chemoattractant by binding to formyl-peptide-receptor-like-1 (FPRL- 1).
  • FPRL- 1 formyl-peptide-receptor-like-1
  • LL-37 can recruit mast cells, and then be produced by the mast cell to kill bacteria.
  • AMPs exert their effects either individually or as the resultant effect of multiple AMPs. For example, in the menstrual cycle there is a monthly cycle-dependent expression of various AMPs (King A. E. et al., 2003. J. Reprod. Immunol.
  • beta-defensin-2 in the menstrual stage
  • beta- defensin-4 in the proliferative stage
  • beta-defensin-3 in the early secretory stage
  • beta- defensin-1 in the mid secretory stage
  • beta-defensin-3 in the late secretory stage.
  • cathelicidin is associated with inflammation, dysregulated cell proliferation/differentiation, dysregulated cell proliferation/differentiation balance, angiogenesis metastasis, and/or epithelial wounds, the inventor hypothesized that an optimal strategy for treating such diseases would be via methods involving decreasing the levels/activity of such AMPs/AMLs, and/or via methods involving administering such AMPs/AMLs or enhancing their expression.
  • the prior art approaches relating to such methods involve the previous application of the inventors in WO 2004-056307 which show that cathelicidin is an immune regulator in- vivo and therefore poses a target in treating autoimmune diseases.
  • the current application provides in-vivo data for specific diseases such as metabolic diseases and low grade inflammatory diseases, obesity, insulin resistance, diabetes type 2, type 1 diabetes, insulin related diabetes, osteoporosis, periodontitis, osteoarthritis, arthritic diseases, rheumatologic diseases such as rheumatoid arthritis, ankylosing spondylitis, gout and systemic lupus erythematosus, as well as multiple sclerosis, neurological and central nervous system diseases as well as osteoporosis.
  • diseases such as metabolic diseases and low grade inflammatory diseases, obesity, insulin resistance, diabetes type 2, type 1 diabetes, insulin related diabetes, osteoporosis, periodontitis, osteoarthritis, arthritic diseases, rheumatologic diseases such as rheumatoi
  • the current invention shows in-vivo the use of cathelicidin or cathelicidin analogs in the treatment of said diseases.
  • a method of treating a medical condition, such as a disease, in a subject in need of treatment thereof comprising providing to the subject a therapeutically effective amount of a compound in particular a cathelicidin peptide or fragment analog thereof, being capable of treating the disease in the subject in need thereof or of regulating, or increasing or decreasing an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule, thereby treating the disease in the subject in need thereof.
  • AMP antimicrobial peptide
  • administering the compound to the subject is effected by exposing a location of the subject to a carrier which includes the compound at a concentration selected from a range of about 50 nanograms per milliliter to about 2 milligram per milliliter.
  • administering the compound to the subject is effected by administering to the subject a plurality of doses of the compound selected from a range of 2 doses to 30 doses, wherein each inter dose interval of the plurality of doses is selected from a range of about 2.4 hours to about 30 days.
  • administering the compound to the subject is effected via a route selected from the group consisting of the topical, intravenous, intranasal, transdermal, intradermal, oral, buccal, parenteral, rectal and inhalation route.
  • the disease is associated with a biological process in a cell and/or tissue, wherein the biological process is selected from the group consisting of growth, differentiation, autoimmunity or inflammation.
  • the subject is human.
  • an article of manufacture comprising packaging material and a pharmaceutical composition, the article of manufacture being identified for treatment of a disease being associated with a biological process in a cell and/or tissue, the biological process being selected from the group consisting of growth, differentiation or diseases associated with inflammation or autoimmunity; the pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, a compound being capable of regulating an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule.
  • AMP antimicrobial peptide
  • the pharmaceutically acceptable carrier is selected so as to enable administration of the pharmaceutical composition via a route selected from the group consisting of the topical, intranasal, transdermal, intradermal, intravenous, oral, buccal, parenteral, rectal and inhalation route.
  • the pharmaceutical composition is formulated as a solution, suspension, emulsion or gel.
  • the pharmaceutical composition is composed so as to enable exposure of a cell and/or tissue of a subject having the disease to the compound at a concentration selected from a range of about
  • the pharmaceutical composition is further identified for administration to a subject of a plurality of doses of the pharmaceutical composition selected from a range of 2 doses to 30 doses, wherein each inter dose interval of the plurality of doses is selected from a range of about 2.4 hours to about 30 days
  • the cell and/or tissue is selected from the group consisting of skin cells, bone cells beta cells and synovial tissue.
  • the disease is selected from the group consisting of an autoimmune disease, a bone resorption disease, a neurological disease, a metabolic disease including diabetes, obesity, and a diabetes related disease.
  • a method of regulating a biological process in a cell and/or tissue comprising exposing the cell and/or tissue to a compound in particular a cathelicidin peptide or its analog, being capable of regulating the biological process in the cell and/or tissue or of increasing or decreasing an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule, thereby regulating the biological process in the cell and/or tissue.
  • a compound such as for example, a cathelicidin peptide or its analog
  • the providing to the subject the compound is effected by administering the compound to the subject and/or by expressing the compound in the subject.
  • the exposing the cell and/or tissue to the compound is effected by exposing the cell and/or tissue to the compound at a concentration selected from a range of about 50 nanograms per milliliter to about one milligram per milliliter.
  • the cell and/or tissue is bone or nerve tissue or synovial tissue, wherein the exposing the cell and/or tissue to the compound (such as for example, a cathelicidin peptide or its analog) is effected by exposing the cell and/or tissue to the compound at a concentration selected from a range of about 0.4 microgram per milliliter to about 100 micrograms per milliliter.
  • the compound such as for example, a cathelicidin peptide or its analog
  • a method of identifying a compound being capable of regulating a biological process in a cell and/or tissue comprising: (a) exposing the cell and/or tissue to a test compound which is: (i) capable of decreasing an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule, and/or (ii) the AMP and/or AMP-like molecule; and (b) evaluating a capacity of the test compound to regulate the biological process in the cell and/or tissue, thereby identifying the compound being capable of regulating the biological process in the cell and/or tissue.
  • the cell and/or tissue is a cultured cell and/or tissue.
  • the cell and/or tissue is derived from a human.
  • the exposing the cell and/or tissue to the test compound is effected by providing the test compound to a subject.
  • the exposing the cell and/or tissue to the test compound is effected by exposing the cell and/or tissue to a cell which produces the test compound.
  • the cell which produces the test compound is a B-cell hybridoma.
  • the providing the test compound to the subject is effected by administering the test compound to the subject and/or by expressing the test compound in the subject.
  • administering the test compound to the subject is effected via a route selected from the group consisting of the topical, intranasal, intravenous, transdermal, intradermal, oral, buccal, parenteral, rectal and inhalation route.
  • the test compound is selected from the group consisting of: (a) a molecule capable of binding the AMP and/or AMP-like molecule; (b) an enzyme capable of cleaving the AMP and/or AMP- like molecule; (c) an siRNA molecule capable of inducing degradation of an mRNA encoding the AMP and/or AMP-like molecule; (d) a DNAzyme capable of cleaving an mRNA or DNA encoding the AMP and/or AMP-like molecule; (e) an antisense polynucleotide capable of hybridizing with an mRNA encoding the AMP and/or AMP-like molecule; (f) a ribozyme capable of cleaving an mRNA encoding the AMP and/or AMP-like molecule; (g) a non-functional analog of at least a functional portion of the AMP and/or AMP-like molecule; (h)
  • the molecule capable of binding the AMP and/or AMP-like molecule is an antibody or an antibody fragment.
  • the antibody fragment is selected from the group consisting of a single-chain Fv, an Fab, an Fab', and an F(ab')2.
  • the AMP and/or AMP-like molecule is selected from the group consisting of a defensin, a cathelicidin, a cationic peptide, a hydrophobic peptide, a human AMP and a human AMP-like molecule.
  • AMP is any one of the cathelicidin and/or cathelicidin fragments listed below as SEQ. ID NOS. 1-59.
  • the cell and/or tissue is selected from the group consisting of an synovial cell and/or tissue, a nerve cell and/or tissue, a beta cell and/or tissue, an osteoblast, osteocyte or osteoclast cell and/or tissue and an endothelial cell and/or tissue.
  • the biological process is selected from the group consisting of growth, differentiation, and associated with an inflammatory disease or autoimmunity.
  • a method of treating a disease in a subject comprising providing to the subject a therapeutically effective amount of an antimicrobial peptide (AMP) and/or AMP-like molecule (and in particular a cathelicidin, active fragment thereof or active cathelicidin analog of the cathelicidin or the fragment thereof), thereby treating the disease in the subject in need thereof.
  • AMP antimicrobial peptide
  • AMP-like molecule and in particular a cathelicidin, active fragment thereof or active cathelicidin analog of the cathelicidin or the fragment thereof
  • administering the AMP and/or AMP-like molecule to the subject is effected by exposing a location of the subject to a carrier which includes the AMP and/or AMP-like molecule at a concentration selected from a range of about 2 nanograms per milliliter to about
  • administering the AMP and/or AMP-like molecule to the subject is effected via a route selected from the group consisting of the topical, intranasal, transdermal, intradermal, oral, buccal, intravenous, parenteral, rectal and inhalation route.
  • an article of manufacture comprising packaging material and a pharmaceutical composition, the article of manufacture being identified for treatment of a disease being associated with a biological process in a cell and/or tissue, the biological process being selected from the group consisting of growth, differentiation, or inflammation associated with a disease; the pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, an antimicrobial peptide (AMP) and/or AMP-like molecule.
  • AMP antimicrobial peptide
  • the pharmaceutically acceptable carrier is selected so as to enable administration of the pharmaceutical composition via a route selected from the group consisting of the topical, intranasal, transdermal, intravenous, intradermal, oral, buccal, parenteral, rectal and inhalation route.
  • the pharmaceutically acceptable carrier may, for example, be of the sort of carriers known in the art for the delivery of therapeutic peptides.
  • the pharmaceutically acceptable carrier may, for example, be other than water alone or other than water altogether.
  • the pharmaceutical composition is formulated as a solution, suspension, emulsion or gel.
  • the pharmaceutical composition is composed so as to enable exposure of a cell and/or tissue of a subject having the disease to the compound at a concentration selected from a range of about 2 nanograms per milliliter to about 10 micrograms per milliliter.
  • a method of treating an autoimmune disease, chronic inflammatory disease, an inflammatory disease, a cancer comprising of delivering the AMP or analog thereof, in particular a cathelicidin AMP to a human subject or mammal, thereby regulating the biological process in the subject.
  • a method of regulating a biological process in a cell and/or tissue comprising exposing the cell and/or tissue to an antimicrobial peptide (AMP) and/or AMP-like molecule, thereby regulating the biological process in the cell and/or tissue.
  • exposing the cell and/or tissue to the AMP and/or AMP-like molecule is effected by providing the AMP and/or AMP-like molecule to a subject.
  • the providing to the subject the AMP and/or AMP-like molecule is effected by administering the AMP and/or AMP-like molecule to the subject and/or by expressing the AMP and/or AMP- like molecule in the subject.
  • the exposing the cell and/or tissue to the AMP and/or AMP-like molecule is effected by exposing the cell and/or tissue to the AMP and/or AMP-like molecule at a concentration selected from a range of about 2 nanograms per milliliter to about 10 micrograms per milliliter or from about 10 micrograms per milliliter to about 30 micrograms per milliliter.
  • the AMP and/or AMP-like molecule is selected from the group consisting of and LL-37 or analogs of LL-37 or other cathelicidins and cathelicidin fragments or analogs as listed below.
  • the cell and/or tissue is derived from a human.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing: (i) a method of treating a disease which is associated with a biological process in a cell/tissue such as growth, differentiation, inflammation, metastasis and/or angiogenesis by using a compound which is capable of regulating levels/activity of an AMP and/or an AMP-like molecule, of decreasing olevels/activity of an AMP and/or an AMP-like molecule; and/or by using an AMP and/or an AMP-like molecule or by increasing levels/activity of an AMP and/or an AMP-like molecule; (ii) an article of manufacture including such a compound and being labeled for treatment of such a disease; and (iii) a method of identifying such a compound.
  • FIG. 1 is a graph depicting incidence of arthritis in mouse model of collagen induced arthritis.
  • FIG. 2 is a graph depicting Severity of Arthritis - The severity of arthritis was analyzed on the basis of degree of inflammation scored as follows and the number of affected limbs.
  • FIG. 3 is a graph depicting the number of Arthritic Limbs/Mouse. Similar to the Severity/Mouse score as in FIG. 2, the number of Arthritic Limbs/Mouse was also generally lower in the experimental group, although the appearance of arthritic limbs followed similar kinetics as the control group, but at a delayed incidence.
  • FIG. 3 is from the same experiment described in FIG. 1 and FIG. 2.
  • FIG. 4 is a graph showing a follow-up of clinical score from the day of incidence of arthritis until day 19 after incidence. This follow-up is required since each arthritic mouse develops an incidence of inflammation on any one of four paws at a varying number of days since the beginning of the experiment. Therefore in order to determine the significance level between the groups it is necessary to run a follow-up test statistic.
  • Data in FIG. 4 is from the same experiment described in FIG. 1 and FIG. 2.
  • FIG. 5 is a graph showing the sum of the severity index of clinical score in all mice of control versus treatment group during the time in days since immunization.
  • a clear trend is shown of greater severity of disease in the control group from day 27 onwards.
  • Data in FIG. 5 is from the same experiment described in FIG. 1 and FIG. 2.
  • FIG. 6 is a graph showing the incidence of arthritic paws in treatment versus control groups. Any one mouse may be included in this data up to four times corresponding to four different paws in any one mouse. A trend line is computed for each of the treatment and control groups using Microsoft excel technology.
  • FIG. 6 is from the same experiment described in FIG. 1 and FIG. 2.
  • FIG. 7 shows a table listing the results of the mouse Experimental Autoimmune Encephalitis (EAE) model.
  • EAE Experimental Autoimmune Encephalitis
  • C57BL/6 mice were purchased from Harlan (Jerusalem, Israel). Female, 9 week old mice were used in the experiment. The mice were housed in the specific-pathogen free (SPF) animal facility of the Hebrew University and all experiments were approved by the institutional animal care and use committee (IACUC).
  • SPF specific-pathogen free animal facility of the Hebrew University and all experiments were approved by the institutional animal care and use committee (IACUC).
  • MOGB35-55B peptide (MEVG WYRSPFSRVVHLYRNGK) 1.25mg/ml in PBS was emulsified in complete Freund's adjuvant (CFA) supplemented with 400 ⁇ g M. tuberculosis (Mt) H37RA (Difco). Mice were immunized s.c. in the flank with 250 ⁇ g MOGB35-55B/CFA using a 25G needle. 200ng Pertussis Toxin (Sigma) was injected i.v. at the time of immunization and 48h later.
  • EAE was scored on a scale of 0-6: 0, no impairment; 1, limp tail; 2, limp tail and hind limb paresis; 3, >1 hind limb paralysis; 4, full hind limb and hind body paralysis; 5, hind body paralysis and front limb paresis; 6, death.
  • Mice were treated with the cathelicidin peptide supplied by Biosight Ltd. Karmiel, Israel diluted in PBS, vs. PBS as a control.
  • Cathelicidin GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ
  • mice were treated by intraperitoneal (i.p.) injection of roughly 200ul volume (adjusted for weight) 3 times a week (Sun-Tues-Thurs) starting the day of immunization with MOG/CFA and through day 48. Clinical EAE scores were evaluated through day 60. Dosage of Cathelicidin injections (IP) was 2mg/Kg and 0.2 mg/Kg. There were six mice in each group (total of 18 mice).
  • IP Cathelicidin injections
  • FIG. 8 shows a graph of the average clinical score for each day after immunization for the three groups in the EAE experiment as described in FIG. 7.
  • FIG. 9 shows photographs taken on day 60 of the three remaining healthy mice in the control group, all six remaining live mice in the low dose group, and two examples of EAE affected mice having paralyzed hind legs and tail.
  • FIG. 10 shows a Western blot analysis of 4 different scFv developed that bind LL-37.
  • FIG. 1 1 shows the inhibitory effect of scFv on LL-37 in bacteria killing assays. In order to find out the concentration of LL37 at which 50% of the bacteria could be killed
  • IC50 Basically the activity protocol follows the ability of the antibody to block the antimicrobial activity of LL-37.
  • the bacteria used were Pseudomonas that was isolated from a wound.
  • the growth medium was LB.
  • OD optical density
  • FIG. 12 shows the percentage change in glucose levels two hours following an LPS injection in treatment vs. control.
  • FIG. 13 shows the average weight gain in male DBA/1 mice about ten weeks of age being fed on a normal no-high fat diet for 21 days. Two groups of mice, 10 in each group were weighed. The control increased in weight at an average of 0.0536 gms per day whereas the treatment (cathelicidin mCRAMP at 0.8 ug/ml) increased at 0.0488 per day.
  • FIG. 14 shows a similar experiment as in Fig. 13 only that the mice were given 0.4 ug/ml and were weighed 3 times a week while being fed a high fat diet of 60%Kcal.
  • FIG. 15 shows a list of mouse paws selected for analysis for bone resortion, deformation, immunohistology and osteoclast analysis and counting. Mouse paws were obtained from experiment in example 1.
  • FIG. 16 to FIG. 19 shows the histology analysis showing a beneficial effect of cathelicidin on bone with reduced bone resorption in treatment group even the inflamed treatment group had less osteoclast than the non-inflamed control. Staining was done with H&E and for tartrate-resistant acid phosphatase (TRAP).
  • TRIP tartrate-resistant acid phosphatase
  • FIG. 20 shows the effect of human beta defensin 2 given for a duration of 7 weeks on human psoriatic skin. Inhibition by dominant negative peptide analogues or fragments is suggested as a viable mode of treatment for this disease
  • FIG. 21 shows a histogram depicting significant BETATC beta cell line proliferation brought about by cathelicidin LL-37.
  • Murine beta cell line were treated for 3.5 days with LL-37 at 2 microgram/ml (red/dark bars) and compared to PBS control (red/dark bars). Cell proliferation was estimated by measuring [3(H)]-thymidine incorporation and was expressed as percent of control untreated cells. A representative experiment is shown.
  • the present invention provides methods of using compounds capable of increasing activities/levels of antimicrobial peptides (AMP)/antimicrobial peptide-like molecules (AMLs) and/or of decreasing activities/levels of antimicrobial peptides, (AMP)/antimicrobial peptide-like molecules (AMLs) and/or of using AMPs/AMLs or analogs or fragments thereof for regulating in cells/tissues biological processes such as growth, differentiation, growth/differentiation balance, of methods of using such molecules for treating diseases associated with such biological processes and/or which are amenable to treatment via regulation of such biological processes; for treating autoimmunity, inflammation, metastasis and angiogenesis; of articles of manufacture which include such molecules and which are labeled as being for use in treating such diseases; and of methods of identifying such compounds capable of regulating or increasing or decreasing activities/levels of AMPs/AMLs and/or of identifying such AMPs/AMLs.
  • AMP antimicrobial peptides
  • AMLs antim
  • the present invention can be used to optimally treat a vast range of diseases associated with such biological processes, including inflammatory diseases/diseases associated with cellular proliferation/differentiation imbalance, autoimmune and inflammatory diseases such as multiple sclerosis, arthritis, obesity, insulin resistance, osteoporosis, periodontitis, and other diseases associated with autoimmunity and/or cellular proliferation/differentiation imbalance.
  • diseases/diseases associated with cellular proliferation/differentiation imbalance autoimmune and inflammatory diseases such as multiple sclerosis, arthritis, obesity, insulin resistance, osteoporosis, periodontitis, and other diseases associated with autoimmunity and/or cellular proliferation/differentiation imbalance.
  • autoimmune and inflammatory diseases such as multiple sclerosis, arthritis, obesity, insulin resistance, osteoporosis, periodontitis, and other diseases associated with autoimmunity and/or cellular proliferation/differentiation imbalance.
  • the AMP involved is endogenous cathelicidin or its analogs or fragments thereof.
  • the prior art approach relating to such methods involves the invention of the current inventors in WO 2004-056307 incorporated by reference herein.
  • the prior art approach relates to autoimmune diseases in general and provides in-vivo data on psoriasis showing that cathelicidin is indeed an immune regulator in psoriasis.
  • the present invention therefore, a continuation in part to the previous invention of the current inventors, shows in-vivo data for various specific diseases associated with autoimmunity, inflammation including low grade inflammation found in metabolic diseases as well as bone cell differentiation/proliferation leading to bone degradation.
  • cathelicidin and therefore AMPs could be used to significantly regulate growth of cultured mouse beta-cells.
  • the method according to the present invention enables use of compounds capable of regulating by either increasing or decreasing levels/activity of cathelicidin or of other AMPs/AMLs, and/or the use of such cathelicidins or analogs or fragments thereof or other AMPs/AMLs for regulating biological processes such as growth, differentiation, inflammation, and for the treatment of numerous specific diseases such as for example type 1 diabetes and other diseases such as those which are associated with inflammation, dysregulated cell proliferation/differentiation, chronic inflammatory diseases and autoimmune diseases.
  • the present invention provides a method of regulating a biological process in a cell and/or tissue associated with a disease.
  • the method is effected by exposing the cell and/or tissue to: an AMP and in particular a cathelicidin peptide, an analog of a cathelicidin peptide, an analog of a cathelicidin peptide that has been designed to be more stable in-vivo so as not to break down into pro-inflammatory fragments of itself, a cathelicidin analog functioning as a dominant negative or a cathelicidin peptide that competes on binding to cognate receptors with an AMP without inducing disease, a compound being capable of decreasing or increasing an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule (AML).
  • AMP antimicrobial peptide
  • AML AMP-like molecule
  • the method can be used to regulate in a cell/tissue a biological process such as growth, differentiation, autoimmunity and inflammation.
  • a biological process such as growth, differentiation, autoimmunity and inflammation.
  • the method can be used for treating a disease which is associated with such a biological process, and can be used for identifying the regulator, as described in further detail herein below.
  • Diseases associated with such biological processes include, for example, autoimmune diseases, diseases associated with dysregulated cell/tissue growth/proliferation balance, wound-associated diseases, and tumors.
  • the term “regulator” refers to the compound which is capable of decreasing an activity and/or level of an AMP/AML, increasing an activity and/or level of an AMP/AML, and/or to an AMP which is used for practicing any aspect of the present invention.
  • the phrases "the compound”, “compound of the present invention”, and “AMP/AML inhibitor” interchangeably refer to the compound which is capable of regulating, decreasing or increasing an activity/level of an AMP/AML.
  • Any of various types of AMP/AML or AMP/AML inhibitors may be employed according to the teachings of the present invention for regulating the biological process, depending on the application and purpose.
  • the term "AMP” includes any cathelicidin, and/or including any naturally occurring variant of such a molecule, such as a natural mutant/polymorphic variant/allele of such a molecule, or any synthetic variant of such a molecule.
  • AML includes any molecule having a biological activity which is substantially similar to that of a cathelicidin, includes any molecule which substantially promotes the biological activity of a cathelicidin, and/or includes any molecule which is substantially structurally homologous to a cathelicidin. In such case, homology implied may, for example, vary between 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 99%, 90%- 100%, or at least 95%.
  • the method may be effected using a single regulator of the present invention, or using any combination of multiple regulators of the present invention.
  • the AMP/AML inhibitor may be: a molecule capable of binding the AMP/AML; an enzyme capable of cleaving the AMP/AML; an siRNA molecule capable of inducing degradation of an mRNA encoding the AMP/AML; a DNAzyme capable of cleaving an mRNA or DNA encoding the AMP/AML; an antisense polynucleotide capable of hybridizing with an mRNA encoding the AMP/AML; a ribozyme capable of cleaving an mRNA encoding the AMP/AML; a non-functional analog of at least a functional portion of the AMP/AML; a molecule capable of inhibiting activation or ligand binding of the AMP/AML; and a triplex-forming oligonucleotide capable of hybridizing with a DNA
  • the AMP/AML inhibitor may be any small molecule, AMP/AML dominant negative, or polypeptide that competes with the AMPs for cognate cell receptors without inducing disease.
  • the AMP/AML inhibitor may be a topological analog of an AMP/AML that has been engineered to remain anti microbial yet lose its chemoattracting ability.
  • Engineering of disulfide bridges to dissect antimicrobial and chemotactic activities of AMPs/AMLs such as human beta-defensin-3 can be performed as previously described (Wu Z. et al., 2003. Proc. Natl. Acad. Sci. U. S. A. 100:8880-5).
  • dominant negative mutant refers to a polypeptide or a nucleic acid coding region sequence which has been changed with regard to at least one position in the sequence, relative to the corresponding wild type native version at a position which changes an amino acid residue position at an active site required for biological and/or pharmacological activity of the native peptide.
  • dominant negative mutants or fragments of the cathelicidin peptide as listed below and contemplated herein include, but are not limited to, polypeptide species which manifest any change (substitution and/or deletion) with regard to at least one amino acid of the AMP or cathelicidin peptide.
  • Dominant negative mutant embodiments of the invention are moreover nucleic acids which encode peptides, as well as the peptides themselves, which comprise fragments of the AMP or more specifically of the cathelicidin hCAP-18 and are listed as in (SEQ. ID NOS: 1-59).
  • the AMP/ AML inhibitor may be a synthetic antibody mimic in which multiple peptide loops are attached to a molecular scaffold (described in U.S. Patent No. 5,770,380).
  • Such an AMP/AML mimic can be obtained, for example, by molecule imprinting. This technique may be performed by preparing a polymer by cross-linking a monomer around a "template molecule" (the AMP/AML).
  • the molecule capable of inhibiting activation or ligand binding of the AMP/AML may advantageously inhibit binding of a receptor expressed on cell, such as a leukocyte, which binds the AMP/AML to inhibit a biological process mediated by binding of the AMP/AML to the receptor. Examples of such AMPs/AMLs and cognate receptors thereof are shown in Table 1.
  • cathelicidin antimicrobial peptides block dendritic cell
  • TLR4 activation J Immunol. 2007 Feb 1 ;178(3): 1829-344 and therefore cathelicidins would act as inhibitors to beta-defensin activation of TLR4.
  • receptors of AMPs/AMLs such as chemokines
  • the cells in which such receptors are expressed and the diseases in which the interaction between such
  • the AMP/AML inhibitor may be gelsolin.
  • Serpins and their analogs or fragments are inactivators of AMP by formation of complexes with AMP (Panyutich, AV. et al., 1995. Am.J.Respir.Cell Mol.Biol. 12:351-357; alpha- 1 antichymotrypsin, the antimicrobial proteins alpha PI, SLPI and elafin are serpins that form complexes with other AMPs) thereby reducing specific types of inflammation (Hiemstra, PS, 2002.
  • the AMP/AML inhibitor may be serpins and their analogs or fragments.
  • the AMP/AML inhibitor may be SIC, a secreted protein of streptococcus pyogenes that inactivates antibacterial peptides.
  • Other AMP inhibitors or specifically cathelicidin inhibitors include, Alpha 2- Macroglobulin-Proteinase Complexes (Patrik Nyberg et al THE JOURNAL OF
  • the molecule capable of binding the AMP/AML is an antibody or an antibody fragment.
  • the molecule capable of binding the AMP/AML may be any of various type of molecule, including non-immunoglobulin peptides and polypeptides, [000123]
  • the antibody fragment is selected from the group consisting of a single-chain Fv, an Fab, an Fab', and an F(ab')2.
  • antibody refers to a substantially intact antibody molecule.
  • the antibody may, for example, be an IgG, IgA or IgM.
  • Antibodies used according to the invention may be monoclonal antibodies or polyclonal antibodies.
  • the antibodies may, for example, be non-human, human or humanized antibodies.
  • antibody fragment refers to a functional fragment of an antibody that is capable of binding to an AMP/AML.
  • Suitable antibody fragments for practicing the present invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a CDR of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv, an Fab, an Fab', and an F(ab') 2 .
  • CDR complementarity-determining region
  • Fv defined as a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains;
  • scFv single chain Fv
  • Fab a fragment of an antibody molecule containing a monovalent antigen- binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and C H 1 domains thereof;
  • Fab' a fragment of an antibody molecule containing a monovalent antigen- binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab' fragments are obtained per antibody molecule);
  • F(ab') 2 a fragment of an antibody molecule containing a monovalent antigen- binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds).
  • pepsin i.e., a dimer of Fab' fragments held together by two disulfide bonds.
  • haptens can be coupled to antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or serum albumin [e.g., bovine serum albumin (BSA)] carriers (see, for example, US. Pat. Nos. 5, 189,178 and 5,239,078].
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the carrier can be coupled using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
  • Condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents.
  • Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, 111.
  • the resulting immunogenic complex can then be injected into suitable mammalian subjects such as mice, rabbits, and the like. Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum.
  • the titers of the immune serum can readily be measured using immunoassay procedures which are well known in the art.
  • the antisera obtained can be used directly or monoclonal antibodies may be obtained as described hereinabove.
  • Antibody fragments can be obtained using methods well known in the art. [(see, for example, Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, New York, (1988)].
  • antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g., Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • an (Fab') 2 antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages to produce 3.5 S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages to produce 3.5 S Fab' monovalent fragments.
  • pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Ample guidance for practicing such methods is provided in the literature of the art (for example, refer to: Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647; Porter, RR., 1959. Biochem. J. 73: 1 19- 126).
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • an Fv is composed of paired heavy chain variable and light chain variable domains. This association may be noncovalent (see, for example, Inbar et al., 1972. Proc. Natl. Acad. Sci. USA. 69:2659-62).
  • the variable domains can be linked to generate a single chain Fv by an intermolecular disulfide bond, or alternately, such chains may be cross-linked by chemicals such as glutaraldehyde.
  • the Fv is a single chain Fv.
  • Single chain Fv's are prepared by constructing a structural gene comprising DNA sequences encoding the heavy chain variable and light chain variable domains connected by an oligonucleotide encoding a peptide linker.
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two variable domains.
  • Ample guidance for producing single chain Fv's is provided in the literature of the art (for example, refer to: Whitlow and Filpula, 1991. Methods 2:97-105; Bird et al., 1988.
  • Isolated complementarity determining region peptides can be obtained by constructing genes encoding the complementarity determining region of an antibody of interest. Such genes may be prepared, for example, by RT-PCR of mRNA of an antibody- producing cell. Ample guidance for practicing such methods is provided in the literature of the art (for example, refer to Larrick and Fry, 1991. Methods 2:106-10). [000137] It will be appreciated that for human therapy or diagnostics, humanized antibodies are preferably used.
  • Humanized forms of non human (e.g., murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having-preferably minimal-portions derived from non human antibodies.
  • Humanized antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementarity determining region of a non human species (donor antibody) such as mouse, rat or rabbit having the desired functionality.
  • donor antibody such as mouse, rat or rabbit having the desired functionality.
  • Fv framework residues of the human antibody are replaced by corresponding non human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence.
  • Humanized antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321 :522-525; Riechmann et al., 1988. Nature 332:323-329; and Presta, 1992. Curr. Op. Struct. Biol. 2:593-596).
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non human. These non human amino acid residues are often referred to as imported residues which are typically taken from an imported variable domain. Humanization can be essentially performed as described (see, for example: Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988. Science 239:1534-1536; U.S. Pat. No. 4,816,567) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions.
  • humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non human species.
  • humanized antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.
  • antibodies used in the treatments of the invention may be humanized antibodies against LL-37 or against an epitope of hCAP-18.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [see, for example, Hoogenboom and Winter, 1991. J. MoI. Biol. 227:381 ; Marks et al., 1991. J. MoI.
  • Humanized antibodies can also be made by introducing sequences encoding human immunoglobulin loci into transgenic animals, e.g., into mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed in such animals which closely resembles that seen in humans in all respects, including gene rearrangement, chain assembly, and antibody repertoire. Ample guidance for practicing such an approach is provided in the literature of the art (for example, refer to: U.S.
  • Suitable antibodies may in many cases be purchased ready for use from commercial suppliers, such as Pharmingen, Dako, Becton-Dickinson, Sigma-Aldrich, and the like. Algae can be used to industrially mass-produce antibodies (Proc Natl Acad Sci U S A. 2003, 100:438-42).
  • the AMP/AML inhibitor may be a small interfering RNA (siRNA) molecule.
  • RNA interference is a two step process, the first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner.
  • nt nucleotide small interfering RNAs
  • RNA 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].
  • the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC.
  • the active RISC targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and
  • RNAi molecules suitable for use with the present invention can be effected as follows.
  • siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239- 245].
  • siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91 /912.html).
  • the term "about” refers to plus or minus 10%. Wherever the term “about” occurs, it should be understood that the invention also provides corresponding embodiments wherein the degree of variation is plus or minus 5%.
  • Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out. [000149] Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • the AMP/AML inhibitor may be a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the AMP/AML.
  • DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R.R. and Joyce, G. Chemistry and Biology 1995;2:655; Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 1997;943:4262).
  • a general model (the "10-23" model) for the DNAzyme has been proposed.
  • DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate- recognition domains of seven to nine deoxyribonucleotides each.
  • This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S.W. & Joyce, G.F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, LM [Curr Opin Mol Ther 4: 119-21 (2002)].
  • Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al.
  • the AMP/AML inhibitor may be an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the AMP/AML.
  • Design of antisense molecules which can be used to efficiently decrease levels/activity of an AMP/AML must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
  • antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin MoI Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin MoI Ther 1 :297-306 (1999)].
  • the AMP/AML inhibitor may be a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the AMP/AML.
  • Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486- 96 (1998)].
  • the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
  • ribozymes In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10: 163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials.
  • ANGlOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway.
  • Ribozyme Pharmaceuticals, Inc. as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models.
  • HEPTAZYME a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated - WEB home page).
  • the AMP/AML inhibitor may be a triplex forming oligonucleotides (TFOs).
  • TFOs can be used for regulating the expression of an AMP/AML gene in cells.
  • Recent studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypyrimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, L. J., et al., Science,1989;245:725-730; Moser, H. E., et al., Science, 1987;238:645-630; Beal, P.
  • the triplex-forming oligonucleotide has the sequence correspondence: oligo, 3'-A G G T; duplex, 5'-A G C T; and duplex, 3'--T C G A.
  • triplex-forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
  • TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).
  • Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No. 5,721 , 138 to Lawn.
  • the method of regulating the biological process of the present invention comprises the step of exposing the cell/tissue to the regulator.
  • Exposing the cell/tissue to the regulator may be effected in various ways depending on the application and purpose. In cases where the cell/tissue form part of a human or an animal subject, exposing the cell/tissue to the regulator is preferably effected by providing the regulator to the subject.
  • Administering the regulator to a subject may be effected via any suitable route facilitating exposure of the cell/tissue with the regulator, including a route selected from the group consisting of the topical, intravenous, intranasal, transdermal, intradermal, oral, buccal, parenteral, rectal and inhalation route.
  • subcutaneous and/or local injection of the regulator in saline solution is used for treating a disease such as arthritis.
  • oral delivery in combination with aspirin or with NSAID of the regulator is used for treating a disease such as arthritis or other inflammatory diseases regularly treated by NSAID or aspirins such as for example atherosclerosis, osteoarthritis, or rheumatic diseases.
  • NSAID of the regulator such as for example cathelicidin or its analog or fragments or analogs of its fragments
  • aspirins such as for example atherosclerosis, osteoarthritis, or rheumatic diseases.
  • topical application of the regulator in lipid or saline solution, or in a cream on the skin is used for treating a cutaneous disease such as a psoriasis legion.
  • the regulator is dissolved in a solution or provided in an inhalable powder form and administered using an inhaler.
  • the cells may be exposed to regulator by expressing the regulator in the human or animal.
  • exposing the regulator to the cell/tissue is preferably effected by providing the regulator to the cell/tissue in-vitro using standard tissue culture methods.
  • providing the regulator to the cell/tissue in-vitro is effected as described in the Examples section which follows.
  • the regulator can be expressed in a subject by directly administering to the subject a nucleic acid construct configured so as to suitably express the regulator in-vivo.
  • a nucleic acid construct for expressing the regulator may be introduced into a suitable cell ex-vivo via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.), and using a suitable genetic expression system as needed.
  • the modified cells may be expanded in culture and administered to the subject where they will produce the regulator in-vivo.
  • a nucleic acid construct which encodes the regulator preferably includes at least one cis acting regulatory element, most preferably a promoter which is active in the specific cell population transformed.
  • the nucleic acid construct can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription there from.
  • Suitable in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems, polylysine based systems and dendrimers.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems, polylysine based systems and dendrimers.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems, polylysine based systems and dendrimers.
  • AAV adeno-associated virus
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • the construct may include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • such a constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • the various aspects of the present invention may be practiced by using, increasing or by decreasing the activity/level, of any of various types of AMPs/ AMLs, depending on the application and purpose.
  • the AMP/AML is a cationic and/or hydrophobic peptide.
  • peptide refers to a polypeptide which is composed of less than 51 amino acid residue.
  • a cathelidin may be more that 51 a.a. such as for example hCAP-18 which contains and includes the pro-region of the LL-37 peptide.
  • the AMP/AML is a cathelicidin.
  • the cathelicidin is LL-37.
  • the AMP/AML is of human origin. Alternately, it may be of non- human origin, in which case it is preferably of mammalian origin.
  • Numerous examples of AMPs/AMLs which may be used, and/or whose activity/levels may be decreased, for practicing the various aspects of the present invention are described in further detail herein below.
  • the method may be practiced so as to regulate the biological process in any of various cells/tissues of the present invention.
  • the cell/tissue is preferably from bone, synovial fluid or beta cells.
  • the method may be used to regulate the biological process in any of various types of cells/tissues involved in disease included in the present invention.
  • the method may be affected by exposing the cell/tissue to the regulator at any of various concentrations, depending on the application and purpose.
  • exposing the cell/tissue to the AMP/AML inhibitor is effected by exposing the cell/tissue to the AMP/AML inhibitor at a concentration selected from a range of about 50 nanograms per milliliter to about one milligram per milliliter.
  • Exposing the cell/tissue to the AMP/AML inhibitor may advantageously be effected, depending on the application and purpose, by exposing the cell/tissue to the AMP/AML inhibitor at a concentration selected from a range of about 50 ng/ml to about 100 micrograms/ml, from a range of about 100 micrograms/ml to about 200 micrograms/ml, from a range of about 200 micrograms/ml to about 300 micrograms/ml, from a range of about 300 micrograms/ml to about 400 micrograms/ml, from a range of about 400 micrograms/ml to about 500 micrograms/ml, from a range of about 500 micrograms/ml to about 600 micrograms/ml, from a range of about 600 micrograms/ml to about 700 micrograms/ml, from a range of about 700 micrograms/ml to about 800 micrograms/ml, from a range of about 800 micrograms/ml to about
  • exposing the cell/tissue to the AMP/AML is effected by exposing the cell/tissue to the AMP/AML at a concentration selected from a range of about 2 ng/ml to about 50 micrograms/ml or from 50 micrograms/ml to 100 micrograms/ml.
  • Exposing the cell/tissue to the AMP/AML and in particular to cathelicidin may advantageously be effected, depending on inhibitor at a concentration selected from a range of about 2 ng/ml to about 1 micrograms/ml, from a range of about 1 micrograms/ml to about 2 micrograms/ml, from a range of about 2 micrograms/ml to about 3 micrograms/ml, from a range of about 3 micrograms/ml to about 4 micrograms/ml, from a range of about 4 micrograms/ml to about 5 micrograms/ml, from a range of about 5 micrograms/ml to about 6 micrograms/ml, from a range of about 6 micrograms/ml to about 7 micrograms/ml, from a range of about 7 micrograms/ml to about 8 micrograms/ml, from a range of about 8 micrograms/ml to about 9 micrograms/ml, from a range of about 9 microgram
  • the method can be used to regulate in the cell/tissue a biological process such as growth, differentiation, inflammation or autoimmunity.
  • the regulator may advantageously be an AMP/AML inhibitor of the present invention and/or an AMP/AML (in particular, a cathelicidin or a cathelicidin analog) of the present invention.
  • AMP/AML in particular, a cathelicidin or a cathelicidin analog
  • the phrase "cathelicidin inhibitor” refers to a compound of the present invention which is capable of decreasing an activity and/or level of a cathelicidin.
  • the present invention can be used for regulating biological processes such as growth, differentiation, inflammation, chronic inflammation and autoimmunity.
  • disease refers to any medical disease, disorder, condition, or syndrome, or to any undesired and/or abnormal physiological morphological, cosmetic and/or physical state and/or condition.
  • the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or substantially preventing the appearance of clinical symptoms of a disease.
  • the method can be used for treating any of various diseases.
  • the method can be used for treating any of various diseases which are associated with: (i) inflammation; (ii) dysregulation of growth/differentiation of a cell/tissue; (iv) dysregulation of growth/differentiation balance in bone (v) dysregulation of growth/differentiation balance in beta cell function and insulin production (iii) dysregulation of growth/differentiation balance of a cell/tissue; and (iv) autoimmunity.
  • the phrase "subject in need thereof refers to a subject having the disease.
  • the subject is a mammal, most preferably a human.
  • the method described above for inducing or inhibiting such growth is particularly suitable for treating any of various diseases associated with dysregulated/diminished growth of insulin producing cells, and hence can be used for treating any of various diseases associated with insulin depletion.
  • diseases notably include diabetes mellitus type 1 or insulin dependant diabetes or type 2 diabetes, and other metabolic diseases including low grade inflammatory diseases.
  • the method of cell growth regulation as required in the treatment of cancer is also demonstrated.
  • EAE Experimental Autoimmune Encephalitis
  • diseases notably include inflammatory or autoimmune diseases, such as neurodegenerative disease, central nervous system diseases, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease, paraneoplastic cerebellar atrophy, non- paraneoplastic stiff man syndrome, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, optic neuritis, spongiform encephal
  • Such diseases notably include inflammatory or autoimmune diseases, such as arthritis, rheumatic diseases and connective tissue/inflammatory diseases include arthritis, rheumatoid arthritis, pyogenic arthritis, mixed connective tissue disease, cholesteatoma, lupus, relapsing polychondritis, autoimmune myositis, primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis, tendinitis, a ligament inflammation, chondritis, a joint inflammation, a synovial inflammation, carpal tunnel syndrome, osteoarthritis, ankylosing spondylitis, a skeletal inflammation, an autoimmune ear disease, osteoporosis, fibromyalgia, periodontitis, and an autoimmune disease of the inner ear, Diseases diagnosed or managed by the rheumatologist include, Rheumatic diseases such as systemic Lupus Erythematosus, scleroderma (systemic sclerosis), dermatomyositis, polymyo
  • Such diseases notably include inflammatory or autoimmune diseases, such as obesity, diabetes, insulin resistance, type 2 diabetes, Phenylketonuria (PKU), Metabolic syndrome, Sodium metabolism disorders, Calcium metabolism disorders, Hypercalcemia, Hypocalcemia, Potassium metabolism disorders, Hyperkalemia, Hypokalemia, Phosphate metabolism disorders, Magnesium metabolism disorders, Acid-Base metabolism disorders, atherosclerosis, cardio vascular diseases including; Aneurysm, Angina, Arrhythmia, Atherosclerosis, Cardiomyopathy, Cerebrovascular Accident (Stroke), Cerebrovascular disease, Congenital heart disease, Congestive Heart Failure, Myocarditis, Valve Disease, Coronary Artery Disease, Dilated cardiomyopathy, Diastolic dysfunction, Endocarditis, High Blood Pressure (Hypertension), Hypertrophic cardiomyopathy, Mitral valve prolapse, Myocardial infarction (Heart Attack), Venous Thromboembolism.
  • inflammatory or autoimmune diseases such as obesity, diabetes, insulin resistance, type
  • the method described above for inhibiting such inflammation is particularly suitable for treating any of various diseases associated with such inflammation.
  • Such diseases notably include inflammatory or autoimmune diseases, such as Fatigue, Intestinal bloating, Sleepiness, Weight gain, Increased blood triglyceride levels, Increased blood pressure, depression, Hyperglycemia, hyperglycaemia, or high blood sugar, diabetes mellitus, type 1 diabetes, type 2 diabetes, Polycystic ovarian syndrome (PCOS), Hypertension, Dyslipidemia that includes high triglyceride levels, glandular/inflammatory diseases that include type B insulin resistance, Schmidt's syndrome, Cushing's syndrome, thyrotoxicosis, benign prostatic hyperplasia, pancreatic disease, Hashimoto's thyroiditis, idiopathic adrenal atrophy, Graves' disease, androgenic alopecia, thyroid disease, thyroiditis, spontaneous autoimmune thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis, Addison's disease, and Type I autoimmune polygla
  • the method described above for inhibiting such inflammation is particularly suitable for treating any of various diseases associated with such inflammation.
  • Such diseases notably include inflammatory or autoimmune diseases, such as osteoporosis, Osteogenesis imperfecta, Paget's disease, Osteochondroma, Osteomalacia, Osteomyelitis, Osteopetroses, Renal Osteodystrophy, Unicameral Bone Spurs, Bone Tumor, Craniosynostosis, Enchondroma, Fibrous Dysplasia, Giant Cell Tumor of Bone, Infectious Arthritis, Osteomyelitis, Klippel-Feil Syndrome, Limb Length Discrepancy, Osteochondritis Dissecans, periodontitis, bone loss in periodontitis, connective tissue/inflammatory diseases which include arthritis, rheumatoid arthritis, pyogenic arthritis, mixed connective tissue disease, cholesteatoma, relapsing polychondritis, autoimmune myositis, primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis
  • the method described above for inhibiting such inflammation is particularly suitable for treating any of various diseases associated with such inflammation or for wound healing.
  • diseases notably include inflammatory or autoimmune diseases, such as cutaneous/inflammatory diseases include psoriasis, rosacea, dandruff, pemphigus vulgaris, lichen planus, atopic dermatitis, excema, scleroderma, dermatomyositis, alopecia, blepharitis, skin carcinoma, melanoma, squamous cell carcinoma, acne vulgaris, erythema toxicum neonatorum, folliculitis, skin wrinkles, autoimmune bullous skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis, and drug eruption.
  • the regulator such as for example an AMP/AML or the cathelicidin peptide or cathelicidin peptide analog or a cathelicidin protein or a cathelicidin dominant negative analog or a cathelicidin fragment or inhibitor may be administered via any of various suitable regimens.
  • each inter dose interval of the plurality of doses may advantageously be selected from a range of about 2.4 hours to about 3 days, from a range of about 3 days to about 6 days, from a range of about 6 days to about 9 days, from a range of about 9 days to about 12 days, from a range of about 12 days to about 15 days, from a range of about 15 days to about 18 days, from a range of about 18 days to about 21 days, from a range of about 21 days to about 24 days, from a range of about 24 days to about 27 days, or from a range of about 27 days to about 30 days.
  • the inter dose interval of the plurality of doses is about 1 day. This bearing in mind that the half-life of LL-37 peptide in blood of humans is approximately 3.4 days during which the peptide is usually protected from degradation by LDL and HDL. (Infection and immunity, Nov. 1999, p. 6084-6089).
  • the cathelicidin analog or peptide can be delivered to the gastro intestinal tract (GIT).
  • GIT gastro intestinal tract
  • Peptides are usually delivered by injection or infusion due to their limited bioavailability and stability when delivered by other routs.
  • the major problems to overcome in the development of oral peptide delivery are enzymatic degradation and denatuartion in the GIT environment and their poor penetration through physiological barriers (E.C. Lavelle et al. Vaccine 15 (1997), pp. 1070-1078).
  • nanoparticles will protect the peptide from deterioration in the GIT and will allow adhesion and penetration to the Gl mucosal tissue and being released in its active form.
  • Such formulations should be in a nanometer scale where the peptide is fully protected when in the GI fluid but being able to release the peptide within the GI mucosal tissue after absorption.
  • PLA based polymers Two types of systems has been developed, encapsulation in PLA based polymer using the Liposphere technology and stereointeraction of the peptide with stereoregular PLA.
  • the first system is based on the formation of PLA nanoparticles coated with phospholipids by emulsion evaporation method.
  • the second system is based on a physical interaction between the peptide and a common biodegradable stereoregular PLA.
  • PLA based polymers are used. These polymers are FDA approved for the delivery of drugs as well as peptides and proteins.
  • the peptide is dissolved in a safe solvent along with the polymer (D-PLA) which upon mixing the solution, a precipitate of the peptide-PLA sterocomplex is formed.
  • the precipitate can be of nanometer scale and contain a high load of the peptide.
  • the peptide is released from the streocomplex as a result of hydrolysis of the PLA chain that intertwined along the peptide chain.
  • This system has been investigated extensively for the injectable delivery of insulin, somatostatin and LHRH for extended release of weeks after injection. (Macromol Biosci. 2006 Dec 8;6(12): 1019-25, J Control Release 2005 Oct 20;107(3):474-83, Biomaterials. 2002 Nov;23(22):4389-96, Macromol Biosci. 2006 Dec 8;6(12):977-90)
  • topical treatments may advantageously include cell proliferation regulators such as retinoid - vitamin A - analog which modulates or changes the cellular differentiation of the epidermis.
  • Such polytherapy may be effected using anti-inflammatory drugs/treatments as a precautionary measure against relapse of psoriasis or other auto-immune disease.
  • drugs/treatments include tazarotene, methotrexate, acitretin, bexarotene, ploralem, etretinate, corticosteroid creams and ointments, synthetic vitamin D3, IL-10, IL-4 and IL-IRA (receptor antagonist).
  • a cathelicidin inhibitor/vitamin D combination is particularly claimed as a treatment modality for cancer.
  • vitamin D pathway can skew cancer cells into a desired differenciating state
  • cathelicidin which is also expressed via the vitamin D pathway, skews cancer cells into the undesired proliferative state.
  • the maximum desired differentiating non-proliferating pathway is achieved.
  • the regulator is preferably included as an active ingredient in a pharmaceutical composition which includes a suitable carrier and which is suitably packaged and labeled for treatment of the disease.
  • the regulator according to the present invention can be administered to a subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a pharmaceutical composition refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of active ingredients to an organism.
  • active ingredients refers to the regulator of the present invention accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered active ingredients.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated as described above using PLA based polymers or can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. [000232] Dragee cores are provided with suitable coatings.
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofiuoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofiuoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the active ingredients and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • Cream solutions can include any lipids or organic alcohols or chemicals including for example benzyl alcohol, macrogol, hexylene glycol, carbomer, ascorbic acid, butyl hydroxyainisole, butyl hydroxytoluene, disodium edentate, water, trometamol, poxoamer.
  • lipids or organic alcohols or chemicals including for example benzyl alcohol, macrogol, hexylene glycol, carbomer, ascorbic acid, butyl hydroxyainisole, butyl hydroxytoluene, disodium edentate, water, trometamol, poxoamer.
  • the active ingredients may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (regulator of the present invention) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., psoriasis or a carcinoma) or prolong the survival of the subject being treated.
  • a disorder e.g., psoriasis or a carcinoma
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et ah, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • Dosage amount and interval may be adjusted individually to provide plasma or brain levels of the active ingredients which are sufficient to achieve a desired therapeutic effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredients.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.
  • the present invention provides an article of manufacture which comprises packaging material identified for treatment of the disease, and a pharmaceutical composition which includes a pharmaceutically acceptable carrier and, as an active ingredient, the regulator.
  • the pharmaceutical composition is formulated as a solution, suspension, emulsion or gel.
  • the pharmaceutically acceptable carrier is selected so as to enable administration of the pharmaceutical composition via a route selected from the group consisting of the topical, intravenous, intranasal, transdermal, intradermal, oral, buccal, parenteral, rectal and inhalation route.
  • the pharmaceutical composition is composed so as to enable exposure of an affected cell/tissue of the subject having the disease, to the regulator at a suitable concentration, as described hereinabove, for treating the disease.
  • the pharmaceutical composition is further identified for administration to the subject according to a suitable regimen, as described hereinabove.
  • the present invention provides a method of identifying a compound capable of treating autoimmune or inflammatory diseases by using a method of regulating the biological process in a cell/tissue involved in disease. The method is effected in a first step by exposing the cell/tissue to a test compound which is: a compound capable of decreasing or increasing an activity and/or level of an antimicrobial peptide (AMP) and/or AMP-like molecule (AML); and/or which is itself the AMP and/or AML.
  • AMP antimicrobial peptide
  • AML AMP-like molecule
  • the method is effected by evaluating a capacity of the test compound to regulate the biological process in the cell and/or tissue.
  • the method involves the regulation of the AMP cathelicidin or by using the cathelicidin peptide or its analog or fragments.
  • the method of identifying the compound can be used for screening a plurality of compounds so as to identify a compound having a desired capacity for regulating a biological process.
  • the method is preferably used to identify a compound capable of regulating a biological process as described hereinabove with respect to the method of the present invention of regulating a biological process.
  • the test compound is a regulator as described hereinabove with respect to the method of the present invention of regulating a biological process.
  • the method is preferably used to identify a compound capable of regulating the biological process in the cell/tissue as described hereinabove with respect to the method of the present invention of regulating a biological process, and as described in the Examples section which follows.
  • the method is preferably employed for identifying a compound which is capable of: inducing growth in an epithelial, bone cells, osteoclasts or osteoblasts, nerve, synovial cell/tissue, beta cell, skin, keratinocytic and/or gastrointestinal cell/tissue; inhibiting growth in a tumor, epithelial, skin, keratinocytic and/or bone cells, osteoclasts or osteoblasts, nerve, synovial cell/tissue, beta cell, gastrointestinal cell/tissue; inhibiting angiogenesis/endothelial cell/tissue growth; inhibiting metastasis in a tumor cell/tissue; correcting dysregulated balance of proliferation
  • the test compound may be exposed to the cell/tissue in any of various ways.
  • the test compound is exposed to the cell/tissue in-vitro as described in the Examples section which follows.
  • the test compound may be exposed to the cell/tissue by exposing the test compound to a cultured cell/tissue.
  • the cell which produces the test compound is a B-cell hybridoma.
  • the cell which produces the test compound may be of any of various types, depending on the application and purpose.
  • a B-cell hybridoma is an antibody producing cell, and hence that exposing the cell/tissue to a B-cell hybridoma can be used for identifying a B-cell hybridoma which expresses an antibody which is capable of regulating the biological process.
  • Exposing the cell/tissue to the test compound may be effected by providing the test compound to a subject which includes the cell/tissue (in-vivo model). Preferably providing the test compound to the test subject is effected as described hereinabove with respect to providing the regulator to a subject.
  • the identification method may be effected by exposing the test compound to: lesions of any of various diseases associated with bone resorption, CNS disease including multiple sclerosis, arthritis, fat cells of the obeseepithelial wounds included in the present invention; a lesion in an animal model as included in the examples of this invention; or a lesion in a human having the associated disease; a human biopsy of a normal or pathological involved lesion maintained in an organotypic culture containing plasma and lymphocytes of patients suffering from the disease having and not having polymorphism on AMPs or their genes and promoters or that are induced to express an AMP (and in particular a cathelicidin)using known technologies such as transfection (Graham FL Virology 52 (2): 456-67m, Bacchetti S Proc Natl Acad Sci U S A 74 (4): 1590-4); and/or to a cell/tissue of a disease in which the disease inductive isoforms are ApoE4 and the non inductive isoform is Apo
  • the identification method may be effected by exposing the test compound to a human lesion biopsy grafted onto an animal (xenograft model), whereby the biopsy is taken with informed consent.
  • the biopsy may be transplanted onto an immunodeficient mouse (for example, NIHS-bg-nu-xid or BNX).
  • an immunodeficient mouse for example, NIHS-bg-nu-xid or BNX.
  • PBMCs may be isolated from the blood obtained from the biopsy donor and activated (for example, using a superantigen), and the animals injected with the activated PBMCs.
  • Ample guidance for practicing the identification method using such animal models is provided in Examples 6-8 of the Examples section below and in the literature of the art (refer, for example, to U.S. Patent Application No.
  • Evaluating regulation of the biological process may be effected using quantitative evaluation or lessional thickness when using an in-vivo model, cell count or histological evaluation.
  • data obtained from the evaluation is processed using statistical analysis and ANOVA for maximum informativity.
  • the identification method may involve exposing the test compound to cultured microbes/bacteria and evaluating regulation of the biological process is effected by measuring survival of the microbes/bacteria. This may be effected by a colony-forming unit assay performed with Staphylococcus aureus (isolated from clinical sample), GAS (NZl 31), and enteroinvasive Escherichia coli O29 as described (Porter et al, 1997). Before analysis, the concentration of the bacteria in culture will be determined by plating different bacterial dilutions. The protocol may be performed as follows.
  • S. aureus GAS
  • E. coli 200,000 cells per milliliter
  • S. aureus and E. coli are incubated for 4 hours at 37 degrees centigrade with various concentrations of an AMP/AML in the presence of various concentrations of the test compound to be examined, in 50 microliters of buffer in 96 well round bottom tissue culture plates (Costar 3799, Corning inc., NY).
  • GAS are incubated for 1 hour due to the poor ability of GAS to grow in such buffers.
  • the cells are diluted from 10x to 100,00Ox , and each of 20 ml of those solutions are plated in triplicate on tryptic soy broth (for S. aureus) and Todd Hewitt broth (for GAS and E. coli), and the mean number of colonies is determined.
  • the number of cfu per ml is calculated, and the blocking activity of the examined test compounds to block the bactericidal activities of the AMP/AML will be calculated as follows: (cell survival after AMP/AML incubation)/(cell survival after incubation without AMP/AML)xl00, which represents the percentage of cells that are alive, as compared to those which are not (cell survival after AMP/AML+test compound incubation)/(cell survival after incubation with test compound alone)xl00.
  • All compounds identified will be screened for one or all of the following effects: their ability to inhibit or regulate the antimicrobial activity of the AMP (cathelicidin in particular) or to which they were raised against; their ability to affect the proliferation or differentiation or other cellular processes of cultured cells of the affected target tissue, originally isolated from normal or diseased individuals or models, for example HaCaT, primary human or murine keratinocytes or fibroblasts for screening for psoriasis; nerve cells, bone cells or osteoclasts or osteoblasts and the effects of the inhibitors on activation of the immune system.
  • Identified compounds may be further screened for their effects on organotypic cocultures and animal models so as to identify inhibitors that will be able to effectively inhibit a desired biological effect or combination of biological effects. This may include, where suitable, identifying compounds that will inhibit or regulate the effects of AMPs/AMLs on proliferation/differentiation balance but which maintain their antibacterial/antimicrobial activity.
  • the test compound or regulator may be any of various type of molecule, such as a small synthetic/non-polypeptidic molecule.
  • the test compound or regulator may advantageously be a peptide, a protein or a glycosylated protein.
  • Test compounds and regulators of the present invention of any of various suitable types may be obtained from a commercial chemical library such as, for example, one held by a large chemical company such as Merck, Glaxo Welcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartis, Pharmacia UpJohn, and the like.
  • Test compounds and regulators of the present invention of any of various suitable types may also be ordered via the World Wide Web (Internet) via companies such as Chemcyclopedia (http://www.mediabrains.com/client/chemcyclop/BG 1/search.asp).
  • test compounds and regulators of the present invention of any of various suitable types may be synthesized de novo using standard chemical and/or biological synthesis techniques.
  • Cathelicidins or cathelicidin peptides include the hCAP-18 pro-peptide and its following fragments and/or analogs of these fragments sequences (SEQ ID NOs: 1 -59) listed in order as follows: [000277] SEQ ID 1 : fdiscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 2 discdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 3 iscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 5 cdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 6 dkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 8 dnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 10 krfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 11 rfallgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 12 fallgdffrkskekigkefl ⁇ vqrikdflrnlvprtes
  • SEQ ID 14 llgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 15 lgdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 16 gdffrkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 20 rkskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 21 kskekigkefkrivqrikdflrnlvprtes
  • SEQ ID 22 skekigkefkrivqrikdflrnlvprtes
  • SEQ ID 25 llgdffrkskekigkefkrivqrikdflrnlvpr
  • SEQ ID 26 llgdffrkskekigkefkrivqrikdflrnlvp
  • SEQ ID 30 llgdffrkskekigkefkrivqrikdflr
  • SEQ ID 31 llgdffrkskekigkefkrivqrikdfl
  • SEQ ID 34 llgdffrkskekigkefkrivqrik [000311]
  • SEQ ID 35 llgdffrkskekigkefkrivqri
  • SEQ ID 39 llgdffrkskekigkefkri [000316]
  • SEQ ID 40 efkriv
  • SEQ ID 44 kigkefkrivqrik [000321]
  • SEQ ID 45 ekigkefkrivqrikd
  • SEQ ID 46 kekigkefkrivqrikdf
  • SEQ ID 48 skekigkefkrivqrikdflrnlvprtes
  • SEQ ID 49 kskekigkefkrivqrikdflr
  • SEQ ID 50 rkskekigkeflcrivqrikdflrn
  • SEQ ID 54 gdffrkskekigkefkrivqrikdflrnlvpr
  • SEQ ID 55 lgdffrkskekigkefkrivqrikdflrnlvprt
  • AMPs compositions comprising the same, and methods of use thereof are provided.
  • the antimicrobial peptide has at least 90% homology with amino acid sequence fallgdffrksk.Xi (SEQ ID NO:
  • the antimicrobial peptide has at least 90% homology with amino acid sequence X.sub. l igkefkrivq.sub.2 (SEQ ID NO: 57), wherein Xi is 0, 1, 2, 3,
  • the antimicrobial peptide has at least 90% homology with amino acid sequence XiffrkskekigkX 2 (SEQ ID NO: 57), wherein Xi is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids and X 2 is 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.
  • the antimicrobial peptide has at least 90% homology with amino acid sequence Xivqrikdflrn X 2 (SEQ ID NO: 58) where X 2 is 0, 1, 2, 3, 4, 5, 6,7 amino acids and Xi is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18,or 19 amino acids.
  • the antimicrobial peptide has at least 90% homology with amino acid sequence XigkefkrivqrikdflrnX 2 (SEQ ID NO: 59) where X 2 is 0, 1, 2, 3, 4, 5, 6,7 amino acids and Xi is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids.
  • the antimicrobial peptide has at least 60%, 70%, 80%, 90% or 95% homology (or identity) with amino acids in cathelicidin peptides from mammals as described in (Curr Issues MoI Biol. 2005 Jul;7(2): 179-96) namely: [000334] RL-37: RLGNFFRKVKEKIGGGLKKVGQKIKDFLGNLVPRTAS Rhesus monkey, CAP 18: GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLAPRTDY Rabbit, CRAMP: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE Mouse, rCRAMP: GLVRKGGEKFGEKLRKIGQKIKEFFQKLALEIEQ rat, CAPl 1 : (GLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI) Guinea, [000335] Canine cathelicdin: K
  • BMAP-27 GRFKRFRKKFKKLFKKLSPVIPLLHL-NH 2 COW
  • BMAP-28 GGLRSLGRKILRAWKKYGPIIVPIIRI-NH 2 COW
  • BMAP-34 GLFRRLRDSIRRGQQKILEKARRIGERIKDIFR-NH 2 COW
  • Indolicidin ILPWKWPWWPWRR-NH2 Cow, Dodecapeptide: RLCRIVVIRVCR COW, Water buffalo cath GLPWILLR WLFFR-NH2 Water buffalo, OADode: RYCRIIFLRVCR Sheep
  • SMAP-29 RGLRRLGRKIAHGVKKYGPTVLRIIRIA- NH2 Sheep
  • SMAP-34 GLFGRLRDSLQRGGQK1LEKAERI
  • ChBac5 RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFP-NH2
  • Protegrin-4 RGGRLCYCRGWICFCVGR-NH 2 Pig
  • Protegrin-5 RGGRLCYCRPRFCVCVGR-NH 2 Pig
  • PMAP-23 RIIDLLWRVRRPQKPKFVTVWVR Pig
  • PMAP-36 GRFRRLRKKTRKRLKKIGKVLKWIPPIVGSIPLGC-NH 2 Pig
  • PMAP-37 GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQS
  • Peptides with enhanced LPS neutralization and reduced pro-inflammatory activity are also included.
  • Such peptides for example 18-mer LLKKK or GKE and P60, P60.4,
  • Treatment using analogs of these above peptides, peptide fragments and proteins can be formed by modification as described below so as to make the analog (modified cathelicidin peptide or modified peptide fragment) more stable in blood (as described below) while preventing their degradation into their pro- inflammatory fragments by extracellular endogenous protease.
  • Methods of evaluating and discovering suitable analogs or fragments of cathelicidin and other AMPs can, for example, be through the use of assays as disclosed in example 9 below.
  • peptide includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into target cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • non-natural acid such as TIC, naphthylelanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • the peptides of the present invention can be utilized in a linear or cyclic form.
  • a peptide can be either synthesized in a cyclic form, or configured so as to assume a cyclic structure under suitable conditions.
  • a peptide according to the teachings of the present invention can include at least two cysteine residues flanking the core peptide sequence. In this case, cyclization can be generated via formation of S-S bonds between the two Cys residues.
  • cyclization can be obtained, for example, through amide bond formation, e.g., by incorporating GIu, Asp, Lys, Orn, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (-CO-NH or -NH-CO bonds).
  • any of various AMPs/AMLs or combinations of different AMPs/AML may be employed and/or regulated so as to practice the various embodiments of the present invention.
  • Numerous examples of AMPs/AMLs suitable for use in the present invention are listed on the Internet/World Wide Web at http://www.bbcm.units.it/ ⁇ tossi/pagl .htm, and are described hereinbelow.
  • Examples of AMPs/AMLs include defensins, cathelicidins, and thrombocidins
  • defensins include alpha-defensins, beta-defensins, and neutrophil defensins.
  • alpha-defensins examples include alpha-defensin-1 to -6 (MoI Immunol. 2003
  • beta-defensins examples include beta-defensin-1 (Genomics. 1997 Aug l ;43(3):316-20; Biochem Biophys Res Commun. 2002 Feb 15;291(1): 17-22; FEBS Lett.
  • Beta-defensins include those encoded by five conserved beta-defensin gene clusters identified using a computational search strategy (Schutte BC. et al, 2002. Proc Natl
  • neutrophil defensins examples include neutrophil alpha-defensins and neutrophil beta-defensins.
  • neutrophil alpha-defensins examples include neutrophil alpha-defensin- l//human neutrophil peptide (HNP)-I (J Clin Invest. 1985 Oct;76(4): 1436-9; Paulsen F et al,
  • cathelicidins include LL-37/hCAP18 (LL-37) in humans (Curr Drug Targets Inflamm Allergy. 2003 Sep;2(3):224-31 ; Eur J Biochem. 1996 Jun l ;238(2):325-32; Paulsen F et al, J Pathol. 2002 Nov;198(3):369-77).
  • LL-37 is a 37 amino acid residue peptide corresponding to amino acid residue coordinates 134-170 of its precursor hCAP18/human cathelicidin antimicrobial peptide protein (GenBank: ACCESSION
  • NP 004336 VERSION NP 004336.2 GI:39753970; REFSEQ: accession NM 004345.3
  • the proliferation and angiogenesis pathway of LL-37 can be inhibited using pertussis toxin, an inhibitor of G-protein coupled receptors (Koczulla, R. et al., 2003. J.Clin. Invest 1 1 1 : 1665- 1672).
  • Similar AMPs/AMLs are listed in the following patent applications: US 2003120037, US 200309626, US20020141620, US20020507, CA 2383172, US 20020072495 and are incorporated by reference herein.
  • the human antibacterial cathelicidin precursor hCAP-18 is synthesized in myelocytes and metamyelocytes and localizes to specific granules in neutrophils (Blood. 1997 Oct l ;90(7):2796-803).
  • Examples of AMP-like molecules include chemokines or fragments thereof.
  • Examples of such chemokines include CC chemokines and CXC chemokines. Considerable overlap of chemokine and AMP functions has been demonstrated (Cole et al, 2001. J. Immunol. 167:623), and certain chemokines and defensins have actually been shown to bind to the same chemokine receptor, CCR6.
  • chemokines and certain chemokines strikingly share similar characteristics, including size, disulfide bonding, interferon (IFN) inducibility, cationic charge, and more.
  • IFN interferon
  • chemokines and AMPs are described in the literature (refer, for example, to Durr and Peschel, 2002. Infection and Immunity 70:6515).
  • various chemokines and antibodies specific for such chemokines may be employed in various applications of the present invention.
  • Examples of such CC chemokines include CCLl , CCL5/RANTES (Infect Immun.
  • CXC chemokines include CXCL 1 , CXCL2, CXCL3, CXCL4 (PF-4), CXCL7/NAP-2, CXCL8/IL-8, CXCL9 (MIG; Yang D et al, Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55), CXCLlO/IP-10 (The Journal of Immunology, 2001, 167: 623-627), CXCLl 1/ IP-9/I-TAC (The Journal of Immunology, 2001, 167: 623-627), CXCL12/SDF-1 (Yang D et al, Journal of Leukocyte Biology Volume 74, September 2003;74(3):448-55), CXCL13, CXCL14, connective tissue activating peptide 3 (CTAP-3; Infect Immun.
  • CXCL13, CXCL14 connective tissue activating peptide 3
  • fibrinopeptides include fibrinopeptide-A (Infect Immun. 2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr l ;237(l):86-92), fibrinopeptide-B (Infect Immun. 2002 Dec;70(12):6524-33; Eur J Biochem 1996 Apr l ;237(l):86-92).
  • AMPs/AMLs further include XCLl (Yang D et al , Journal of
  • AMPs/AMLs include adrenomedullin (Regul Pept. 2003 Apr 15;1 12(l-3): 147-52; J Biol Chem 1998 JuI 3;273(27):16730-8), alpha-melanocyte stimulating hormone (Cutuli M et al, J Leukoc Biol.
  • LAK lymphokine- activated killer
  • MIP macrophage inflammatory protein
  • MIP-lalpha MIP-l beta
  • MIP-3alpha a mast cell granule serine proteinase
  • MMP matrix metalloproteinase
  • MMP-7 Paulsen F et al, J Pathol. 2002 Nov;198(3):369-77
  • migration inhibitory factor J Immunol. 1998 Sep l ;161(5):2383-90; Scand J Infect Dis. 2003;35(9):573-6
  • MMP-9 Behring Inst Mitt. 1992
  • the AMP/ AML may any one of 28 potential candidates for defensin like peptides which were computationally discovered. (Am J Respir Cell MoI Biol. 2003 Jul;29(l):71-80). [000387] As described hereinabove, the present invention can be used to treat any of various diseases which are associated with: (i) a tumor; (ii) inflammation; (iii) a wound; (iv) autoimmunity; (v) dysregulation of growth/differentiation of a cell/tissue; (vi) dysregulation of growth/differentiation balance of a cell/tissue; and/or (vii) angiogenesis. [000388] Examples of diseases which can be treated according to the present invention are listed in International Pub. No. WO 2004-056307. [000389] Examples of diseases which can be treated according to the present invention are also as follows.
  • tumors include a skin tumor, Osteosarcoma , Ewing's sarcoma, Chondrosarcoma, Malignant fibrous histiocytoma, Fibrosarcoma, Chordoma , osteoid osteoma, osteoblastoma, osteochondroma, enchondroma, chondromyxoid fibroma, and giant cell tumor, lymphoma and multiple myeloma, a keratinocytic tumor, a gastrointestinal tumor, a carcinoma, a melanoma, a squamous cell tumor, oral squamous cell carcinoma, lymphoma, a malignant tumor, a benign tumor, a solid tumor, a metastatic tumor and a non-solid tumor.
  • Urokinase-type plasminogen activator (uPA)
  • uPA Urokinase-type plasminogen activator
  • has antimicrobial properties Gyetko, MR. et al., 2002. J.Immunol. 168:801-809
  • MMPs matrix metalloproteinases
  • BMP-2/4 and BMP-5 but not BMPR-IA might be involved in the metastasis of oral carcinoma cells (Overexpression of BMP-2/4, -5 and BMPR-IA associated with malignancy of oral epithelium Oral Oncol.
  • diseases include an idiopathic/inflammatory disease, a chronic/inflammatory disease, an acute/inflammatory disease, an inflammatory cutaneous disease, an inflammatory gastrointestinal disease, a tumor associated with inflammation, an allergic disease, an autoimmune disease, an infectious disease, a malignant disease, a transplantation related disease, an inflammatory degenerative disease, an injury associated with inflammation, a disease associated with a hypersensitivity, an inflammatory cardiovascular disease, an inflammatory glandular disease, an inflammatory hepatic disease, an inflammatory neurological disease, an inflammatory musculoskeletal disease, an inflammatory renal disease, an inflammatory reproductive disease, an inflammatory systemic disease, an inflammatory connective tissue disease, an inflammatory neurodegenerative disease, necrosis, an inflammatory disease associated with an implant, an inflammatory hematological disease, an inflammatory eye disease, an inflammatory respiratory disease.
  • Examples of cutaneous/inflammatory diseases include psoriasis, dandruff, pemphigus vulgaris, lichen planus, atopic dermatitis, excema, scleroderma, dermatomyositis, alopecia, blepharitis, skin carcinoma, melanoma, squamous cell carcinoma, acne vulgaris, erythema toxicum neonatorum, folliculitis, skin wrinkles, autoimmune bullous skin disease, bullous pemphigoid, pemphigus foliaceus, dermatitis, and drug eruption.
  • Examples of gastrointestinal/inflammatory diseases include Crohn's disease, chronic autoimmune gastritis, autoimmune atrophic gastritis, primary sclerosing cholangitis, autoimmune achlorhydra, colitis, ileitis, chronic inflammatory intestinal disease, inflammatory bowel syndrome, chronic inflammatory bowel disease, celiac disease, an eating disorder, gallstones and a gastrointestinal ulcer.
  • Crohn's disease is an inflammatory bowel disease. Since the bowel is exposed to the outer environment, the importance of AMPs as part of its defense and normal cellular regulation is important, as in skin, and the activity of the AMPs plays an important role in the normal physiology as well as pathological conditions in these tissues. Abnormalities in the expression and/or activity of the AMPs will contribute to pathologies in these tissues. Paneth cells (a specific type of cell in the intestine) are required to help promote normal vessel formation in cooperation with bacteria - mice absent Paneth cells were incapable of appropriate blood vessel formation. Of note, colonization by one particular type of bacteria commonly found in normal mouse and human intestine, called Bacteroides thetaiotaomicron, or B.
  • thetaiotaomicron stimulated blood vessel development as efficiently as implantation of a whole microbial society.
  • B. thetaiotaomicron and Paneth cells work together to stimulate postnatal blood vessel formation.
  • the ability of AMPs to act as chemoattractants for cells of the innate- and adaptive-immune system plays an important role in perpetuating chronic inflammation in the gastrointestinal tract (Cunliffe, RN, Mahida, YR., 2003. J Leukoc Biol. Oct 2 [Epub ahead of print]).
  • the AMP LL-37, beta-defensins, human alpha-defensins, beta-defensins (including HD5), HN-6, lysozyme and secretory PLA2, TLlA, are expressed in Paneth cells and intestine, secretory epithelial cells in the small intestine (Ghosh, D. et al, 2002. Nat.Immunol. 3:583-590; Fellermann, K. et al, 2003. Eur. J. Gastroenterol. Hepatol. 15:627-634).
  • alpha-defensins are chemoattract naive T and immature dendritic cells and dendritic cells and monocytes (Yang, D. et al, 2000. J. Leukoc. Biol. 68:9-14; Risso, A., 2000. J. Leukoc. Biol. 68:785-792; Territo, MC. et al, 1989. J.Clin.Invest 84:2017-2020).
  • Human alpha-defensins as well as other AMPs contribute to local intestinal host defense as part of innate immunity and may be of major relevance in microbial infection and chronic inflammatory bowel disease
  • alpha-defensins convert an acute inflammation to a chronic inflammation by downregulating human polymorphonuclear leukocyte chemotaxis, for example, alpha-defensin-1 /human neutrophil protein- 1, acts as an antichemotactic agent for human polymorphonuclear leukocytes). It is known that chronic inflammation is commonly characterized by the presence of increased cell proliferation and connective tissue than exudate with the presence of lymphocytes and plasma cells rather than polymorphonuclear leukocytes.
  • Gastritis is an inflammatory condition of the stomach.
  • gastritis There are two main forms of gastritis, A and B. Gastritis type A is considered to develop in an autoimmune process. In both types there is a role for infectious agents such as Helicobacter pylori.
  • AMPs are involved in both processes. Defensins are involved in pathogenesis of gastritis (Bajaj-Elliott, M. et al, 2002. Gut 51 :356-361).
  • suitable regulation of such AMPs/AMLs can be used to treat diseases such as gastritis.
  • Examples of allergic/inflammatory diseases include asthma, hives, urticaria, a pollen allergy, a dust mite allergy, a venom allergy, a cosmetics allergy, a latex allergy, a chemical allergy, a drug allergy, an insect bite allergy, an animal dander allergy, a stinging plant allergy, a poison ivy allergy, anaphylactic shock, anaphylaxis, atopic allergy and a food allergy.
  • hypersensitivity examples include Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity, delayed type hypersensitivity, helper T lymphocyte mediated hypersensitivity, cytotoxic T lymphocyte mediated hypersensitivity, TH l lymphocyte mediated hypersensitivity, and TH2 lymphocyte mediated hypersensitivity.
  • cardiovascular/inflammatory and/or inflammatory hematological diseases include atherosclerosis, Takayasu's arteritis, polyarteritis nodosa, Raynaud's phenomenon, temporal arteritis, inflammatory anemia, inflammatory lymphopenia, pernicious anemia, occlusive disease, myocardial infarction, thrombosis, Wegener's granulomatosis, lymphoma, leukemia, Kawasaki syndrome, anti-factor VIII autoimmune disease, necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis, antiphospholipid syndrome, antibody induced heart failure, thrombocytopenic purpura, autoimmune hemolytic anemia, cardiac autoimmunity, Chagas' disease, iron-deficiency anemia, and anti-helper T lymphocyte autoimmunity
  • Inflammation is part of the pathological process leading to the development of atherosclerosis. Chlamydia pneumonia as well as other various microorganisms serve as potential etiological factors, linking inflammation and atherosclerosis. Inflammation is a predisposing factor as well as a consequence of several CNS pathologies. Inflammation is part of the pathophysiologic processes occurring after the onset of cerebral ischemia in ischemic stroke, as well as other CNS pathologies such as head injury and subarachnoid hemorrhage. In addition, inflammation in the CNS or in the periphery by itself is considered as a risk factor for the triggering the development of cerebral ischemia. Endothelial cells express and secrete AMPs.
  • Cationic antimicrobial protein of 37 kDa also termed heparin binding protein, originally isolated from human neutrophils, is an important multifunctional inflammatory mediator is expressed within the vascular endothelium associated with atherosclerotic plaques (Lee, TD. et al., 2002. Am.J.Pathol. 160:841-848).
  • Human beta-defensin-2 is expressed by astrocytes and its expression is increased in response to cytokines and LPS (Hao, HN. et al, 2001. J.Neurochem. 77: 1027-1035). Therefore, AMP regulation can be used for treatment or prevention of these conditions.
  • Anemia associated with inflammatory chronic diseases is one of the body's methods of fighting pathogens by reducing available inter cellular iron uptake of pathogens. Iron is absorbed by neutrophils. Sometimes chronic inflammation can occur without the presence of pathogens. Under chronic inflammatory conditions, cytokines induce a diversion of iron traffic leading to hypoferremia. Such as in chronic bacterial endocarditis, osteomyelitis, juvenile rheumatoid arthritis, rheumatic fever, Crohn's disease, and ulcerative colitis and Chronic renal failure. Transferrin bound iron transports to monocytes causing anemia. This "transport" is thought to be related to AMP activity.
  • Cytokines IL-I, IL-6 and TNF-beta initiate defensin production and defensin initiate the cytokine production, the result being iron over absorption by monocytes.
  • the regulation of iron transport by cytokines is a key mechanism in the pathogenesis of anemia of chronic disease (Ludwiczek, S. et al, 2003. Blood 101 :4148-4154). Therefore, regulation of AMPs can be used to regulate iron level homeostasis.
  • Hepcidin AMP is known to regulate iron uptake, therefore inhibiting hepcidin can be used to increase iron absorption (Nicolas, G. et al, 2002. Blood Cells MoI. Dis. 29:327-335).
  • HNP-I is a non-specific defensive peptide present in neutrophils, it plays an important role in the protection against diseases such as oral lichen planus, leukoplakia, and glossitis associated with iron deficiency (Mizukawa, N. et al, 1999. Oral Dis. 5: 139-142).
  • diseases such as oral lichen planus, leukoplakia, and glossitis associated with iron deficiency (Mizukawa, N. et al, 1999. Oral Dis. 5: 139-142).
  • all cationic neutrophil derived AMPs would induce iron hypoferremia when over expressed. Therefore regulating of these AMPs can be used to treat such diseases.
  • Leukocyte SLPI secretory leukocyte proteinase inhibitor (SLPI)
  • SLPI secretory leukocyte proteinase inhibitor
  • Examples of glandular/inflammatory diseases include type I diabetes, type II diabetes, type B insulin resistance, Schmidt's syndrome, Cushing's syndrome, thyrotoxicosis, benign prostatic hyperplasia, pancreatic disease, Hashimoto's thyroiditis, idiopathic adrenal atrophy, Graves' disease, androgenic alopecia, thyroid disease, thyroiditis, spontaneous autoimmune thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti- sperm infertility, autoimmune prostatitis, Addison's disease, and Type I autoimmune polyglandular syndrome.
  • Diabetes mellitus is a systemic disease with several major complications affecting both the quality and length of life.
  • Periodontal disease Periodontal disease (periodontitis).
  • Periodontitis is much more than a localized oral infection.
  • periapical and other lesions heal as readily as in nondiabetics (Bender, IB, Bender, AB. et al, 2003. J.Endod. 29:383-389).
  • Recent studies on diseases which involve insulin insensitivity e.g. obesity, type 2 diabetes and atherosclerosis
  • Proliferative retinopathy is one of the chronic complications of diabetes. The process includes the development of abnormal blood vessels that might lead to retinal detachment and blindness. LL37 and other AMPs are involved in angiogenesis (Koczulla, R. et al, 2003. J.Clin.Invest 1 11:1665-1672), therefore LL-37 regulation can be used to prevent the development of newly formed blood vessels and therefore for preventing diabetes related eye diseases.
  • hepatic inflammatory diseases include primary biliary cirrhosis, active chronic hepatitis, lupoid hepatitis, autoimmune hepatitis, and hepatic cirrhosis.
  • neurological inflammatory diseases include neurodegenerative disease, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, optic neuritis, spongiform encephalopathy, migraine, headache, cluster headache, and stiff-man syndrome.
  • MS multiple sclerosis
  • CSF cerebrospinal fluid
  • connective tissue inflammatory diseases include arthritis, rheumatoid arthritis, pyogenic arthritis, mixed connective tissue disease, cholesteatoma, relapsing polychondritis, autoimmune myositis, primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis, tendinitis, a ligament inflammation, chondritis, a joint inflammation, a synovial inflammation, carpal tunnel syndrome, osteoarthritis, ankylosing spondylitis, a skeletal inflammation, an autoimmune ear disease, osteoporosis, fibromyalgia, periodontitis, and an autoimmune disease of the inner ear.
  • AMPs are expressed and produced in healthy and inflamed human synovial membranes.
  • mRNA for the AMPs bactericidal permeability-increasing protein (BPI), heparin binding protein, LL37, human alpha-defensin-5, human alpha-defensin-6, and human beta-defensin-1, -2, and -3 was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).
  • RT-PCR revealed CAP37 and human beta-defensin-1 mRNA in samples of healthy synovial membrane.
  • human beta-defensin-3 and/or LL37 mRNA was detected in synovial membrane samples from patients with pyogenic arthritis (PA), osteoarthritis (OA) or rheumatoid arthritis (RA).
  • Microbial mixed keratin-biofilms in cholesteatomas are caused by AMPs which are overexpressed (Jung, HH. et al, 2003. Laryngoscope 113:432-435; Chole, RA, Faddis, BT., 2002 Arch.Otolaryngol.Head Neck Surg. 128:1129-1133), AMPs such as LL-37 or other defensins or other AMPs are involved.
  • Suitable regulation of such AMPs can be used for treating diseases such as cholesteatomas.
  • diseases such as cholesteatomas.
  • inflammatory renal diseases include diabetic nephropathy.
  • examples of inflammatory reproductive diseases include repeated fetal loss, ovarian cyst, or a menstruation associated disease.
  • Examples of inflammatory systemic diseases include systemic lupus erythematosus, systemic sclerosis, septic shock, toxic shock syndrome, Reiter's syndrome, and cachexia.
  • Examples of inflammatory infectious diseases include candidiasis, a fungal infection, mycosis fungoides, a chronic infectious disease, a subacute infectious disease, an acute infectious disease, a viral disease, a bacterial disease, a protozoan disease, a parasitic disease, a mycoplasma disease, gangrene, sepsis, a prion disease, influenza, tuberculosis, bacterial pneumonia, malaria, acquired immunodeficiency syndrome, chronic fatigue syndrome, and severe acute respiratory syndrome.
  • transplantation related/inflammatory diseases include graft rejection, chronic graft rejection, subacute graft rejection, acute graft rejection hyperacute graft rejection, rejection of an implant and graft versus host disease.
  • implants include a prosthetic implant, a breast implant, a silicone implant, a dental implant, a penile implant, a cardiac implant, an artificial joint, a bone fracture repair device, a bone replacement implant, a drug delivery implant, a catheter, a pacemaker, an artificial heart, an artificial heart valve, a drug release implant, an electrode, and a respirator tube.
  • Examples of injuries associated with inflammation include a skin wound, an abrasion, a bruise, a cut, a puncture wound, a laceration, an impact wound, a concussion, a contusion, a thermal burn, frostbite, a chemical burn, a sunburn, a desiccation, a radiation burn, a radioactivity burn, a smoke inhalation, a torn muscle, a pulled muscle, a torn tendon, a pulled tendon, a pulled ligament, a torn ligament, a hyperextension, a torn cartilage, a bone fracture, a pinched nerve and a gunshot wound.
  • inflammatory respiratory diseases include asthma, allergic asthma, diffuse panbronchiolitis, emphysema, idiopathic pulmonary fibrosis, cystic fibrosis, influenza, sinusitis, sinusitis and chronic obstructive pulmonary disease.
  • Examples of inflammatory eye diseases include dry-eye disease, phacogenic uveitis, blepharitis and sympathetic ophthalmia.
  • Dry eye disease is a chronic inflammatory eye disease. Is particularly an issue for post- menopausal women, the elderly, and patients with systemic diseases such as Sjogren's syndrome, rheumatoid arthritis, lupus and diabetes (37% of people with diabetes suffer from the disease and 28% of adults having the disease). Defensins act as chemokines to T-cells (Stern, ME, et al, 2002. Invest Ophthalmol. Vis. Sci. 43:2609-2614).
  • kits comprising a reagent useful for identifying the level of cathelicidin in blood for identifying diseases types is included.
  • the kit is compartmentalized to receive one or more of (i) an oligonucleotide for detection of a cathelicidin or fragment thereof; or (ii) an antibody for detection of cathelicidin or a fragment thereof.
  • preventing binding of AMPs/AMLs to cognate receptors by using ananlogues of same AMPs that compete with binding to same receptors without inducing the disease may be used to inhibit a biological process mediated by binding of the AMP/AML to the receptor.
  • AMPs/ AMLs and over 20 receptors thereof are involved disease pathogenesis, therefore inhibiting correct target combinations of ligand and receptors is essential for treatment of such diseases.
  • Examples of such AMPs/ AMLs and cognate receptors thereof, and examples of the types of diseases which can be treated using this approach are shown in Table 1.
  • the present invention enables for the first time relative to the prior art, treatment of any of various diseases by AMPs in particular by cathelicidin.
  • the present invention clearly shows how cathelicidin is associated with biological processes in cells/tissues such as dysregulated growth/differentiation, dysregulated growth/differentiation balance, inflammation, and angiogenesis and autoimmunity.
  • AMPs/ AMLs, and/or inhibitors of pro-inflammatory fragments thereof is needed for treatment of disease.
  • the invention also provides a new medical use for the treatment of obesity and/or excess body weight that includes the administration of a therapeutically effective amount of one or more LPS neutralizing compounds selected from the group consisting of: BPI (bactericidal/permeability-increasing protein) and fragments and variations thereof such as NeuprexTM (rBPI21, opebacan) a modified recombinant fragment of BPI and MycoprexTM (both Xoma Corporation); protegrins such as protegrin-1 ; lactoferrins such as lactoferricin; Nisin(s) and their variants (MoI Microbiol.
  • BPI Bacillicidal/permeability-increasing protein
  • fragments and variations thereof such as NeuprexTM (rBPI21, opebacan) a modified recombinant fragment of BPI and MycoprexTM (both Xoma Corporation)
  • protegrins such as protegrin-1
  • lactoferrins such as lactofer
  • mCRAMP cathelicidin
  • AMPs Antimicrobial peptides
  • Synthetic peptides for the Mouse Cathelicidin mCRAMP 34a.a. was purchased from Biosight Ltd (Israel).
  • IFA Incomplete Freund's adjuvant
  • mice Male DBA/1 mice, 9-1 1 weeks of age were used in these experiments. Mice were divided into two groups, experimental and control, and each mouse was immunized at the base of the tail with 50 ⁇ l containing 100 ⁇ g of bovine CII emulsified in complete Freund's supplemented to 4 mg/ml of heat killed mycobacterium. Mice were anesthetized during the immunization by inhaled isofluorane. On the same day as the immunization, mice also received their first injection of the vehicle (150 mM saline, control group) or experimental peptide (experimental group) at a concentration of 1.5mg/kg.
  • vehicle 150 mM saline, control group
  • experimental peptide experimental group
  • rheumatic diseases and connective tissue/inflammatory diseases include arthritis, rheumatoid arthritis, pyogenic arthritis, mixed connective tissue disease, cholesteatoma, relapsing polychondritis, autoimmune myositis, primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis, tendinitis, a ligament inflammation, chondritis, a joint inflammation, a synovial inflammation, carpal tunnel syndrome, osteoarthritis, ankylosing spondylitis, a skeletal inflammation, an autoimmune ear disease, osteoporosis, fibromyalgia, periodontitis, and an autoimmune disease of the inner ear.
  • Example 2 Cathelicidin for the treatment of Multiple sclerosis and CNS inflammatory disease
  • MS Multiple sclerosis
  • CNS central nervous system
  • C57BL/6 (B6) mice were purchased from Harlan (Jerusalem, Israel). Female, 9 week old mice were used in the experiment. The mice were housed in the specific-pathogen free (SPF) animal facility of the Hebrew University and all experiments were approved by the institutional animal care and use committee (IACUC).
  • SPF specific-pathogen free animal facility of the Hebrew University and all experiments were approved by the institutional animal care and use committee (IACUC).
  • Emulsion preparation MOGB35-55B peptide (MEVGWYRSPFSRVVHLYRNGK) 1.25mg/ml in PBS was emulsified in complete
  • CFA Freund's adjuvant supplemented with 400 ⁇ g M. tuberculosis (Mt) H37RA (Difco).
  • mice were immunized s.c. in the flank with 250 ⁇ g MOGB35-55B/CFA using a 25G needle.
  • EAE was scored on a scale of 0-6: 0, no impairment; 1, limp tail; 2, limp tail and hind limb paresis; 3, >1 hind limb paralysis; 4, full hind limb and hind body paralysis; 5, hind body paralysis and front limb paresis; 6, death. [000470] EAE treatment
  • mice were treated with cathelicidin peptide of sequence GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ was purchased and supplied by Biosight Ltd of Karmiel, Israel and diluted in PBS, vs. PBS as a control. The cathelicidin was diluted in sterile PBS and divided to aliquots kept at -2O 0 C such that each aliquot was thawed once for use. Mice were treated by intraperitoneal (i.p.) injection of roughly 200ul volume (adjusted for weight) 3 times a week (Sun-Tues-Thurs) starting the day of immunization with MOG/CFA and through day 48. Clinical EAE scores were evaluated through day 60. [000472] Results: [000473] Results are displayed in Figures 7, 8, 9, [000474] A graph showing clinical score up to day 50 is shown as graph in FIG. 8. [000475] Conclusion:
  • Cathelicidin peptide treatment lowered EAE severity and protected mice from fatal EAE observed at a late stage of the disease in control animals.
  • the lower dose of peptide, 0.2mg/Kg was more protective than the higher 2mg/Kg dose.
  • Cathelicidin or its analogs or fragments can therefore be used as a drug for the treatment of neurological and CNS inflammatory diseases.
  • neurological/inflammatory diseases include neurodegenerative disease, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton myasthenic syndrome, paraneoplastic neurological disease, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, progressive cerebellar atrophy, Rasmussen's encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de Ia Tourette syndrome, autoimmune polyendocrinopathy, dysimmune neuropathy, acquired neuromyotonia, arthrogryposis multiplex, optic neuritis, spongiform encephalopathy, migraine, headache, cluster headache, and stiff-man syndrome.
  • Example 3 Example 3
  • a library of expression vectors was generated in yeast cells through homologous recombination; and the encoded proteins complexes with high binding affinity to their target molecule LL-37 was selected by high throughput screening in vivo or in vitro. Testing for ability to inhibit LL-37 in-vivo was performed by measuring the ability of the humanized antibody to inhibit bacterial killing by LL-37.
  • FIG. 10 shows a Western blot analysis of 4 different scFv developed that bind LL- 37.
  • FlG. 11 shows the inhibitory effect of scFv on LL-37 in a bacterial killing assays.
  • IC50 concentration of LL37 at which 50% of the bacteria could be killed.
  • the activity protocol follows the ability of the antibody to block the antimicrobial activity of LL-37.
  • the bacteria used was Pseudomonas that was isolated from a wound.
  • the growth medium was LB.
  • LL-37 was added at a concentration of 100 microgram/ml (the final volume or the reaction is 50 microliter).
  • TLR4 and CD 14 are the receptor for LPS and play a critical role in innate immunity. Stimulation of TLR4 activates pro-inflammatory pathways and induces cytokine expression in a variety of cell types. Inflammatory pathways are activated in tissues of obese animals and humans and play an important role in obesity-associated insulin resistance. TLR4 and CD 14 are a molecular link among nutrition, lipids, and inflammation and that the innate immune system participates in the regulation of energy balance and insulin resistance in response to changes in the nutritional environment.
  • Lipopolysaccharide (LPS)-mediated inflammatory response may modulate pathways implicated in insulin resistance (J Clin Endocrinol Metab 85: 3770-3778, 2000).
  • Materials and Methods [000491] Two groups of 6 mice were used. One group was treated with PBS and the other group with the mCRAMP cathelicidin peptide at 0.4mg/Kg , both groups injected (IP) three times a week on Sunday Tuesday and Thursday. Both groups were fed on a high fat diet of 60%Kcal fat diet (Research Diets Inc. New Brunswick USA) for a four week period by which time under normal circumstances they would be insulin resistant.
  • Cathelicidin in the treatment of obesity and overweight as well as related diseases such as periodontitis and diabetes associated diseases and healing deficiencies.
  • a high-fat diet chronically increased insulin resistance, obesity and metabolic diseases. Diabetes and obesity are two metabolic diseases characterized by insulin resistance and low-grade inflammation.
  • the present experiment shows that obesity can be controlled using cathelicidin or cathelicidin fragments or analogues and is therefore a novel drug for the treatment of obesity and obesity related diseases.
  • the in-vivo mouse model used is as described in (Diabetes 56: 1761-1772, 2007).
  • mice were fed on a normal non-high-fat diet for 21 days and their average weight was monitored.
  • Control group were injected with vehicle (150 mM saline) whilst the experimental group were injected with the cathelicidin mCRAMP at a concentration of 1.5mg/kg on day 0. Subsequently on days 2 and 4 , the dose was reduced to 1.0 mg/kg. Starting with day 7 and through to day 21, a dose of 0.8 mg/kg was used. All treatments were performed 3 times per week, on a Monday, Wednesday, and Friday schedule, and the peptide or control vehicle was administered intraperitoneally for each treatment, rotating injection areas. Results shown in
  • mCRAMP sequence is: GLLRKGGEKIGEKLKKIGQKIKNFFQKL VPQPEQ
  • mice contained 2 groups of 6 mice: Group 1 : PBS, Group 2: mCRAMP cathelicidin 0.2mg/Kg for 3 weeks and then 0.4mg/Kg for another 3 weeks. Mice were treated by intraperitoneal injection of PBS vs. peptide (200 ⁇ l per injection) on Sunday,
  • mice were weighed at baseline and three times a week on each day of treatment.
  • mice in treatment group increased in weight at a rate of 0.0536 gm/day while in the treatment the weight gain was 0.0488 gm/day.
  • Cathelicidin was injected (IP) into treatment mice and compared with control. Histological samples of bone taken from the ankle joints of mice paws were analyzed and osteoclasts were counted using the H&E (hematoxylin and eosin stain) Immunohistochemical staining and with TRAP staining technique (Acid Phosphatase, Leukocyte - Procedure No, 387 A from Sigma-Aldrich).
  • control and treatment groups had induced inflammation in their paws but their inflammation levels were dissimilar.
  • IP LPS injections
  • the tissues were decalcified by incubation in 0.5 M EDTA/PBS, pH 7.4, for 10 days, in which the EDTA solution was changed every day. Tissues were embedded in paraffin and 6 ⁇ m sections were made. Deparaffinised, rehydrated sections were either stained with haematoxylin and eosin or preincubated for 2.5 hours at 37°C in a 12.5 mM sodium tartrate solution in 100 mM acetate buffer, pH 5.5.
  • Paws having similar arthritic scores for equal lengths of time were compared for bone resorption and degradation in cathelicidin treatment group versus control group. This type of comparison rules out any influence of inflammation as a determinant of bone degradation or ankylosis leaving the differentiation status of osteoblasts and osteoclasts as the main influence.
  • the materials and methods used for inducing arthritic bone degredation are described for the mouse model in example 1 above. The arthritic paws of grade 3 severity index and above were obtained from this same experiment and placed in fixative for histology measurements.
  • FIG. 15 Other paws having different severity index and durations were also studied.
  • LF Left Front paw
  • RF Right Front paw
  • LH Left Hind paw
  • RH Right
  • FIG. 16 is an example of histology slides between mouse 3 (Right Front) and FIG.
  • FIG. 18 shows the TRAP staining of control mouse 13 having no inflammatory sign in its paw yet still showing more osteoclasts that the inflamed mouse 3 shown above.
  • FIG.19 shows a TRAP staining of inflamed paw of control mouse 17- Right front paw clearly showing a marked increase in osteoclasts.
  • Cathelicidin inhibits bone erosion and deformation as found in either osteoporosis, ankylosing spondylitis, osteoarthritis and periodontitis and can therefore be used as a drug for treating these diseases.
  • cathelicidin was delivered by IP injections. Therefore, it is obviously implied that the drug delivery of cathelicidin can be either orally using a vehicle carrier to the blood steam via the GI tract or by injection i.v. or subcutaneous injections.
  • Cathelicidin is a suitable drug candidate in the treatment of osteoporosis, ankylosing spondylitis, osteoarthritis and periodontitis, Osteomyelitis, bone cancer, Osteogenesis imperfecta, Paget's disease, Osteochondroma, Osteomalacia, Osteomyelitis, Osteopetroses, Renal Osteodystrophy, Unicameral Bone Spurs, Bone Tumor, Craniosynostosis, Enchondroma, Fibrous Dysplasia, Giant Cell Tumor of Bone, Infectious Arthritis, Osteomyelitis, Klippel-Feil Syndrome, Limb Length Discrepancy, Osteochondritis Dissecans, and bone loss in periodontitis .
  • Cathelicidin analog and fragment for optimal treatment of diseases, such as psoriasis, which are associated with inflammation, autoimmunity and/or skin cell/tissue proliferation/differentiation imbalance and wound healing.
  • diseases such as psoriasis
  • psoriasis which are associated with inflammation, autoimmunity and/or skin cell/tissue proliferation/differentiation imbalance and wound healing.
  • Angiogenesis and epithelialization common in psoriatic skin is enhanced by AMPs such as LL-37 (Koczulla, R. et al, 2003. J.Clin.Invest 1 1 1: 1665-1672; Heilborn, JD. et al, 2003.
  • KLK1-KLK15 trypsin-like or chymotrypsin-like secreted serine proteases
  • cathelicidin is an immune regulator in-vivo and plays a major role in psoriasis and skin inflammation. Inhibiting or regulating its activity is essential for treatment of the disease. Inhibition of cathelicidin in skin inflammation was further demonstrated in psoriasis (Nature 2007 Oct
  • Antimicrobial peptides (AMPs): The antimicrobial peptides used were used were the fragment cathelicidin SK29:SKEKIGKEFKRIVQRIKDFLRNLVPRTES or GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ the mouse cathelicidin CRAMP (BIOSIGHT LTD, Karmiel, Israel), [000559] LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Catalogue No. 61302 LL-37 antimicrobial peptide human , AnaSpec, Inc. USA. , Human Beta-Defensin-2 peptide was purchased from Sigma-Aldrich.
  • Antimicrobial peptides The antimicrobial peptides human cathelicidin LL-37: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES was obtained from AnaSpec, USA (Catalogue numbers: 61302). Cathelicidin at 2ug/ml was added to the plate and compared with control.
  • Thymidine incorporation cell proliferation assay Cell proliferation was evaluated by measuring [3(H)]-thymidine incorporation into DNA. Cells were pulsed with [3(H)]-thymidine (1 microcurie/mL, ICN, Irvine, CA) for 1 hour, at 37 degrees centigrade. After incubation, cells were washed 3 times with PBS, incubated for 15 minutes at room temperature in 5 % trichloroacetic acid and solubilized in 1 % triton X-100. The radioactivity incorporated into the cells was counted in the [3(H)]-window of a Tricarb liquid scintillation counter. Mean values were determined from measurements of triplicate samples under each experimental condition for each experiment. Thymidine incorporation was determined as number of disintegrations per minute (DPM) per mg of protein. [000572] Experimental Results:
  • Beta cells are significantly stimulated to proliferate by AMPs:
  • mouse BETATC beta cell line were chronically treated with cathelicidin, and their proliferation was monitored.
  • cathelicidin led to a slight increase in cell proliferation.
  • This data clearly demonstrates that AMPs are involved in the pathogenesis of diabetes and in particular to type 1 diabetes with respect to cellular hyperproliferation.
  • Chemotaxis assays Cells (e.g. neutrophils, monocytes, T cells, HEK293; 25 microliters at a density of 1.0-3.0 x 106 cells/ml) in RPMI medium (Beit Haemek) containing 0.5% BSA (Sigma-Aldrich) are placed on the top of a 96-well ChemoTx disposable chemotaxis apparatus with a 5 micron pore size (Neuroprobe). Tenfold serial dilutions of the tested reagent in RPMI medium with or without 0.5% BSA are placed in the bottom wells of the chamber. The apparatus is incubated for 60-600 min at 37 0 C in an atmosphere of 5% carbon dioxide, and the cells migrating at each concentration of chemoattractant is counted with the use of an inverted microscope.
  • RPMI medium Beit Haemek
  • BSA Sigma-Aldrich
  • Cells (1 ⁇ lO7/mL) are suspended in a buffer containing 0.25% BSA, 145 mM NaCl, 5 mM KCl, 10 mM Na/MOPS, 1 mM CaC12, 1 mM MgC12, 10 mM glucose, 10 mM HEPES (all from Sigma-Aldrich), pH 7.4, and incubated with 2 micromolar Fura-2-AM (Molecular Probes, Eugene, OR), for 40 min at room temperature. The cells are washed once, resuspended in the buffer containing 0.25% BSA, and are kept at room temperature.

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Abstract

La présente invention concerne des procédés permettant de traiter une maladie et d'accélérer la cicatrisation, qui consistent à obtenir une quantité thérapeutiquement efficace d'un peptide antimicrobien mammalien (PAM) ou d'un analogue de celui-ci, en particulier une cathélicidine ou un fragment de cathélicidine ou un analogue de cathélicidine, et de traiter de ce fait la maladie chez le sujet nécessitant le traitement. L'invention concerne également des analogues ou des fragments spécifiques de cathélicidine qui agissent en tant qu'agonistes, ainsi que le font les cathélicidines endogènes, ou en tant que dominants négatifs ou en tant qu'inhibiteurs de cathélicidine endogène ou de tout autre AMP endogène, ou qui font concurrence à des agents pro-inflammatoires ou des fragments d'AMP sur des récepteurs de même origine sans induire une maladie.
PCT/IL2008/000977 2007-07-15 2008-07-15 Traitement de maladie par des peptides antimicrobien ou leurs inhibiteurs WO2009010968A2 (fr)

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