JP2015028022A - Disease treatment using antimicrobial peptide or inhibitor thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapeutic agent of psoriasis or diabetes.SOLUTION: This invention provides cathelicidin that is an antimicrobial peptide, an effective fragment thereof, or a cathelicidin derivative.

Description

The present invention relates to a method for treating diseases using antimicrobial peptides (AMP) and / or antimicrobial peptide-like molecules (AML), and particularly cathelicidin type AMPs (cathelicidin type antimicrobial peptides).

It also relates to a method for identifying compounds that can modulate, amplify, or decrease the activity / level of AMPs / AML to treat disease. More specifically, the present invention relates to bone loss or osteolysis or osteoporosis, and other autoimmune diseases such as multiple sclerosis, arthritis, psoriasis, or malignant tumors such as cancer with inflammation, metabolic diseases Cathelicidin or cathelicidin fragments or cathelicidin analogues (such as obesity, insulin resistance, type 2 diabetes, diseases involving unregulated cell regulation / cell differentiation that cause type 1 diabetes and related diseases) It relates to methods of treating disease using compounds that can modulate the activity / level of cathelicidin analogs) or antimicrobial peptides (AMPs) / antimicrobial peptide-like molecules (AML).

Compared to the prior art, as the data included for the first time in the present invention show, cathelicidin (cathelicidin) forms the main immunoregulatory factor of diseases known to be also regulated by vitamin D3. This includes in particular the following diseases: periodontitis due to bone loss, obesity, true type 2 diabetes, type 1 diabetes, atherosclerosis, hypertension, asthma, allergy, osteoporosis, osteopenia, Autoimmune diseases such as multiple sclerosis, indirect rheumatism, arthritis, Crohn's disease, type 1 diabetes, schizophrenia, muscle fatigue including muscle fatigue due to aging, dermatitis such as cancer, psoriasis, tuberculosis, influenza, chronic pain , Osteoarthritis, common cold, and other known diseases related to vitamin D3 (The Breast Journal, Volume 14 Number 3, 2008 255-260, Photochem Photobiol. 2008 Mar-Apr; 84 (2): 356-65 ). The data shows a common pathway of disease control between cathelicidin and vitamin D3 as presented in the present invention.

CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY Sept. 2002, p. 972-982 (18-mer LLKKK), ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2006, p. 2983-2989 Vol. 50, No. 9 (GKE), Laryngoscope. 2008 May; 118 (5): 816-20, Inflamm Res. 2004 Nov; 53 (11): 609-22, Antimicrob Agents Chemother. 2005 Jul; 49 (7): 2845-50, Protein Expr Purif. 2004 Sep; 37 (1): 229-35, Int J Antimicrob Agents. 2004 Jun; 23 (6): 606-12, Am J Respir Crit Care Med. 2004 Jan 15; 169 (2): 187-94, Eur J Biochem. 2002 Feb; 269 (4): 1181-9, Surgery. 1995 Jun; 117 (6): 656-62, Prog Clin Biol Res. 1995; 392: 317-26, p e p t i d e s 2 7 (2 0 0 6) 6 4 9? 6 6 0 (P60, P60.4, P60.4-Ac), Biotechnol Lett (2008) 30: 1183-1187 P18.

Abnormal cleavage of the cathelicidin hCAP18 propeptide by endogenous prothesis can result in a pro-inflammatory fragment of cathelicidin (Nat Med. 2007 Aug; 13 (8): 975-80). In contrast, anti-inflammatory cathelicidin analogs and peptides can be produced by proper cleavage through proper endogenous pro-thesis processing. That is, methods for modulating cathelicidin processing abnormalities are required as well as methods using the anti-inflammatory cathelicidin analogs, or peptides or propeptide fragments described and exemplified below.
Antimicrobial peptides (AMP) are effective either alone or as a result of a combination of multiple antimicrobial peptides (AMP).

  According to features of the preferred embodiments of the invention, administration of vitamin D3, cathelicidin peptide fragment, or analog thereof to a subject contains a concentration of the compound selected in the range of about 50 ng / ml to about 2 mg / ml. The carrier is administered to the subject's site in 2 to 30 doses, topically, intravenously, nasally, transdermally, intradermally, oral, buccal, parenteral, rectal, inhaled Each administration interval is selected from a range of about 2.4 hours to about 30 days through a route selected from the group consisting of routes.

Similarly, products made of packaging materials, pharmaceutical compositions, and products identified for the treatment of diseases related to in vivo effects in cells and / or tissues, proliferation, differentiation or disease related to inflammation or autoimmunity. In vivo effects selected from the constituent groups are included; through a route selected from the group consisting of administration routes such as topical, intravenous, nasal, transdermal, intradermal, oral, buccal, parenteral, rectal, inhalation, etc. A pharmaceutically acceptable carrier and a compound capable of modulating the activity / level of antimicrobial peptide (AMP) and / or antimicrobial peptide-like molecule (AML) as an active ingredient, the pharmaceutical composition comprising a solution, suspension, emulsion, or A gel dosage form configured to expose a selected concentration of compound ranging from about 50 ng / ml to about 1 mg / ml to diseased subject cells and / or tissues. ing.
According to still further features in the described preferred embodiments, a pharmaceutical composition is identified that is administered 2-30 times up to about 30 days at intervals of about 2.4 o'clock administration for multiple doses to a subject.

 According to further features in preferred embodiments of the invention described below, administration of the compound to the subject is administered to the subject at a concentration selected from about 50 ng / ml to about 1 mg / ml. Or by expressing the compound in a subject.

 According to further features in preferred embodiments of the invention described below, the antimicrobial peptide (AMP) is any one of the cathelicidins or cathelicidin fragments listed below as SEQ ID NO: 1-58 It is.

It is a graph showing the collagen-induced “arthritis incidence” in a mouse model. The horizontal axis is the number of days after immunization. Cathelicidin 34a.a.mCRAMP peptide was administered (to the experimental group) at a concentration of 1.5 mg / kg. Following immunization, the dose was reduced to 1.0 mg / kg for 2 and 4 consecutive days. From day 7 to day 72, the dose was 0.8 mg / kg. All treatments were performed three times a week on Monday, Wednesday, and Friday, and the vehicle was administered intraperitoneally to the peptide or control group while changing the administration site for each treatment. The body weight of all mice was measured at the start of the experiment, and the dose was calculated. On day 49, body weight was measured again (average 1.6 gram increase). The dose was adjusted according to body weight. The severity of arthritis in all mice was measured three times weekly from the first day of the first day, the degree of arthritis inflammation in each mouse limb was observed, and a numerical score was assigned according to the following criteria. If any of the limbs shows tarsal bone, metatarsal bone, foot, finger, joint stiffness, or erythema or mild swelling in the ankle joint, it is added to the incidence of arthritis. As shown in the figure, the incidence of the control group is high. Details of the analysis results of the incidence of inflammatory limbs in all mice are shown in FIG. mCRAMP is as follows: gllrkggekigeklkkigqkiknffqklvpqpeq It is a graph showing the severity of arthritis. The horizontal axis is the number of days after immunization. The severity of arthritis was analyzed based on the degree of inflammation and the number of affected limbs as follows. • No erythema or swelling is observed. 1- Mild swelling with erythema is found in tarsal bones or ankle joints. 2- Swelling with erythema extends from the ankle joint to tarsal bone. 3-moderate swelling with erythema extends from the ankle joint to the metatarsal joint. 4- Severe swelling with erythema is observed in the ankles, feet, fingers, and joint stiffness. As seen in FIG. 2, when the average value of the severity score per mouse was analyzed, a significant difference was observed between the two groups. Although these data show some differences in the incidence of arthritis, the differences in severity are clearly seen more than the incidence of arthritis. The data in FIG. 2 is from the same experiment described in FIG. It is a graph which displays the average value of the number of affected limbs. The horizontal axis is the number of days after immunization. Similar to the average value of the mouse severity score as shown in FIG. 2, the experimental group also generally shows a low value. However, the development of affected limbs is similar to that in the control group. However, the onset is slow. The data in FIG. 3 is from the same experiment described in FIG. 1 and FIG. It is a graph which displays the follow-up of the clinical score from the arthritis onset day to the 19th day. The horizontal axis is the date from the onset of arthritis. Follow-up is required when each arthritic mouse has inflammation in any one of the limbs on various days since the start of the experiment. Therefore, to measure significant differences between the two groups, the follow-up test statistics need to be met. The data in FIG. 4 is from the same experiment described in FIG. 1 and FIG. FIG. 5 is a graph showing the total severity index measured in all mice in the control group versus the treatment group for several days after immunization. The horizontal axis is the number of days after immunization. From day 27, the control group clearly shows a tendency to increase the severity of the disease. The data in FIG. 5 is from the same experiment described in FIG. 1 and FIG. It is a graph which displays the onset rate of the extremity arthritis of a treatment group versus a control group. The vertical axis is the incidence, and the horizontal axis is the number of days after immunization. Any mouse can record up to 4 data for each limb. Trend lines for the treatment and control groups are calculated using Microsoft Excel. The data in FIG. 6 is from the same experiment described in FIG. 1 and FIG. 2 shows a table listing the results of a mouse experimental autoimmune encephalitis (EAE) model. Cathelicidin vs. PBS treatment results, the box in the figure is from left to right: group number, test material, incidence, mortality on day 50, summary of clinical score (mean), first peak average of disease It is a score. C57BL / 6 (B6) mice were purchased from Harlan in Jerusalem, Israel. Female, 9 week old mice were used in the experiments. Mice were bred in the SPF (specific pathogen free) area of the animal facility at Hebrew University, and all experiments were approved by the Institutional Animal Care and Use Committee (IACUC). 35-55 peptide (peptide sequence: MEGGWYRSPFSRVVHLYRNGK) diluted to 1.25 mg / ml in phosphate buffered saline (PBS) was added to 400 μg of M. tuberculosis (Mt) H37RA (Difco) complete Freund An emulsion was made in addition to adjuvant (CFA). 250 μg MOGB35-55B peptide / CFA was subcutaneously injected into the mouse flank with a 25G needle (immunization). Pertussis Toxin (pertussis toxin) purchased from Sigma was injected intravenously at the time of vaccination and 48 hours later. Experimental autoimmune encephalomyelitis (EAE) was assessed on a scale from 0 to 6: 0, no dysfunction; 1, dragging the tail; 2, dragging the tail, paralyzing the hindlimb 3, complete paralysis of the hind limbs of more than 1 degree; 4, complete paralysis of the entire hind limbs and back; 5, complete paralysis and paralysis of the forelimbs of the back; 6, death; in Carmiel, Israel Mice were each administered with a PBS dilution of cathelicidin peptide purchased from Biosight Ltd and PBS for the control group. Cathelicidin (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) was diluted with sterile PBS, and a single use was thawed and stored separately at −20 ° C. for use. Mice were intraperitoneally administered at a dose of about 200 ul (adjusted according to body weight) 3 times a week (Sunday-Tuesday-Thursday) for 48 days from the day of immunization with MOG peptide / CFA. The 60-day EAE clinical score was calculated. A dose of cathelicidin 2 mg / Kg and a dose of 0.2 mg / Kg were administered to each group. Six mice (18 total) were used for each group. Of note is the fact that mice with advanced EAE eventually died by day 50, whereas none of the treatment group mice died after 60 days. There was no clear difference between the mean clinical score and mean score at the first peak. The group with a lower dose of peptide (0.2 mg / Kg) was more protective than the group with a higher dose (2 mg / Kg). As described in FIG. 7, the mean clinical scores of the three groups measured daily after immunization are shown. It is a clinical score chart of EAE. The vertical axis is the clinical score, and the horizontal axis is the number of days after immunization. It is a photograph of 3 healthy mice in the PBS control group on the 49th day. Photo taken on day 60 of all 6 low dose groups of healthy mice that survived to day 49. The severity of EAE decreased with time. Two cases of EAE mice with completely paralyzed hind limbs and tail are shown. Shown is a Western blot analysis of four different scFv expressed in association with the LL-37 peptide LL-37. FIG. 5 shows the inhibitory effect of scFv on LL-37 in a bacterial lethal assay. The concentration of LL37 that can kill 50% of the bacteria is examined (referred to as “IC50”). Basically, the activity protocol follows up the ability of antibodies to block the antimicrobial activity of LL37. The bacteria used is Pseudomonas bacteria isolated from the wound. The growth medium is LB medium. LL-37 was added at a concentration of 100 μl // ml (final volume or reaction is 50 μl /). Blocking antibody with 1 or 5 μl of antibody (diluted to 1:50 or 1:10, respectively) Low antibody titers have non-specific effects. The bacterial concentration was estimated to be 490 by optical density (OD) measurement. The rate of change in blood glucose level for 2 hours after administration of (lipopolysaccharide) to the treatment group versus the control group is shown. LPS 0.2 mg / kg was administered to C57BL / 6 mice. Blood was collected approximately 2 hours after LPS administration (T = 0), and the change in blood glucose level was measured. Shown is the average weight gain of male DBA / 1 mice about 10 weeks old who had been fed a normal non-high fat diet for 21 days. The body weights of both groups of 10 mice were measured. The control group gained an average of 0.0536 grams per day, whereas the treatment group (cathelicidin mCRAMP 0.8 ug / ml) was one day. As shown in FIG. 13, 0.4 ug / ml was administered to mice fed with a high-fat diet with 60% kcal and body weight was measured three times a week. A list of mouse limbs selected for statistical analysis of bone resorption, bone deformation, immune tissue, and osteoclasts is shown. Mouse limbs were collected from the experimental procedure of Example 1. A list of mouse limbs selected for statistical analysis of bone resorption, bone deformation, immune tissue, and osteoclasts is shown. Mouse limbs were collected from the experimental procedure of Example 1. Shows histological analysis of the effective effect of cathelicidin in reducing bone resorption in the treatment group and the effect of cathelicidin in the treatment group with inflammation and fewer osteoclasts than the control group without inflammation . Histological analysis was performed with hematoxylin and eosin staining, and tartrate-resistant acid phosphatase (TRAP) was measured. Shows histological analysis of the effective effect of cathelicidin in reducing bone resorption in the treatment group and the effect of cathelicidin in the treatment group with inflammation and fewer osteoclasts than the control group without inflammation . Histological analysis was performed with hematoxylin and eosin staining, and tartrate-resistant acid phosphatase (TRAP) was measured. Shows histological analysis of the effective effect of cathelicidin in reducing bone resorption in the treatment group and the effect of cathelicidin in the treatment group with inflammation and fewer osteoclasts than the control group without inflammation . Histological analysis was performed with hematoxylin and eosin staining, and tartrate-resistant acid phosphatase (TRAP) was measured. Shows histological analysis of the effective effect of cathelicidin in reducing bone resorption in the treatment group and the effect of cathelicidin in the treatment group with inflammation and fewer osteoclasts than the control group without inflammation . Histological analysis was performed with hematoxylin and eosin staining, and tartrate-resistant acid phosphatase (TRAP) was measured. Shows 7 weeks effect of human beta defensin 2 on human psoriatic skin. Inhibiting with dominant negative peptide analogs or fragments suggests a method that can be applied to treat the disease. FIG. 5 shows a histogram representing the marked proliferation of BETATC beta cell line caused by cathelicidin LL-37. A murine beta cell line was administered 2 micrograms of LL-37 for 3.5 days and compared to a PBS-treated control group (red / black bars). Cell proliferation was measured by the [3 (H)]-thymidine incorporation method and expressed as a percentage (%) of untreated cells in the control group. A representative experiment is shown.

Specifically, the present invention includes autoimmunity and / or multiple sclerosis, arthritis, obesity, insulin resistance, osteoporosis, periodontitis, or other diseases related to cell proliferation / cell differentiation imbalance, etc. It may be used as an optimal treatment for a wide range of diseases related to in vivo effects, including inflammatory diseases associated with cell proliferation / cell differentiation imbalances. More specifically, the relevant antimicrobial peptide (AMP) competes with the antimicrobial peptide (AMP) without causing cathelicidin or an analog or fragment thereof, antimicrobial peptide (AMP), in particular cathelicidin peptide, disease. Analogues of cathelicidin peptides designed to be more stable in vivo so that they do not break down into pro-inflammatory fragments, cathelicidin analogs that function as dominant negative peptides or cathelicidin peptides that bind to cognate receptors A compound capable of reducing or amplifying the activity / level of antimicrobial peptide (AMP) and / or antimicrobial peptide-like molecule (AML).
As used herein, the term “antimicrobial peptide (AMP)” includes any cathelicidin and / or a naturally occurring variant of the molecule, the natural mutant / polymorphic variant / allele of the molecule Or any synthetic variant of the molecule. As used herein, the term “antimicrobial peptide-like molecule (AML)” includes any molecule that possesses a biological activity that is substantially similar to that of cathelicidin, and any molecule that greatly promotes the biological activity of cathelicidin. And / or any molecule that is structurally highly homologous to cathelicidin. In such cases, the defined homology is between 0% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 99%. Can be classified.

 The method can be accomplished using a single modulator of the present invention or can be accomplished using any combination of multiple regulatory molecules.

 An inhibitor of AMP / AML may be a small molecule, a dominant inhibitor of AMP / AML, or a polypeptide that binds to a cognate receptor in competition with an antimicrobial peptide (AMP) without inducing disease. For example, an inhibitor of AMP / AML may be a topological analog of AMP / AML designed to maintain antibacterial properties. However, the ability to chemically induce may be lost. As used herein, a dominant negative mutant is a position that alters the amino acid residue position at the active site required for the biological and / or pharmacological activity of the natural peptide, relative to the corresponding wild-type natural version, A sequence of a polypeptide or coding region that is altered with respect to at least one position in the sequence. Therefore, the dominant negative mutant or cathelicidin peptide fragment listed below or envisaged herein is a polypeptide species that exhibits an alteration (replacement or / or deletion) with respect to at least one amino acid of AMP or cathelicidin peptide. Including, but not limited to. In addition, examples of dominant negative mutants of the present invention are nucleic acids encoding peptides, or peptides themselves. It consists of an AMP fragment or a fragment of cathelicidin hCAP-18 and is described in (SEQ. ID. NOs: 1-58).

 Of particular note is that the cathelicidin antibacterial peptide inhibits dendritic cell TLR4 activation (J Immunol. 2007 Feb 1; 178 (3): 1829-34), so that cathelicidin is a TLR4 β- It plays a role of suppressing defensin activity.

 Administration of the modulator is intended for the treatment of diseases such as arthritis usually treated with non-steroidal anti-inflammatory drugs such as atherosclerosis, osteoarthritis, rheumatic diseases or aspirin, or other inflammatory circle diseases. In a tablet type, preferably by oral administration of a modulator (eg, cathelicidin or an analog or fragment thereof, or an analog of the fragment) together with aspirin or a non-steroidal anti-inflammatory drug (NSAID). The In the treatment of skin diseases such as psoriasis, the modulators preferably in lipids or saline are applied topically. Alternatively, the regulator in cream is used topically on the skin. In the treatment of respiratory diseases such as cystic fibrosis and asthma or chronic obstructive pulmonary disease, the regulator is preferably dissolved in a solvent or administered using an inhaler. Preferably, the antimicrobial peptide (AMP) and / or antimicrobial peptide-like molecule (AML) is a cationic or / or hydrophobic peptide.

  Because the growth of pancreatic beta cells / tissues can be reliably regulated, the aforementioned methods of promoting or inhibiting the growth are particularly useful for any of a variety of diseases related to dysregulation of insulin producing cell growth or reduced growth rate. Any of a variety of diseases that are suitable for treatment and thus associated with depletion of insulin secretion can be used for treatment. Such diseases include, among others, true type 1 diabetes or insulin-dependent diabetes or second diabetes, and other metabolic diseases such as low-grade inflammatory diseases. Furthermore, cell growth regulation as required in cancer therapy has also been demonstrated.

  Depending on the application and purpose, each dose interval for multiple doses is 2.4 hours to 3 days, 3 days to 6 days, 6 days to 9 days, 9 days to 12 days, 12 days to 15 days, 15 days You can choose from various ranges from days to 18 days, 18 to 21 days, 21 to 24 days, 24 to 27 days, and 27 to 30 days.

 For oral administration, the cathelicidin analog or peptide can reach the gastrointestinal tissue (GIT). Microencapsulation technology using synthetic polyesters such as poly (L-lactide) (L.PLA) used for drug delivery to humans and co-treatment with poly (D, L-lactide co-glycolide) (PLG) Polymers have been investigated for oral vaccines and peptides (M. Manocha et al Vaccine 23 (2005) (48-49), pp. 5599-5617). PLA nanoparticles are coated with phospholipids by emulsion evaporation method. The second tissue is based on physical interactions between peptides and common degradable and stereoregular PLA. When compounding with a solvent, the peptide is dissolved in a safe solvent together with a polymer (D-PLA) that produces a peptide-PLA stereocomplex precipitate. The precipitate may have a nanometer (nm) unit size and contain a highly loaded peptide. The peptide is released from the stereocomplex by hydrolysis of the PLA chain entangled along the peptide chain.

 The pharmaceutical composition of the present invention is produced using, for example, conventional preparation, decomposition method, granulation, sugar-coated tablet production, powdering, emulsification method, encapsulation, inclusion method, lyophilization treatment, as is well known in the art. You may do it.

 Pharmaceutical compositions that can be used for oral administration include push-fit gelatin capsules, soft sealed capsules made of gelatin, and plasticizers made of glycerol or sorbitol. Push-in capsules may contain active ingredients with a bulking agent such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate and optionally a stabilizer. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. A stabilizer may be added. All dosage forms for oral administration should contain dosages suitable for the appropriate route of administration. The composition for buccal administration may be a tablet or lozenge dosage form devised by conventional methods.

 In accordance with the present invention, the active ingredient for administration by nasal inhalation can be aerosolized from a pressurized pack or nebulizer with the use of a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane). Conveniently Administered in Dosage Forms The pharmaceutical compositions described herein may be in parenteral dosage forms, such as by bolus injection or continuous infusion. The dosage form for injection may be a dosage form for example in an ampoule or a container for large doses optionally using a preservative. The pharmaceutical composition may be a suspension, solution, oily or aqueous emulsion. Moreover, you may contain formulation agents, such as a suspension agent, a stabilizer, and / or a dispersing agent.

 Pharmaceutical compositions for parenteral administration also include aqueous solutions (solvents) of water-soluble active formulations. In addition, the suspension of the active ingredient may be formulated as an aqueous suspension injection. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl orenate, triglycerides or liposomes. Aqueous suspension injections may contain substances that increase the viscosity of the suspension, such as carboxymethyl cellulose sodium, sorbitol, or dextran. It may also optionally contain stabilizers suitable to produce a high concentration solution in the suspension or agents that increase the solubility of the active ingredient. Creamy solutions may include lipids or organic alcohols, or chemicals such as benzyl alcohol, sodium edetate, water, trometalol, poloxamer, and the like. Alternatively, the active ingredient may be in the form of a powder containing a suitable vehicle, such as a sterile pyrogen-free aqueous solution.

 The pharmaceutical composition of the present invention may also be in the form of a composition for rectal administration such as a suppository or a retention enema using a conventional suppository base such as cocoa butter or other glycerides.

 Cathelicidin or cathelicidin peptides include hCAP18 propeptide and fragments and / or analogues of these fragment sequences (SEQ ID NOs 1-58) listed in sequence as follows: SEQID1: fdiscdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes, SEQID2: discdkdnkrfallke SEQID3: Iscdkdnkrfallgdffrkskekigkefkrivqrikd flrnlvprtes, SEQ ID 4: Scdkdnkrfallgdffrkskekig kefkrivqrikdflrnlvprtes, SEQ ID 5: Cdkdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes SEQ ID 6: Dkdnkrfallgdffrkskekig kefkrivqrikdflrnlvprtes, SEQ ID 7: kdnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 8: dnkrfallgdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 9: nkrfallgdffrk skekig kefkrivqrikdflrnlvprtes, SEQ ID 10: krfallgdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 11: rfallgdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 12: fallgdffrkskekig kefkrivqrikdflrnlvprtes, SEQ ID 13: allgdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 14: llgdffrkskekigkefkprqtesd SEQ D 15: lgdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 16: gdffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 17: dffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 18: ffrkskekigkefkrivqrikdflrnlvprtes, SEQ ID 19: frkskekigkefkrivqrikdflrnlvprtes, SEQ ID 20: rkskekigkefkrivqrikdflrnlvprtes, SEQ ID 21: kskekigkefkrivqrikdflrnlvprtes, SEQ ID 22: skekigkefkrivqrikdflrnlvprtes, SEQ ID 23 : llgdffrkskekigkefkrivqrikdflrnlvprte, SEQ ID 24: llgdffrkskekigkefkrivqrikdflrnlvprt, SEQ ID 25: llgdffrkskekigkefkrivqrikdflrnlvpr, SEQ ID 26: llgdffrkskekigkefkrivqrikdflrnlvp, SEQ ID 27: llgdffrkskekigkefkrivqrikdflrnlv, SEQ ID 28: llgdffrkskekigkefkrivqrikdflrnl, SEQ ID 29: llgdffrkskekIgkefkrivqrikdflrn, SEQ ID 30: llgdffrkskekigkefkrivqrikdflr, SEQ ID 31: llgdffrkskekigkefkrivqrikdfl , SEQ ID 32: llgdffrkskekigkefkrivqrikdf, SEQ ID 33: llgdffrkskekigkefkrivqrikd, SEQ ID 34: llgdffrkskekigkefkrivqrik, SEQ ID 35: llgdffrkskekigkefkrivqrik SEQki 36, llgdffrkskekiki ID 39: llgdffrkskekigkefkri, SEQ ID 40: efkriv, SEQ ID 41: kefkrivq, SEQ ID 42: gkefkrivqr, SEQ ID 43: igkefkrivqri, SEQ ID 44: kigkefkrivqrik, SEQ ID 45: ekigkefkrivqrikd, SEQ ID 46: kekigkefkrivqrik : skekigkefkrivqrikdfl, SEQ ID 48: skekigkefkrivqrikdflrnlvprtes, SEQ ID 48: kskekigkefkrivqrikdflr, SEQ ID 50: rkskekigkefkrivqrikdflrn, SEQ ID 51: frkskekigkefkrivqrikdflrnl, SEQ ID 52: ffrkskekigkefkrivqrikdflrnlv, SEQ ID 53: dffrkskekigkefkrivqrikdflrnlvp, SEQ ID 54: gdffrkskekigkefkrivqrikdflrnlvpr, SEQ ID 55: lgdffrkskekigkefkrivqrikdflrnlvprt .

In accordance with the present invention, AMPs, compositions using the same, and methods of use thereof are provided. In certain embodiments, the antimicrobial peptide is at least 90% homologous to the amino acid sequence fallgdffrksk.Xsub.1 (SEQ ID NO: 56), where X.sub.1 is 0, 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids. Moreover, the amino acid sequence of the peptide may be arranged in the opposite orientation. In other embodiments, the antimicrobial peptide is at least 90% homologous to the amino acid sequence X.sub.1igkefkrivq.sub.2 (SEQ ID NO: 57), and X.sub.1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, X.sub.2 is 0, 1, 2, 3, 4, 5 , 6, 7 or 8 amino acids. In other embodiments, the antimicrobial peptide further has at least 90% homology with the amino acid sequence X.sub.ffrkskekigk.sub.2 (SEQ ID NO: 57), wherein X.sub.1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, where X.sub.2 is 0, 1, 2, 3, 4, 5, It is characterized by 6, 7 or 8 amino acids. It also has at least 90% homology with X.sub.1 vqrikdflrn X.sub.2, where X.sub.2 is the amino acids 0, 1, 2, 3, 4, 5, 6, 7. .sub.1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 It is a feature. Also, X.sub.1 gkefkrivqrikdflrn X.sub.2 is at least 90% homologous, X.sub.2 is 0, 1, 2, 3, 4, 5, 6, 7 amino acids, .sub.1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 It is a feature.

In other embodiments, the antimicrobial peptide includes at least amino acids in a cathelicidin peptide from mammals as described below (Curr Issues Mol Biol. 2005 Jul; 7 (2): 179-96). 60%, 70%, 80% or 90% homology: RL-37: RLGNFFRKVKEKIGGGLKKVGQKIKDFLGNLVPRTAS Rhesus monkey, CAP18: GLRKRLRKFRNKIKEKLKKIGQKLQQLPLPAPPR ), CAP11: (GLRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI) Guinea, dog (canine) cath: KKIDRLKELITTGGQKIGEKIRRIGQRIKDFFKNLQPREEKS, Bac5: RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLGPFP-NH2 cow (cow), Bac7: RRIRPRPPRLPRPRPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPLPFPRPGPRPIPRPL cow (cow), BMAP-27: GRFKRFRKKFKKLFKKLSPVIPLLHL-NH2 cow (cow), BMAP-28 : GGLRSLGRKILRAWKKYGPIIVPIIRI-NH2 cattle (Cow), BMAP-34: GLFRRLRDSIRRGQQKILEKARRIGERIKDIFR-NH2 cattle ( Cow), Indolicidin: ILPWKWPWWPWRR-NH2 Cattle (Cow), Dodecapeptide: RLCRIVVIRVCR Cattle (Cow), Water buffalo cath GLPWILLRWLFFR-NH2 Water buffalo, OADode: RYCRIIFLRVRL RL VGP -NH2 sheep (sheep), SMAP-34: GLFGRLRDSLQRGGQKILEKAERIWCKIKDIFR-NH2 sheep (sheep), OaBac5 RFRPPIRRPPIRPPFRPPFRPPVRPPIRPPFRPPFRPPIGPFP-NH2 sheep (sheep), OaBac6: RRLRPRHQHFPSERPWPKPLPLPLPRPGPRPWPKPLPLPLPRPGLRPWPKPL sheep (sheep), OaBac7.5: RRLRPRRPRLPRPRPRPRPRPRSLPLPRPQPRRIPRPILLPWRPPRPIPRPQIQPIPRWL sheep (sheep), OaBac11: RRLRPRRPRLPRPRPRPRPRPRSLPLPRPKPRPIPRPLPLPRPRPKPIPRPLPLPRPRPRRIPRPLPLPRPRPRPIPRPLPLPQPQPSPIPRPL sheep (Sheep), ChBac5: RFRPPIRRPPIRPPFNPPFRPPVRPPFRPPFRPPFRPPIGPFP-NH2Goat, eCATH-1: LAKSFLRMRILLPRRKILLAS, eCATH-2: KRRHWFPLSFQEFLEQLRRFRDQLPGIFRP EPRHPRKRP (Pig), AFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPWFPPPPPFRPPPFGPPRFP- NH2 pigs (Pig), Prophenin-2: AFPPPNVPGPRFPPPNVPGPRFPPPNFPGPRFPPPNFPGPRFPPPNFPGPPFPPPIFPGPWFPPPPPFRPPPFGPPRFP- NH2 pigs (Pig), Protegrin-1: RGGRLCYCRRRFCVCVGR-NH2 Pig, RGGRLCYCRRRFCICV-NH2 pigs (Pig), Protegrin-3: RGGGLCYCRRRFCVCVGR-NH2 pigs ( Pig), Protegrin-4: RGGGLCYCRRRFCVCVGR-NH2 Pig (Pig), Protegrin-5: RGGRLCYCRPRFCVCVGR-NH2, Pig (Pig), PMAP-23: RIIDLLWRVRRPQKPKFVTVWVR Pig (Pig), PMAP-36: GRFRRLRKKTRKRLPPIGGPLPLK , PMAP-37: GLLSRLRDFLSDRGRRLGEKIERIGQKIKDLSEFFQS chCATH-B1: (Proc Natl Acad Sci US A. 2007 Sep 18; 104 (38): 15063-8) chicken, Canine cathelicidin (K9CATH) :( Dev Comp Immunol.2007; 31 (12) : 1278-96), Fowlicidin-3: (FEBS J. 2007 Jan; 274 (2): 418-28.), (J Biol Chem. 2006 Feb 3; 281 (5): 2858-67), (Immunogenetics. 2004 Jun; 56 (3): 170-7.) Chicken, CMAP27: (Vet Immunol Immunopathol. 2005 Jul 15; 106 (3-4): 321-7), Fish (cathelicidin from Atlantic cod and Atlantic salmon) Maier VH et al. Mol Immunol. 2008 Jul 7.
It also describes the promotion of the LPS (lipopolysaccharide) neutralization action of peptides and the reduction of pro-inflammatory activity. Such peptides, eg 18-mer LLKKK or GKE and P60, P60.4, P60.4-Ac, CAP11 (11 kDa cationic antibacterial polypeptide), CAP18, GSLL-39, SMAP-29 In addition, the discovery method of the peptide is described in Non-Patent Document 1.

In particular, the peptide having LPS (lipopolysaccharide) neutralizing activity is described as a therapeutic agent for metabolic diseases, obesity and insulin resistance, and is displayed together with cathelicidin in Example 4 below.

Therapies utilizing analogs, peptide fragments and proteins of these above peptides stabilize the analog (modified cathelicidin peptide or modified peptide fragment) in the blood (as described below) In order to prevent degradation by proteolytic fragments by endogenous proteases, they can be formed by modification as described below.

For modification, N-terminal modification, C-terminal modification, CH2-NH, CH2-S, CH2-S = O, O = C-NH, CH2-O, CH2-CH2, S = C-NH, CH = CH or Peptide bond modifications such as CF = CH, backbone modifications and residue modifications are included, but are not limited. Methods for formulating peptidomimetic compounds are well known in the art and are specified, for example, in the following literature: Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

 The peptide bond (-CO-NH-) in the peptide is, for example, an N-methylated bond (-N (CH3) -CO-), an ester bond (-C (R) HCOOC (R) -N-), ketomethylene ( Ketomethylen) bond (-CO-CH2-), α-aza bond (-NH-N (R) -CO-) may be substituted. Where R is, for example, methyl (carba) bond (-CH2-NH-), hydroxyethylene bond (-CH (OH) -CH2-), thioamide bond (-CS-NH-) All alkyl groups such as olefin double bond (-CH = CH-), retro-amide bond (-NH-CO-), peptide derivative (-N (R) -CH2-CO-) And R 2 is conventionally a “normal” side chain on a carbon atom.

 Modifications can occur at any bond along the peptide chain and at the same time multiple (2-3).

 The natural flavoring amino acids Trp, Tyr and Phe are substituted with synthetic unnatural amino acids such as TIC, naphthylalanine (Nol), ring-methyl derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. May be. In addition to the above, the peptides of the present invention also include one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.). The term “amino acid” includes both D-amino acids and L-amino acids. Furthermore, experimental support for the various embodiments and aspects of the invention described hereinabove and described in the claims is found in the following examples.

 Reference is now made to the following examples, which, like the description of the invention above, do not limit the invention. All animal experiments in the following examples are from the Hebrew University of the Jerusalem Animal Care and Ethics Committee (EAE model, obesity model, insulin resistance and periodontitis model) and the University of Tennessee (CIA model and osteoporosis model). Enforced under approved guidelines.

Optimal treatment cathelicidin use for rheumatoid arthritis such as rheumatoid arthritis associated with inflammation or autoimmunity We assume that by adjusting cathelicidin, it can be used for diseases such as arthritis. For the implementation of the present invention, as an optimal treatment of a mouse model of collagen-induced arthritis in human diseases associated with inflammation or autoimmunity such as rheumatoid arthritis, Sjoegren's disease and scleroderma, dermatomyositis, systemic lupus erythematosus, sarcoidosis, etc. .a. Cathelicidin (mCRAMP) (GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ) was demonstrated for the first time as follows, thereby overcoming the limitations of the prior art.
This in-vivo experiment uses a collagen-induced arthritis mouse model.

Experimental materials:
Antimicrobial peptides (AMPs): Synthetic peptides for mouse cathelicidin mCRAMP 34a.a. were purchased from Biosight Ltd (Israel). Chick native CII (purchased from Sigma or Chondrex), a1 (II) chain or CB11 fragment of CII. Freund's incomplete adjuvant (FIA, eg Difco) filtered through 10 mM acetic acid, 0.2-um filter. Mycobacterium tuberculosis (strain H37Ra; heat-sterilized; Ministry of Agriculture, Fisheries, and Food, Central Veterinary Laboratory (address: Waveridge, Surrey, UK) ). DBA / 1JLacJ mouse (Jackson Labs) method: The protocol for this model is described in the published literature Nature Protocols (Brand DD et al. 2007; 2 (5): 1269-75 ). Male 9-11 week old DBA / mouse 9-11 were temporarily used in these facilities. Mice were divided into two groups, the experimental group and the control group, and 50 μl of bovine CII containing 100 μg of emulsified Freund's complete adjuvant supplemented with 4 mg / ml of heat-killed Mycobacterium butyricum was immunized to the tail base of all mice. did. While the mice were immunized, isoflurane inhalation anesthesia was performed. On the same day as the day of immunization, mice also received vehicle (150 mM) in the control group or experimental peptide in the experimental group at a concentration of 1.5 mg / kg. Reduce dose to 1.0 mg / kg for 2 and 4 days after immunization. From day 7 to day 72, the dose was 0.8 mg / kg. All treatments were performed three times a week on Monday, Wednesday, and Friday, and the vehicle was administered intraperitoneally to the peptide or control group while changing the administration site for each treatment. The body weight of all mice was measured at the start of the experiment, and the dose was calculated. Mice were weighed on days 0, 21, 46. Body weight was measured on day 46 (average 1.6 gram increase). The dose was adjusted according to body weight.

The severity of arthritis in all mice was measured three times weekly starting from the 11th day, the degree of arthritis inflammation in each mouse limb was observed, and a numerical score was assigned according to the following criteria. Severity scoring is as described in published literature (Rosleniec E et al. Current protocols in immunology, 1997). Briefly, score 0-no erythema or swelling. Score 1 Mild swelling with erythema is found in tarsal bone or ankle joint. Score 2 Swelling with erythema extends from the ankle joint to the tarsal bone. Score 3 Moderate swelling with erythema extends from ankle joint to metatarsal joint. Score 4 Severe swelling with erythema is found in the ankles, feet, fingers, and joint stiffness. In this experiment, mice in both groups (10 in the treatment group and 11 in the control group) were compared for comparison. MCRAMP (0.8 mg / Kg) was intraperitoneally administered to the treatment group three times a week, and physiological saline was administered to the control group in parallel with the treatment group on the same day.

Experimental results and statistical analysis:
Results of statistical analysis: There was a significant difference between the treatment group and the control group in the progression of arthritis (p = 0.0037). (Significant difference was found by t-test determined from the difference in mean values of severity scores measured from onset on day 1 to onset on day 19). Arthritis incidence—In the control group, the rate and rate of progression of autoimmune arthritis was higher than in DBA / 1 mice. The control group reached 100% by day 44. On the other hand, the incidence of the treatment group (peptide administration) is slightly lower than that of the control group, and the arthritis progression rate seems to be slow. Similarly, the number of affected limbs per mouse in the treatment group was significantly lower than in the control group when shown with a 95% confidence interval. Arthritis Severity-Arthritis severity was analyzed based on inflammation and number of affected limbs (scored as above). As seen in the figure below, when the average severity score per mouse was analyzed, a significant difference was observed between the two groups. Although these data show some differences in the incidence of arthritis, the differences in severity are clearly seen more than the incidence of arthritis.

A 95% confidence interval was calculated with the t-test statistic of the residual distribution obtained by subtracting the expected value-realized value. Expected values were calculated using a linear regression model of actual data. In CIA (collagen-induced arthritis), weight loss is usually observed, which is a judgment factor of disease severity. The control group did not gain more weight than the treatment group. Therefore, between the two groups (control group vs. treatment group) calculated by dividing the body weight from day 21 to 46 and comparing the weight on day 46 by the weight on day 21 with the Mann-Whitney significance test There was a slight difference in weight (P <0.05, two-sided test). The average weight gain from the control group (from day 21 to day 46) was 2.1%, and the treatment group was 5.4% Met.
Results and statistical analysis of the severity and incidence of arthritic limbs are shown in FIGS.

Results and Discussion: The results described in FIGS. 1-6 above clearly demonstrate for the first time compared to the prior art the disease treatment using antimicrobial peptides, specifically cathelicidin. In particular, the results described above clearly demonstrate for the first time in comparison with the prior art the optimal in vivo treatment of a mouse model for arthritis associated with autoimmunity. When treated with cathelicidin, a reduction in arthritis incidence and severity was observed in the collagen-induced arthritis model.

In this experiment, cathelicidin was administered intravenously or subcutaneously or intraperitoneally, arthritis, rheumatic diseases, and arthritis, rheumatoid arthritis, purulent arthritis, mixed connective tissue disease, cholesteatoma, relapsing polychondritis , Autoimmune myositis, primary Sjogren's syndrome, smooth muscle autoimmune disease, myositis, tendonitis, ligament inflammation, chondritis, joint inflammation, synovitis, carpal tunnel syndrome, osteoarthritis, ankylosing spondylitis Suggests a viable treatment for connective tissue / inflammatory diseases such as skeletal inflammation, autoimmune inner ear disease, osteoporosis, fibromyalgia, periodontal disease, and inner ear autoimmune disease .

Experiments also show that oral, intravenous, or subcutaneous or intraperitoneal administration of cathelicidin is associated with systemic lupus erythematosus, systemic sclerosis, septic shock, toxic shock syndrome, Reiter syndrome, and cachexia This suggests a viable treatment for inflammatory / systemic diseases.
Example 2

Cathelicidin multiple sclerosis (MS) for the treatment of multiple sclerosis and CNS inflammatory disease is an immune-mediated demyelinating disease of the central nervous system (CNS), an unknown etiology. is there. The dosage form of cathelicidin in this experiment was injection (IP).
Materials and Methods: Protocol for myelin protein (MOG) -peptide-induced EAE by C57BL / 6 mice

Mice: C57BL / 6 (B6) Mice were purchased from Harlan, Jerusalem, Israel. Female, 9 week old mice were used in the experiments. Mice were bred in the SPF (specific pathogen free) area of the animal facility at Hebrew University, and all experiments were approved by the Institutional Animal Care and Use Committee (IACUC).

EAE induction: Emulsion formulation: MOGB35-55 peptide (peptide sequence: MEGGWYRSPFSRVVHLYRNGK) diluted to 1.25 mg / ml with phosphate buffered saline (PBS) was added to 400 μg M. tuberculosis (Mt) H37RA ( Difco) Complete emulsion was made in addition to complete Freund's adjuvant (CFA). 250 μg MOGB35-55 peptide / CFA was subcutaneously injected into the flank of the mouse with a 25 G needle (immunization).

200 ng Pertussis Toxin (pertussis toxin) purchased from Sigma was injected intravenously at the time of immunization and 48 hours later. Experimental autoimmune encephalomyelitis (EAE) score: Experimental autoimmune encephalomyelitis (EAE) was evaluated on a scale from 0 to 6: 0, no dysfunction; 1, dragging tail 2, dragged tail, hind limb paralysis observed; 3, complete paralysis of hind limbs observed more than once; 4, complete paralysis of entire hind limb and back; 5, complete paralysis and paralysis of hind limbs; 6, death. Experimental autoimmune encephalomyelitis (EAE) treatment: Sequence purchased from Biosight Ltd, Carmiel, Israel: Treatment group with PBS dilution of cathelicidin peptide of GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ In addition, PBS was administered to each control group. Cathelicidin was diluted with sterile PBS, and a single use was thawed and stored separately at −20 ° C. so that it could be used. Mice were intraperitoneally administered at a dose of about 200 ul (adjusted according to body weight) three times a week (Sunday-Tuesday-Thursday) for 48 days from the day of MOG / CFA immunization. The 60-day EAE clinical score was calculated.
Results: The results are shown in Figs.
As in the graph of FIG. 8, a graph showing clinical scores up to the 50th day is displayed.

Conclusion: Treatment with cathelicidin peptide reduces the severity of EAE and protects mice in the treatment group from the severe end-stage symptoms of EAE seen in control mice. The group with a lower dose of peptide (0.2 mg / Kg) was more protective than the group with a higher dose (2 mg / Kg). Therefore, cathelicidin or an analog or fragment thereof can be used as a therapeutic agent for nervous system and central nervous system inflammatory diseases.

The diseases include the following: neurodegenerative diseases, multiple sclerosis, Alzheimer's disease, Parkinson's disease, myasthenia gravis, motor neuropathy, Guillain-Barre syndrome, autoimmune neuropathy, Lambert-Eaton muscle Helplessness syndrome, myasthenia syndrome, neurological paraneoplastic disease, progressive cerebellar atrophy, Rasmussen encephalitis, amyotrophic lateral sclerosis, sydenam chorea, Gilles de la Tourette syndrome, autoimmune multiglandular Nervous / inflammatory diseases such as endocrine insufficiency, autoimmune peripheral neuropathy, congenital polyarthritis, optic neuritis, spongiform encephalopathy, migraine, headache, cluster headache, and stiff man syndrome.
Example 3

Development of fully humanized antibodies against LL-37: Single chain variable fragment antibody (scFv), a fully humanized antibody to cathelicidin LL-37, is a yeast two-hybrid technology as described in US Pat. Developed with. Briefly, the expression vector library was generated by homologous recombination in yeast cells; high affinity encoded protein complexes of the target molecule LL-37 were screened in vivo or in vitro Selected with. We measured the ability of humanized antibodies to block the bactericidal action of LL-37 and tested its ability to suppress LL-37 in-vivo.

FIG. 10 shows a Western blot analysis of four different scFvs expressed by binding to the LL-37 peptide LL-37. FIG. 11 shows the inhibitory effect of scFv on LL-37 in a bacterial lethal assay. The concentration of LL37 that can kill 50% of the bacteria is examined (referred to as “IC50”). Basically, the activity protocol follows up the ability of antibodies to block the antimicrobial activity of LL37. The bacteria used is Pseudomonas bacteria isolated from the wound. The growth medium is LB medium. LL-37 was added at a concentration of 100 μl // ml (final volume or reaction is 50 μl / ml). Blocking antibody with 1 or 5 μl of antibody (dilute 1:50 or 1:10, respectively).

Nonspecific effects are seen in low antibody titers. Two fractions of the eluate containing 100 mM imidazole were used. The antibody and LL-37 mixture was incubated at room temperature for 30 minutes. Bacteria were added (40 microliter capacity). The mixture was incubated for 3 hours at 37 ° C. with shaking. Since the volume used for culturing the bacteria for a long time was small, LB medium was added at that time and the mixture was further cultured for 2-3 hours. The bacterial concentration was estimated to be 490 by optical density (OD) measurement.

Example 4
Cathelicidins for treatment-related diseases such as hyperglycemia or hypoglycemia, hypotension or hypertension, gland / inflammatory diseases, obesity, atherosclerosis, and poor wound healing due to periodontitis and diabetes : In this experiment, we demonstrated that cathelicidin can be used to block insulin resistance and blood glucose levels, indicating that it is a new drug for diabetes and diabetes-related diseases.

The in-vivo mouse model used is described in the following literature. “LPS-induced biomarkers in mice: A potential model for identifying insulin sensitizers”-Biochemical and Biophysical Research Communications 361 (2007) 140-145. Lipopolysaccharide (LPS) -mediated inflammatory response may modulate pathways implicated in insulin resistance (J Clin Endocrinol Metab 85: 3770-3778, 2000).

Materials and methods: Two groups of 6 mice per group were used. One group received PBS (phosphate buffered saline), and the other group received mCRAMP cathelicidin (0.4 mg / Kg) 3 times a week (Sunday-Tuesday-Thursday). Both groups were fed a 50% kcal calorie high-fat diet (Research Diets Inc. New Brunswick, USA) for 4 weeks to ensure normal conditions in which the test mice were prone to insulin resistance. At the end of the fourth week, blood glucose was measured using a blood glucose meter with blood collected from the mouse tail. LPS 0.2 mg / kg was administered to C57BL / 6 mice. Blood was collected approximately 2 hours after LPS administration (T = 0). Insulin sensitivity (or insulin-independent) insulin sensitivity was measured while adjusting glucose uptake, and the increase or decrease in glucose was shown by a curve. The rate of decrease in glucose over time can be determined by the degree of the gradient. mCRAMP sequence: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ. In order to obtain the proportion of each mouse after 2 hours, the glucose level at the time of administration (t = 0) and 2 hours after each subject (T = 2hrs.) The statistics were calculated by dividing by 0. Average values of all proportions were calculated and Student's t-test was performed.

Results: A significant difference was observed in the rate of change of glucose value (Student t test <0.05). The mean glucose level after 2 hours after LPS administration in the control group increased by 5.31%, whereas the mean value in the cathelicidin treatment group decreased to 90.05% from the initial glucose value measured 2 hours ago doing. The treatment group is protected from loss of insulin sensitivity, and the glucose level drops in 2 hours compared to the control group. Since the control group ingests a high fat diet, the glucose level remains high and shows insulin resistance. Insulin resistance, which is normally expressed when the high-fat diet is ingested for 4 weeks, is protected from being expressed in the therapeutic group as in the control group. Figure 12 shows the data in a graph.

Conclusion: In this experiment, we show that cathelicidin, its analogs or fragments thereof can be administered intravenously, subcutaneously or intraperitoneally to suppress insulin resistance and hyperglycemia and inhibit LPS-induced blood glucose dysregulation. It has been demonstrated that it can be used for the treatment of diseases such as metabolic diseases or glandular / inflammatory diseases such as the following diseases: Type 1 diabetes, Type 2 diabetes, Type B insulin resistance, Schmidt syndrome, Cushing syndrome, Hyperthyroidism, benign prostatic hyperplasia, pancreatic disease, Hashimoto thyroiditis, idiopathic adrenal atrophy, Graves' disease, androgenic alopecia, thyroid disease, thyroiditis, spontaneous autoimmune thyroiditis, idiopathic myxedema, Ovarian autoimmune damage, infertility due to anti-sperm antibody, autoimmune prostatitis, Addison's disease, multigland autoimmune syndrome type 1, type 2 diabetes mellitus, obesity, high blood Diseases or hypoglycemia, diabetic complications such as skin ulceration and proliferative retinopathy diabetes-related ocular diseases such as.

Example 5
Cathelicidin efficacy in the treatment of related diseases such as obesity, overweight and periodontitis and diabetes related diseases Background: Chronic high fat diet promotes the development of insulin resistance, obesity and metabolic diseases. Diabetes and obesity are metabolic diseases characterized by insulin resistance and mild inflammation. In this experiment, it was demonstrated that cathelicidin or cathelicidin fragments or analogs can be used to prevent obesity, and it is shown that this is a new drug for obesity and obesity-related diseases. The in-vivo mouse model used is described in (Diabetes 56: 1761? 1772, 2007).

Materials and Methods: Two experiments were conducted, one experiment with a normal diet and the other with a high fat diet.
Initial experiment: Mice were temporarily fed a normal non-high fat diet for 21 days and the average weight of the mice was measured. 2 groups of 10 week old DBA / 1 mice (treatment group and control group), 10 mice each. On the day of experiment start, vehicle (150 mM saline) was administered to the control group, and cathelicidin mCRAMP peptide was administered to the experiment group at a concentration of 1.5 mg / kg. (In the experimental group) at a concentration of 1.5 mg / kg. Thereafter, the dose was reduced to 1.0 mg / kg for 2 days and 4 days. From the 7th day to the 21st day, the dose was 0.8 mg / kg. All treatments were performed three times a week on Monday, Wednesday, and Friday, and the vehicle was administered intraperitoneally to the peptide or control group while changing the administration site for each treatment.

Second experiment: C57BL / 6 mice were temporarily fed a 60 %% kcal calorie high fat diet (Research Diets Inc. New Brunswick, USA) for 6 weeks to experimentally induce obesity. In the experiment, 2 groups of 6 mice were used: 1 group: 2 groups (PBS): mCRAMP cathelicidin 0.2 mg / Kg for 3 weeks, then 0.4 mg / Kg for the next 3 weeks. PBS was administered to the control group and the peptide (200 ml per administration) was intraperitoneally administered every Sunday, Tuesday and Thursday, respectively. The body weight of the test mice was measured at baseline and three times a week on the treatment day.

Results: In the first experiment using a non-high fat diet and as shown in Figure 13, the control group gained an average of 0.0536 grams per day, whereas the treatment group (cathelicidin mCRAMP 0.8 ug / ml) increased 1 It was a day.

Second experiment: calculated by dividing the average body weight of the control group measured for each measurement (three times a week) with that of the treatment group while giving a high fat diet. As can be seen in FIG. 14, certain trends are depicted in the graph. This trend is prominent when analyzed using linear regression and statistical analysis of residual analysis. When treatment was continued until day 50, and the weights of both groups on day 50 were compared, a statistically significant difference was found between both groups by Student's t-test (<0.05). Thus, long-term use of cathelicidin at normal endogenous levels will significantly reduce the weight of obese mice.

Conclusion: In this experiment, cathelicidin intravenously, subcutaneously or intraperitoneally was suggested to be applicable to obesity treatment, 60% Kcal diet and 0.4 mg / Kg 3 times a week By volume, the weight of obese mice was significantly reduced and obesity was prevented.
Example 6

Use of cathelicidin to prevent bone erosion or resorption for the treatment of osteoporosis, ankylosing spondylitis, osteoarthritis and periodontitis
Background: The effects of cathelicidin on bone resorption, bone degradation or bone formation, and comparison of affected limbs treated with cathelicidin in the control group in a mouse model of collagen-induced arthritis. We have also examined whether all are different, and the comparison of the cathelicidin-treated versus control group for LPS-induced osteoporosis in periodontitis (J Clin Periodontol 2004; 31: 596-603). In addition, joints and bones in both groups that were not inflamed were examined and compared.
Cathelicidin was intraperitoneally administered to the treatment group and compared with the control group. Bone tissue samples collected from the ankle joints of mouse limbs were analyzed using hematoxylin and eosin staining (H & E) immunohistochemical staining and TRAP staining technology (acid phosphatase, leukocyte product number, 387 A manufactured by Sigma-Aldrich) The number of osteoclasts was counted. The inflammatory status of the eight groups was compared: two groups: inflammation was induced in the limbs of the control and treatment groups, but the degree of inflammation was the same. In addition, there was no inflammation in the limbs of two groups: the control group and the treatment group. In addition, inflammation was induced in the limbs of two groups: the control group and the treatment group, but the degree of inflammation was different. In addition, LPS was administered to the limbs of two groups: the control group and the treatment group to induce inflammation, and the bone morphology of the mandible was found to be different. By utilizing this method, it became possible to examine osteolysis, inflammation or bone resorption by examining the activity of osteoclasts and immune cells or inflammatory cells.

Materials and Methods: Collagen-induced arthritis (CIA) limb experiments were studied. Of the 80 limbs of the 20 mice available in this study, only 50 were examined according to the inflammation / arthritis severity score. A CIA-induced protocol has been reported in Experiment 1 above.

Tissues: To detect TRAP + cells on joint tissue slides, amputated limbs were fixed with 1% paraformaldehyde for several weeks and washed with PBS. Tissue was removed by culturing with 0.5 M solution of ethylenediaminetetraacetic acid diluted with PBS. 6 μm sections were made from paraffin-embedded tissue. Rehydrated paraffin sections were stained with hematoxylin and erosine, or 12.5 mM sodium tartrate solution in 100 mM pH 5.5 acetate buffer and preincubated for 2.5 hours at 37 ° C.

In addition, the sections were incubated with acid phosphatase for 1 hour at 37 ° C (0.05% naphthol AS-BI phosphate 50 mM sodium tartrate, 0.16% p-roseaniline, 0.16% sodium nitrite (NaNO2), 25% Michaelis 0.14. M acetate / barbital buffer, pH 5.0, with distilled water). Sections were washed with distilled water, counterstained with 0.15% Lightgreen SF Yellowish mixed with 0.2% acetic acid, incubated in 1% acetic acid for 10 minutes and dried at 37%. Red-stained cells are said to contain TRAP and TRAP-positive multinucleated cells (3 or more nuclei) that are considered osteoclasts.

Mice limbs with the same arthritis severity for the same length of time were examined by comparing bone resorption and bone degradation in the treatment group treated with cathelicidin treatment and the control group. This type of comparative study considers the differentiation state of osteoblasts and osteoclasts as the main determinant, and eliminates inflammation from osteolysis or joint stiffness. The materials and methods used to induce osteolysis of arthritis are described in the mouse model of Example 1 above. Arthritic limbs with a severity index of grade 3 were collected from the same experimental procedure, placed in a place where they could be fixed, and tissue was measured.

Experimental results: Stained with H & E and TRAP to examine osteoclasts such as periodontitis, osteoarthritis and inflammatory osteolysis. We examined several mouse limb tissues of equal severity over the same length of time (see table in Figure 15). In addition, we examined non-inflamed limbs in the control and treatment groups (Fig 15). Other mouse limbs with different severity numbers and the same period were also examined. In the figure, LF = left forelimb, RF = right forelimb, LH = left hindlimb, and RH = right hindlimb.

A clear significant difference in bone erosion and bone resorption seen between the treatment group and the control group is that there is less bone degradation and bone resorption in the treatment group. Osteolysis and bone deformation were mainly observed in the control group. Similarly, when mice with the same severity and length of time were selected and divided into two groups, similar clear differences in bone resorption / erosion were observed.

FIG. 16 is an example of a tissue slide between mouse 3 (right forelimb), and FIG. 17 shows H & E staining of mouse 3 limb (right forelimb). FIG. 18 shows TRAP staining of a control group in which no signs of inflammation are observed in the limbs in which more osteoclasts are observed than in the above-mentioned inflammatory mouse 3.

FIG. 19 shows TRAP staining of the inflamed limb of control mouse 17—the right forelimb shows a significant increase in osteoclasts. Even when mice in both groups have the same inflammation, the control group mice have more active osteoclasts, and bone resorption and osteolysis are prominent. Results and Discussion: Cathelicidin prevents bone erosion or resorption and can be used as a therapeutic for these diseases, as seen in examples of osteoporosis, ankylosing spondylitis, osteoarthritis and periodontitis.

The dosage form of cathelicidin in this experiment was intraperitoneal administration (IP). Therefore, the dosage form of cathelicidin can be orally administered to the bloodstream via the gastrointestinal tract via a carrier, or can be administered by injection or subcutaneous injection.
The data shows that cathelicidin is acceptable as an appropriate drug candidate for use in the following treatments: osteoporosis, ankylosing spondylitis, osteoarthritis, periodontitis, osteomyelitis, osteosarcoma, osteogenesis imperfecta Disease, budget disease, osteochondral species, osteomalacia, osteomyelitis, marble osteopathy, renal osteodystrophy, single chamber osteophyte, bone tumor, skull fusion, endochondroma, fibrous bone dysplasia, bone giant Decreased bone mass due to cell tumor, infectious arthritis, osteomyelitis, Krippel-Fille syndrome, leg length inequality, severe osteochondrosis, periodontitis.
Example 7

Use of cathelicidin analogues and fragments for optimal treatment of diseases such as psoriasis associated with inflammation or autoimmunity or skin cell / tissue proliferation / differentiation imbalance and wound healing
Background: Diseases related to inflammation, autoimmunity and / or skin cell / tissue proliferation / differentiation imbalances include many diseases such as psoriasis and dandruff. However, it does not exist. Angiogenesis and epithelialization in psoriatic skin is enhanced by AMPs such as LL-37. (Koczulla, R. et al., 2003. J. Clin. Invest 111: 1665-1672; Heilborn, JD. Et al., 2003. J Invest Dermatol 120: 379-389). To identify a factor involved in abnormal regulation of skin cell / tissue proliferation / differentiation and to use a compound capable of blocking the activity of the factor to treat the disease.

Human tissue kallikreins are homologous to 15 trypsin-like or chymotrypsin-like or secreted serine proteases (KLK1-KLK15). Many KLKs were identified from the normal stratum corneum and sweat as proteases related to desquamation. Anomalies were found in stratum corneum and serum human tissue kallikrein levels in patients with psoriasis (British Journal of Dermatology 2007 156, pp875-883). These kallikreins are proteases involved in the maturation process of cathelicidin LL-37 from its precursor hCAP-18. An imbalance among many proteases may also determine whether cathelicidin is cleaved into pro-inflammatory or anti-inflammatory fragments. As demonstrated by the inventor in WO 2004-056307, cathelicidin is an in-vivo immunomodulator and plays a major role in psoriasis and skin inflammation. Preventing or adjusting its activity is a very important function for disease treatment. Regarding cathelicidin inhibition in skin inflammation, psoriasis (Nature 2007 Oct 4; 449 (7162): 564-9) and other skin inflammations such as rosacea (Nat Med. 2007 Aug; 13 (8): 975-80) Proven. For the implementation of the present invention, dominant negative cathelicidin peptides or fragments as an optimal treatment in humans for diseases associated with inflammation, autoimmunity and / or skin cell / tissue proliferation / differentiation imbalances such as psoriasis For the first time as demonstrated below, thereby overcoming the limitations of the prior art.

Materials and methods-The antimicrobial peptide used was the cathelicidin fragment SK29: SKEKIGKEFKRIVQRIKDFLRNLVPRTES, or GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ mouse cathelicidin, the analogs of the antimicrobial peptide LL 2 Purchased from Sigma Aldrich.

 Human in-vivo psoriasis lesion treatment: Cathelicidin 10 ug / ml or human β-defensin 2 was diluted with PBS containing 0.1% BSA or buffer and applied to the lesions of human subjects in a blinded manner.

Experimental results: In human β-defensin 2 performed during the 7-week course of treatment, as shown in FIG. 20, the applied psoriatic lesions were exacerbated. On the other hand, cathelicidin LL-37 and SK29 showed slight improvement in the course of treatment for 5 days.

Results and Discussion: Recently, research results on rosacea have been published, and cathelicidin processing abnormalities have been observed due to endogenous proathesis involved in hatefulness of rosacea (Nat Med. 2007 Aug; 13 (8): 975-80). Perhaps a similar mechanism works for other diseases such as psoriasis. In this case, a dominant negative peptide inhibitor that competes with the LL-37 fragment without exacerbating the disease opens up the potential for application in disease treatment. As demonstrated by our previous application in WO 2004-056307 and as demonstrated in the published literature (Nature 2007 Oct 4; 449 (7162): 564-9) There is no doubt that the use of LL-37 can be expected to be effective as well. LL-37 itself may inhibit fragments of LL-37 formed by abnormal endogenous protease action.

Example 8
Use of cathelicidin fragments or analogues thereof for the treatment of diabetes
In-vitro studies of beta-cells Background: No optimal therapy exists for treatment of type 1 diabetes. The optimal treatment for the disease is to identify the factors involved in promoting beta cell growth. For the implementation of the present invention, we have identified the important role of antimicrobial peptides (AMP) and demonstrated the ability of cathelicidin to promote the growth of beta cells in order to achieve optimal treatment of type 1 diabetes Thereby overcoming the limitations of the prior art.
Materials and Methods: The antimicrobial peptide human cathelicidin LL-37: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES was purchased from AnaSpec, USA. , USA (catalog number: 61302). Cathelicidin (2ug / ml) was cultured and compared with the control group.

Cell proliferation assay by thymidine incorporation method: The amount of cell proliferation was measured using [3 (H)]-thymidine incorporation into DNA as an index. ells were pulsed with [3 (H)]-thymidine cells were pulsed with [3 (H)]-thymidine at 37 ° C for 1 hour (1 μcrie / mL, ICN, Irvine, CA). After incubation, the cells were washed with PBS for 3 hours, 5% trichloroacetic acid was added, incubated at room temperature for 15 minutes, and solubilized with 1% triton X-100. Incorporation of radioactivity into the cells was counted in the [3 (H)] window of the Tricarb liquid scintillation counter. Three parts of the sample were measured under the experimental conditions of each experiment, and the average value was obtained. Thymidine incorporation was measured per minute per minute of protein per mg.

Experimental Results: Antibacterial peptides promote beta (β) cell proliferation: Chronic treatment of mouse BETATC beta cell line with cathelicidin and observe its proliferation to investigate the efficacy of antibacterial peptides to promote beta cell growth did. This cell. As can be seen in FIG. 21, some of the epithelial cells exposed to cathelicidin showed some enhancement of cell proliferation. The data clearly demonstrate that antimicrobial peptides (AMPs) are involved in the development of diabetes, particularly the development of type 1 diabetes, in terms of cellular hyperproliferation.

Conclusion: The above results indicate that antimicrobial peptides such as cathelicidin or fragments thereof, or analogs thereof are involved in promoting beta cell proliferation and thus helping to treat diabetes by increasing the number of insulin secreting beta cells. It proves clearly.

Example 9
Chemotaxis assay.
Chemotaxis assay. Cells (eg, neutrophils, monocytes, T cells, HEK293; 1.0-3.0 x) in RPMI medium (Beit Haemek) containing 0.5% BSA (Sigma Aldrich) 10 ⁶ cells / ml at a concentration of 25 μl) is placed on a 96-well ChemoTx disposable chemotaxis assay device with a 5 μ pore size (neuroprobe). Pour a 10-fold serial dilution of the reagent in RPMI medium with or without 0.5% BSA into the bottom of the chamber. The cells were incubated for 60-600 minutes in air at 37 ° C in 5% carbon dioxide, and the number of migrated cells was counted using an inverted microscope at each concentration of chemotactic substance.

Cells (1 x 10 ⁷ / mL) in Buffer solution (contains the following substances: 0.25% BSA, 145 mM sodium chloride (NaCl), 5 mM potassium chloride (KCl), 10 mM sodium) / MOPS (buffer), 1 mM calcium chloride (CaCl ₂₎ , 1 mM magnesium chloride (MgCl ₂ ), 10 mM glucose, 10 mM HEPES (buffer) (all from Sigma-Aldrich), pH 7.4)) suspended and incubated with 2 micromolar Fura-2-AM (Molecular Probes, Eugene, OR) for 40 minutes at ambient temperature. The cells were washed once with a buffer solution containing 0.25% BSA and stored at room temperature. Prior to use, aliquots of cells (4 × 10 ⁵ ) were washed and placed in a stirring cuvette and resuspended with 2 ml of 0.05% BSA containing buffer at 37 ° C. Measurement of intracellular calcium ion (Ca2 +) concentration and mobility assay were performed as described above (Maghazachi, AA. Et al., 1997. FASEB J. 11: 765-774). For clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination with one embodiment. On the contrary, certain features of the invention described in the context of separate embodiments may be considered separately or sub-subject to clarify various features of the invention in the context of one embodiment for the sake of brevity. It may be provided in combination. Although the present invention has been described in conjunction with specific embodiments thereof, it is evident that many changes, modifications and variations will become apparent to those skilled in the art. It is intended to encompass any changes, modifications and variations of the present invention which are limited in its spirit and scope. All publications, patent applications, and sequences identified by accession number referred to herein are specifically identified by the sequence identified by each individual publication, patent application or accession number. Are incorporated herein by reference in their entirety, at the same level as directed to be incorporated herein by reference. In addition, citations or identification information of references in this application shall not be construed as an admission that the invention is available as prior art.

The above description relates to one embodiment of the present invention, and those skilled in the art can consider various modifications of the present invention, all of which are included in the technical scope of the present invention. The

Claims (1)

  Use of LL-37 (SEQ ID NO: 14) in a pharmaceutical formulation for the treatment of psoriasis or diabetes.