Patent Application: US-565307-A

Abstract:
peptides having four to fourteen residues are disclosed that possess biological activity . these peptides constitute short fragments of the peptide hb - 107 , which itself is a fragment of the antimicrobial protein cecropin b , and exhibit cell stimulatory , migratory and anti - inflammatory properties . as keratinocytes are especially sensitive to these effects , the disclosed peptides comprise a useful agent for the medical treatment of injury to the skin , such as from diabetic ulcers . the peptides also are effective in preventing and reversing skin surface damage resulting from various environmental insults . importantly , the therapeutic effects of the peptides manifest at concentrations equal to or greater than those of peptide hb - 107 , and thus represent a less expensive , more versatile means for developing effective therapies . methods for the production and use of these peptides are also disclosed .

Description:
u . s . pat . nos . 5 , 962 , 410 and 5 , 861 , 478 provide disclosures useful for understanding the present invention and are herein incorporated by reference in their entirety . the invention is directed towards short , bio - active peptides that , for example , are derived from the peptide hb - 107 ( seq id no : 1 ), and methods of their use . peptide hb - 107 itself constitutes a fragment of cecropin b , which is an antimicrobial protein present in a species of moth . although hb - 107 does not display the bacteriostatic effects of the protein from which it is derived , it does display epidermal wound healing qualities ( lee at al ., 2004 ). the peptides of the present invention display activities that are important for upregulating healing processes in epidermal tissues such as skin and associated mucosa ( e . g . oral cavity ). not to be limited to any particular mechanism , these activities are drawn towards keratinocytes , the epithelial cells responsible for wound closure ( i . e . epithelialization ) and development of epidermal surfaces . the specific activities that the inventive peptides display towards keratinocytes that are of direct relevance to wound healing are stimulation of cell proliferation and migration , as well as the ability to downregulate inflammatory signaling by the keratinocytes . though hb - 107 is capable of inducing all of these activities in keratinocytes , quite surprisingly , the peptides of the present invention do so at levels equal to or greater than hb - 107 . these results are both surprising and significant , since the inventive peptides are only 26 %- 74 % the length of hb - 107 . because of this size differential , the peptides of the current invention are easier and thus less expensive to prepare compared to production of hb - 107 and full - size proteins such as pdgf - bb . also in contrast to larger peptides , the disclosed peptides are solubilized , manipulated ( e . g . chemical modification ) and stored in a more straightforward manner . their ease - of - handling enables a greater number of drug delivery options , such as the vehicle to be used and how it is to be applied . the size and greater solubility of the inventive peptides permit their increased healing potency through increased absorption and retention at the wound site ; local keratinocytes and other cells are exposed to higher concentrations of the peptides for longer periods of time . the biological activities elicited by the peptides of the present invention are cell proliferation and migration , as well as the inhibition of inflammation . the former two processes play a large role in mediating the wound healing function of the peptides . the peptides are able to first , stimulate migration of keratinocytes bordering the wound edge , and second , stimulate proliferation of these cells so as to create a new epidermal layer over the injury site . the third activity , inhibiting inflammation , is achieved via the disclosed peptides &# 39 ; negative effect on secretion of the cytokine interleukin - 6 ( il6 ) by cells at the wound . il6 has been shown to be a released by epidermal keratinocytes in response to factors associated with tissue damage ( sugawara et al ., 2001 ); this cytokine signals for immune cell infiltration into the wound , a process which can actually aggravate healing and cause scarring ( martin and leibovich , 2005 ; liechty , 2000 ). though inflammation is important to prevent wound infection , the provision of good antiseptic practices during standard wound care negates any drawbacks that may be associated with blocking inflammation . though the above activities are likely those through which the invention effects wound healing , it is noted that the application is not limited by any one particular set of biological mechanisms . with respect to inducing cell proliferation and migration , the peptides seq id no : 3 ( hb - 1061 ) and seq id no : 6 ( hb - 1072 ) are preferred . these peptides produce significant increases in cell proliferation compared to induction by hb - 107 , but yet are shorter than hb - 107 ( refer to examples 1 and 2 ). they are also just as capable as hb - 107 in inducing cell migration . the peptides seq id no : 8 and seq id no : 12 are preferred with respect to induction of cellular anti - inflammatory activity ( refer to example 3 ). the inventive peptides also exhibit salutary effects towards problems associated with aging skin , or skin that has been highly exposed to damaging agents such as solar radiation . the short peptides by themselves unaltered , or via chemical modification and / or specialized delivery , can be made to absorb through the epidermis to effect processes counter to those that cause skin thinning , wrinkles , fragility and roughening / hardening . a major mode by which the invention stimulates skin preservation is through the peptides &# 39 ; positive effect toward keratinocyte growth . as these cells are the main component of epidermal surfaces and are diminished in aged and damaged skin ( enoch and price , 2004 ), replenishment thereof by peptide stimulation is expected to reverse the aforementioned problems . il6 expression is implicated in processes underlying the abnormal thickening of the epidermis in patients with certain autoimmune problems ( sato et al ., 2001 ; oyama et al ., 1998 ); the inventive peptides can block such an inflammation - related outcome by inhibiting il6 expression . solely as a guiding point , the inventive peptides can be derived from the hb - 107 fragment ( table 1 ) of the moth cecropin b protein . the metabolic features associated with these peptides are their capability of inducing cell proliferation , migration , and / or anti - inflammatory activities . all the inventive peptides share the common feature of having four to fourteen contiguous amino acid residues of hb - 107 ( seq id no : 1 ). therefore , peptides of the invention may consist of four , five , six , seven , eight , nine , ten , eleven , twelve , thirteen or fourteen contiguous amino acid residues of hb - 107 ( seq id no : 1 ). aside from having the above amino acid compositions , the above - described peptides may additionally contain the following amino acids ( full name / three letter abbreviation / single letter abbreviation ): alanine / ala / a , arginine / arg / r , asparagine / asn / n , aspartate / asp / d , cysteine / cys / c , glutamine / gln / q , glutamate / glu / e , glycine / gly / g , histidine / his / h , isoleucine / ile / i , leucine / leu / l , lysine / lys / k , methionine / met / m , phenylalanine / phe / f , proline / pro / p , serine / ser / s , threonine / thr / t , tryptophan / trp / w , tyrosine / tyr / y and valine / val / v . these amino acid residues are characterized as follows : aliphatic ( alanine , glycine , isoleucine , leucine , proline , valine ), aromatic ( phenylalanine ), tryptophan , tyrosine ), acidic ( aspartate , glutamate ), basic ( arginine , histidine , lysine ), hydroxylic ( polar ) ( serine , threonine ), sulphur - containing ( polar ) ( cysteine , methionine ), and amidic ( asparagine , glutamine ). non - standard amino acid residues may also be incorporated into the disclosed peptide including , but not limited by , selenocysteine , pyrolysine and various cyclic forms of amino acids . the following peptides are non - limiting examples of the present invention and are shown for illustrative purposes ( table 1 ). the peptides seq id no : 2 , seq id no : 3 , seq id no : 5 , seq id no : 6 , seq id no : 7 , seq id no : 8 , seq id no : 9 , seq id no : 10 , seq id no : 11 , seq id no : 12 and seq id no : 15 are examples of peptides associated with one or more of the activities ( proliferative , migratory , anti - inflammatory ) described above . the peptides seq id no : 2 , seq id no : 3 , seq id no : 5 , seq id no : 6 , seq id no : 7 , seq id no : 9 , seq id no : 10 , seq id no : 11 and seq id no : 12 are examples of peptides that can induce cell proliferation . the peptides seq id no : 3 and seq id no : 6 are examples of peptides that can induce cell proliferation and migration . the peptide seq id : 12 is an example of a peptide that displays both proliferative and anti - inflammatory activities . the peptides seq id no : 8 and seq id no : 15 are examples of peptides having anti - inflammatory activity . each of the above - described peptides can comprise l - or d - amino acid enantiomers , either containing residues of one enantiomeric form or a combination of both forms . the peptides may be further augmented or modified , either chemically or enzymatically , as described in the following non - limiting examples . the carboxy - terminus of the peptides can be acidic (— cooh ) or be amidated ( e . g . — conh 2 , — conhr , or — conr 2 ). amidation of the carboxy - terminus may render the inventive peptides less susceptible to protease degradation and increase their solubility compared to the free acid forms , therefore providing heightened therapeutic potency . the peptides may also be lipidated which may provide for enhanced skin penetration . peptide modifications may be made such that a hydrogen of the n - terminal amino group is replaced , a hydroxyl group ( oh ) of the c - terminal carboxylic group is replaced , the entire n - terminal amino group is replaced , or the entire c - terminal carboxylic group is replaced . one or more of the molecular bonds that link the amino acids of each peptide may be a non - peptide bond . such non - peptide bonds include , but are not limited to , imido , ester hydrazine , semicarbazoide and azo bonds . a variety of modifications can be made to the peptides as long as the characteristic proliferative , migratory and anti - inflammatory activities thereof are retained . some modifications may be used to increase the potency of the peptide , while other modifications may facilitate peptide handling . peptide functional groups that may typically be modified include hydroxyl , amino , guanidinium , carboxyl , amide , phenol , imidazol rings or sulfhydryl . typical , non - limiting reactions of these groups include the following : acetylation of hydroxyl groups by alkyl halides ; esterification , amidation or hydrogenization ( i . e . reduction to alcohol ) of carboxyl groups ; deamidation , acylation , alkylation , arylation of amino groups ( e . g . primary amino group of the peptide or the amino group of lysine residues ); halogenation or nitration of tyrosine phenol groups . peptides may be conjugated to soluble or insoluble carrier molecules to modify their solubility properties as needed and to increase the local concentrations of peptides in targeted tissues . examples of soluble carrier molecules include polymers of polyethyleneglycol ( peg ) and polyvinylpyrrolidone ; examples of insoluble polymers include silicates , polystyrene , and cellulose . peptides may also be micro - encapsulated to enhance their stability during and after therapeutic application ; typically , polyester and peg microspheres are used to encapsulate and stabilize the peptides . various methods of preparing microspheres for peptide encapsulation may be employed depending upon the hydrophilic or hydrophobic nature of the peptide composition to be encapsulated . examples of protocols for such methods are found in wang ht et al . ( 1991 , j . control . release 17 : 23 - 25 ) and u . s . pat . no . 4 , 324 , 683 , both of which are incorporated herein in their entirety . in vitro peptide release studies may be performed to determine the relative availability of the peptide after it has been incorporated into a microsphere . microspheres ( 200 mg ) are suspended in ph 7 . 2 phosphate - buffered saline ( pbs ) ( 2 . 5 ml ) and agitated at 37 degrees c and 100 rpm in an environmental incubator shaker ( g - 24 , new brunswick scientific co ., edison , n . j .). at specific sampling times ( each day for the first 4 days and every other day thereafter ) the buffer solution is completely removed and replaced with fresh pbs . the peptide content of the pbs is measured using the bradford method or other suitable quantitative assay typically used for protein analysis . all the disclosed peptides may be synthesized using standard fmoc ( 9 - fluorenylmethoxycarbonyl ) solid - phase chemistry on an advanced chemtech ® apex 396 ® multiple peptide synthesizer . the apex 396 ® is equipped with a 40 - well reaction block for the production of up to 40 peptides simultaneously at a scale of 0 . 15 mmol . the peptides can be prepared as either amidated or free acid sequences using standard amino acids . the resin was first washed and pre - swelled with n , n - dimethyl formamide ( dmf ). the swelling time was one hour for rink amide resins . the fmoc protecting group was removed with 25 % piperidine in dmf for 25 minutes , after which the piperidine was completely washed from the resin . to control racemization processes , the fmoc amino acid monomers were pre - activated in an equimolar solution of 1 - hydroxy - benzotriazole ( hobt ) or 1 - hydroxy - 7 - aza - benzotriazole ( hoat ) in dmf at a 0 . 5 m concentration . the amide couplings were carried out using o -( 7 - azabenzotriazol - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ( hatu ) pybop ® or 2 -( 1h - benzotriazol - 1 - yl -)- 1 , 1 , 3 , 3 - tetrameth - yluronium hexafluorophosphate ( hbtu ) as an activation agent and 2 . 5 - 5 . 0 fold molar excess of amino acid under basic conditions using a hindered base ( diisopropylethylamine ). the coupling times were 1 - 1 . 5 hours followed by a wash and re - coupling to accomplish a double or triple couple before deprotection and continuation of the growing peptide chain . coupling efficiency was monitored using the standard kaiser test . once the peptide synthesis was completed on the resin , the final fmoc group was removed as above and the sequences were left as the free base . cleavage of the acid - labile linkage of the peptide to the resin was accomplished using 95 % trifluoroacetic acid ( tfa ) and water with the appropriate scavengers added . after cleavage was allowed to proceed for about 30 minutes to one hour , the released peptides were immediately removed from the cleavage block and transferred to tubes for the removal of the tfa under reduced pressure . the peptides were then ready for purification and analysis via high performance liquid chromatography ( hplc ) using a reverse phase c18 column and mass spectrometry . primary sequence confirmation and preparative purification was accomplished using an lc / ms / ms system ( abi api2000 ). general to the above protocol , the peptides may be produced using any method known to those skilled in the art such as those disclosed in merrifield , r . b ., solid phase peptide synthesis i ., j . a m . c hem . s oc . 85 : 2149 - 2154 ( 1963 ); carpino , l . a . et al ., [( 9 - fluorenylmethyl ) oxy ] carbonyl ( fmoc ) amino acid chlorides : synthesis , characterization , and application to the rapid synthesis of short peptides , j . o rg . c hem . 37 : 51 : 3732 - 3734 ; merrifield , r . b . et al ., instrument for automated synthesis of peptides , a nal . c hem . 38 : 1905 - 1914 ( 1966 ); or kent , s . b . h . et al ., high yield chemical synthesis of biologically active peptides on an automated peptide synthesizer of novel design , in : p eptides 1984 ( ragnarsson u ., ed .) almqvist and wiksell int ., stockholm ( sweden ), pp . 185 - 188 , all of which are incorporated by reference herein in their entirety . preferably , the peptides will be produced by a machine capable of sequential addition of amino acids to a growing peptide chain . however , the peptides may also be manufactured using standard solution phase methodology , which can be amenable to large - scale production efforts . additional embodiments of the current invention are directed towards methods of using the above - described peptides , such as in formulations or as therapeutic agents . these methods may involve the use of a single peptide , or multiple peptides in combination . the peptides of the current invention may be used for treating wounds of the skin ( epidermis , dermis and hypodermis ) and associated mucosal tissues . as used herein , the term “ associated mucosal tissues ” relates to any tissue organized in a manner similar to the skin and contains epithelial cells . keratinocytes are a non - limiting example of such epithelial cells . examples of such tissues are oral , nasopharyngeal , aural and urogenital surfaces , as well as the palpebral conjunctiva of the eye . other examples of associated mucosal tissues include the entire lining ( i . e . lumen ) of the alimentary canal , including the esophagus , stomach , small intestine , large intestine ( colon ), and rectum . these latter examples can sustain wounds / lesions much like those that can affect the skin , and as such can be targeted with the present invention . examples of wounds / lesions / injuries that can affect these tissues and are amenable to treatment with the inventive peptides are abrasions , blisters , burns , lacerations , punctures , ulcers , bruises , rashes and scars . post - surgical tissue trauma can also be treated with the peptides . the inventive peptides may also be used to prevent or reverse the effects of aging on all of the abovementioned tissues . in a related manner , the peptides could be applied to tissue that has been damaged by exposure to various external agents such as sunlight . examples of debilitation related to aging and exposure are skin wrinkling , dryness , thinning , sagging and greater susceptibility to bruising . the invention can also be used as a cosmetic in these regards to render a more youthful appearance and texture , and to provide better function . other tissue problems that are effectively treated using the peptides of the present invention are related to allergy or autoimmunity . such maladies include dermatitis , psoriasis , scleroderma , pemphigus and inflammatory bowel disease . the compositions used to deliver the peptides in the above therapeutic method can be an aerosol , emulsion , liquid , lotion , cream , paste , ointment , powder , or foam , or other pharmaceutically acceptable formulation . furthermore , the peptides can be delivered using less involved formulations such as deionized / distilled water , pbs or standard medical saline solutions . generally , a pharmaceutically acceptable formulation would include any carrier suitable for use on human skin . such pharmaceutically acceptable carriers include ethanol , dimethyl sulfoxide , glycerol , silica , alumina , starch , and equivalent carriers and diluents . the formulation may optionally have cosmetic appeal , and / or contain other agents such as retinoids or other peptides that can act as adjuvants for the therapeutic action of the inventive peptides . antibiotics can also be added to the formulation in order to ward off infection , thereby permitting maximal healing processes to occur . the concentration of the peptide in the composition can be about 0 . 1 μg / ml to about 50 μg / ml or about 0 . 1 μg / ml to about 20 μg / ml ; however , the ultimate concentration employed may vary outside these ranges , depending on the nature of the wound / tissue condition , the bio - activity of the inventive peptide and the use of any adjuvant or technique to obtain enhanced composition absorption . the compositions of the present invention can contain one or more additional agents that exert skin care activity . in a preferred embodiment of the instant invention , where the composition is to be in contact with human keratinous tissue , any additional components besides the inventive peptides should be suitable for application to keratinous tissue ; that is , when incorporated into the composition , such other components demonstrate undue toxicity , incompatibility , instability , allergic response , and the like within the scope of sound medical judgment . the ctfa cosmetic ingredient handbook , second edition ( 1992 ) describes a wide variety of non - limiting cosmetic and pharmaceutical ingredients commonly used in the skin care industry , which are suitable for use in the compositions of the present invention . examples of these ingredient classes include : abrasives , absorbents , aesthetic components such as fragrances , pigments , colorings / colorants , essential oils , skin sensates , astringents , etc . ( e . g . clove oil , menthol , camphor , eucalyptus oil , eugenol , menthyl lactate , witch hazel distillate ), anti - acne agents , anti - caking agents , antifoaming agents , antimicrobial agents ( e . g ., iodopropyl butylcarbamate ), antioxidants , binders , biological additives , buffering agents , bulking agents , chelating agents , chemical additives , cosmetic biocides , denaturants , drug astringents , external analgesics , film formers or materials , opacifying agents , ph adjusters , propellants , reducing agents , sequestrants , skin bleaching and lightening agents ( e . g . hydroquinone , kojic acid , ascorbic acid , magnesium ascorbyl phosphate , ascorbyl glucosamine ), skin - conditioning agents ( e . g . humectants ), skin soothing and / or healing agents ( e . g . panthenol and its derivatives , aloe vera , pantothenic acid and its derivatives , allantoin , bisabolol , and dipotassium glycyrrhizinate ), skin treating agents , thickeners , and vitamins and derivatives thereof . the administration of the inventive peptides and associated compositions may be made to humans and animals , including all mammals . application may also be made in combination with typical and / or experimental materials such as tissue grafts , tissue culture products , oxygen and dressings . in general , the composition can be administered topically , orally , transdermally , systemically , or by any other method known to those of skill in the art to be useful to deliver the inventive peptides to the injury site . compositions may also be applied in an in vitro or ex vivo manner , either to cells or patient grafts growing in culture , for example . due to their small size , the peptides are expected to be able to gain by themselves some level of permeability through the skin ; however , certain techniques may be used to amplify this movement . for example , lipophilic ( non - polar ) groups can be added to the peptides , or the peptides can be delivered to the skin in a lipophilic excipient , in order to enhance peptide accessibility to the stratum corneum to allow translocation to the lower epidermal layers . in this manner such lipophilic modifications may be considered as a pro - drug . permeation enhancers such as known solvents and surfactants may be used in the excipient to allow better peptide absorption . special techniques that are anticipated to be useful in enhancing peptide access to the targeted tissue / injury include iontophoresis , electrophoresis and ultrasound . an iontophoretic device consists of two electrodes immersed in an electrolyte solution and placed on the skin . when an electric current is applied across the electrodes , an electric field is created across the stratum corneum that drives the delivery of the peptides . electroporation involves the application of high - voltage electric pulses to increase the permeation through lipid bilayers . this differs from iontophoresis in the duration and intensity of the application of electrical current ( iontophoresis uses a relatively constant low - voltage electric field ). the high - voltage electric pulse of electroporation is believed to induce a reversible formation of hydrophilic pores in the lipid lamellae membranes that can provide a high degree of permeation enhancement . ultrasound applies sound waves having a frequency greater than 16 khz to the skin , which causes compression and expansion of the tissue through which the sound waves travel . the resulting pressure variations cause a number of processes ( e . g ., cavitation , mixing , increase in temperature ) that may enhance permeation of the peptides . all the above peptide formulations and uses are well known in the art . additional modes of preparing and using the inventive peptides are described , for example , in u . s . pat . nos . 6 , 492 , 326 and 6 , 974 , 799 , both of which are incorporated herein by reference in their entirety . the following examples are included to demonstrate certain preferred embodiments of the invention . as the hb - 107 ( seq id no : 1 ) peptide fragment has previously been shown to stimulate wound healing in vivo ( lee et al ., 2004 ), it was hypothesized that within the sequence of hb - 107 there may exist yet smaller peptide fragments that can similarly or better stimulate wound healing and related processes . to examine this question , an overlapping set of peptide fragments of hb - 107 was generated using standard solid phase peptide chemistry . these peptides were then assayed for cell proliferation activity at concentrations of 0 . 22 , 2 . 15 , 21 . 5 and 46 . 4 μg / ml . a number of peptides caused a significant increase in the proliferation of epidermal keratinocytes at low concentrations ( table 2 ), including peptides hb - 1061 ( seq id no : 3 ) and hb - 1072 ( seq id no : 6 ) which , respectively , only contain seven and six amino acids each . several other peptides exhibited stimulatory activity at levels equal to or above that of hb - 107 ( table 2 ). in conclusion , the cell proliferation induced by a number of the inventive peptides exceeded the level induced by the parent hb - 107 peptide . importantly , several of these fragments are significantly shorter than hb - 107 . 3 . neutral red stock solution : neutral red powder is added to dulbecco &# 39 ; s phosphate buffered saline ( dpbs ) at a concentration of 3 mg / ml . the resulting solution is then sterile - filtered . 4 . neutral red media : neutral red stock solution is added to dmem or kbm at a final concentration of 50 μg / ml . 5 . mtt media : mtt ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ) powder is added to dmem or kbm at 1 mg / ml and filtered or centrifuged to remove any precipitate . mtt solution is used within 24 hours . 6 . fixing solution consisting of 1 % formaldehyde and 1 % calcium chloride in an aqueous solution . 8 . lysing solution : 1 . 0 % glacial acetic acid and 50 % ethanol in an aqueous solution for neutral red , or acidified isopropanol for mtt - treated cells . keratinocytes are examined microscopically daily . as the culture becomes 50 - 75 % confluent , the media in the plate is aspirated and 0 . 25 % trypsin / edta is added ( 0 . 05 % trypsin for human cells ). when the cells become rounded , the trypsin is neutralized by addition of an equal volume of dmem or kbm supplemented with approximately 10 % bovine serum or by using trypsin neutralizing solution ( tns , from clonetics ). cells are then centrifuged and the pellet is resuspended in 1 ml of dmem - 1 or kbm . a hemacytometer is used to count the cell suspension and the total number of cells / ml is adjusted to 1 . 5 - 2 . 5 × 10 4 cells / ml with dmem - 1 or kbm . cells are then plated in 96 - well plates at a concentration of 3 . 0 - 5 . 0 × 10 3 cells / well by adding 200 μl of the cell suspension to each well . typically , the central 60 wells are used and the outer wells are filled with dmem or pbs to minimize evaporation effects . a stock solution of the test article is prepared using dmem - 1 or kbm , and all other dilutions are henceforth prepared from this solution . typically , each dilution is filtered through an 0 . 2 - μm filter before application to the cell culture . a 1 . 0 % ( w / v or v / v ) solution or suspension is prepared , and 10 - fold or 3 - fold serial dilutions are made in culture medium . the cells are examined 18 - 24 hours after plating to ensure cell attachment and division , and then 100 μl of the media is aspirated , leaving behind 100 μl in each well . then 100 μl of a 2 × concentration of each test article dilution is added to the replicate wells . for the negative control , 100 μl of the vehicle media ( dmem - 1 or kbm ) is added to the control wells . the microplate is then incubated at 37 degrees c . and 5 . 0 % co 2 for 48 - 72 hours after dosing , and cells are allowed to proliferate undisturbed . following this 48 - 72 hour exposure , all media is aspirated and proliferation is assessed by one of the below methods . option a : neutral red uptake . after exposure and aspiration of media , 200 μl of neutral red media is immediately added to each well . the microplate is returned to the incubator for an additional three hours . following this dye uptake period , the microplate is removed from the incubator and gently inverted , and the neutral red media is decanted into a collection pan . the cells are then fixed with fixing solution for approximately one minute . the fixing solution is decanted off , and the microplate is washed gently three times with washing solution . the washing solution is decanted , and 200 μl of lysing solution is added . after at least 15 minutes , the contents of each well are re - pipetted to ensure even color distribution in each well , and an aliquot is read on the spectrophotometer as detailed below . option b : mtt conversion assay . after exposure and aspiration of media , 200 μl of mtt assay media is immediately added to each well . ( mit should be used only if the test article does not reduce mit directly , as determined by a range - finding experiment or mit compatibility test ). the microplate is returned to the incubator for an additional three hours . following this dye uptake period , the microplate is removed from the incubator and gently inverted , and the mtt media is decanted into a collection pan . the cells are then washed gently three times with washing solution . the washing solution is decanted , and 200 μl of lysing solution is added . after lysing for at least 60 minutes , each well is re - pipetted to ensure even color distribution in each well , and the absorbance is read on the spectrophotometer as detailed below . option c : flow cytometry - based analyses . after exposure , the medium is adjusted to 10 μm bromodeoxyuridine ( brdu ) and incubated at 37 degrees c . for 45 minutes . during this incubation , brdu , a thymidine analog , is incorporated into the dna of proliferating cells . the cells are then be harvested by trypsinization . following centrifugation , the cell pellet is resuspended in 0 . 5 ml of pbs and the cells are fixed by the addition of 70 % ethanol . the cells are then be washed and stained with the dna - specific ( red ) fluorescent dye propidium iodide . the proliferating cells are stained with a brdu - specific fluorescent ( green ) antibody . cell cycle analysis as well as the amount of proliferating cells staining positive for brdu is determined using flow cytometry . ( note : for flow cytometry studies , cells are cultured in 24 - well or 6 - cm plates ). dye uptake studies : for dye - based endpoints the optical density of the wells is read using a titertek multiskan ® mcc / 340 at a wavelength of 540 nm subtracting the absorbance at a reference wavelength of 620 nm for neutral red , or 670 nm to 680 nm reference wavelength for mit . a printout of the absorbance values is generated by the plate reader . the average absorbance and standard deviations are calculated for each treatment group , and the results are expressed as percent of control absorbencies . ec50 calculation : the data is plotted as percent of control absorbencies vs . concentrations for each test sample . the ec50 is extrapolated from the regression line drawn through the data in an excel 5 . 0 graph . in addition , the ec50 is calculated using the equation for the regression line provided by the excel 5 . 0 graph or by using an excel 5 . 0 macro . flow cytometry studies : for flow cytometric analysis the percent positive brdu - labeled will be determined and a proliferation index ( i . e ., % of cells actively proliferating at time of harvest ) will be calculated . optionally , other parameters such as percent cell viability and the percentage of apoptotic cells in the samples can also be determined . cell proliferation alone is not sufficient to aid in wound healing . upon sustaining an injury , cells bordering the wound proliferate ; the migration of such newly formed cells to close the injury ( or chronic lesion of older , dysfunctional tissue ) is of equal importance . to address this issue , hb - 107 and peptide fragments thereof were examined using a keratinocyte cell migration assay based on a simple tissue culture scratch test . this assay demonstrated that peptides such as hb - 1072 ( seq id no : 6 ) and hb - 1061 ( seq id no : 3 ) are capable of increasing the migration of cells in a manner similar to that of the parent hb - 107 peptide ( table 3 ). it is of interest to note that the hb - 1072 and hb - 1061 peptides overlap as part of hb - 107 , and that the analog hb - 1062 ( seq id no : 4 ) exhibits no migratory activity on cells . the protocol for this assay is standard and has been described by shanley et al . ( 2004 , invest . ophthalmol . vis . sci . 45 : 1088 - 1094 ), the article of which is herein incorporated by reference in its entirety . in addition to increasing the rate of healing , peptide hb - 107 has also been shown to decrease the level of inflammation associated with a wound . to identify small peptide fragments that may exhibit such an activity , fragments of hb - 107 were screened for the activity of reducing a key inflammatory cytokine , il6 . it was found that peptide fragments hb - 802 ( seq id no : 12 ) and hb - 1076 ( seq id no : 8 ) decrease the level of il6 expression to the same degree as the hb - 107 parent peptide ( table 4 ). the protocol for this assay is standard and described by murakami et al . ( 2004 , j . immunol . 172 : 3070 - 3037 ), the article of which is incorporated herein by reference in its entirety . given that certain small peptides were able to promote anti - inflammatory activity in resting cells ( see example 3 ), it was of interest to know whether small peptides could promote such activity in cells that were exposed to uvb radiation . uvb radiation damages skin and promotes aging thereof . since inflammation in damaged tissue is a contributor to aging in that tissue , reduction of epidermal inflammation resulting from ultraviolet light - induced damage will lessen the aging effects thereof . an assay was performed to determine whether peptide fragments of hb - 107 can induce anti - inflammatory activity in cells exposed to ultraviolet light . human skin epithelial cells ( atcc crl - 2592 ) were seeded into 6 - well plates and grown to more than 95 % confluence in dmem with 4 mm l - glutamine adjusted to contain 1 . 5 g / l sodium bicarbonate and 4 . 5 g / l glucose ( complete medium ) supplemented with 10 % fetal bovine serum . the cells were serum starved for 5 hours prior to uvb treatment . uvb was generated using a uvlms lamp ( 4w model , 3uv assembly , upland , calif .) with the irradiation wavelength set at 302 nm . the uv lamp was placed 12 cm above the tissue culture plate ( 6 - well plate ) and two wells were treated at one time to allow homogenous uvb irradiation . cells were exposed to uvb irradiation ( 450 μw / cm 2 , measured using a radiometer ) in pbs to avoid ultraviolet light - induced generation of toxic photochemicals . il6 expression was measured as an indicator of cell inflammatory activity in response to uvb . as shown in fig2 , il6 expression in skin epithelial cells was induced in a uvb dose - dependent manner , thus demonstrating that ultraviolet light stimulates cellular inflammatory processes in skin epithelial cells . individual peptides were screened to determine the effects thereof on uvb - induced il6 expression in skin epithelial cells . cells were exposed to uvb ( 450 μw / cm 2 ) for 35 seconds in pbs , after which the pbs was replaced with complete medium without serum in the presence or absence of 40 μg / ml peptide and incubated at 37 degrees c ./ 5 % co 2 for twenty hours . the supernatant media was then collected and centrifuged at 10 , 000 rpm to remove debris , prior to elisa for human il6 ( cellsciences , mass .). it was found that specific sequences within the hb - 107 peptide are capable of significantly reducing il6 expression in cells subjected to a potent inflammatory stimulus ( table 5 , results shown graphically in fig3 ). the results from this study show that peptides such as hb - 1410 ( pkek , seq id no : 15 ) are capable of significantly reducing the level of il6produced by skin epithelial cells in response to uv radiation . this activity is of particular application to skin care after sun exposure , and also has broad application toward reducing associated inflammation in a variety of skin conditions resulting from wounds and aging . it is interesting to note that even minor changes in the peptide sequence can significantly alter peptide inhibitory activity towards il6 expression in skin epithelial cells [ e . g . compare activities of hb - 801 ( pkekv ) and hb - 802 ( mpkek ) with the inhibitory activity of hb - 1410 ]. interestingly , hb - 802 has demonstrated the ability to reduce il6 in resting cells ( refer to example 3 , table 4 ). all of the compositions or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure . while the compositions and methods of this invention have been described in terms of preferred embodiments , it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept , spirit and scope of the invention . more specifically , it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved . all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention . all patents and publications identified in this application are hereby incorporated by reference in their entirety . braff m h , gallo r l ( 2006 ). antimicrobial peptides : an essential component of the skin defensive barrier . curr . top . microbiol . immunol . 306 : 91 - 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