Patent Application: US-201314400299-A

Abstract:
the present invention relates to composition for preventing or treating cachexia , and more specifically , to a composition for preventing or treating cachexia containing a peptide derived from a telomerase . the composition for preventing or treating cachexia , according to present invention , has the advantages of improving symptoms of cachexia , such as weight loss , anemia , edema , and loss of appetite , and has few side effects .

Description:
since the present invention can have adaptability for diverse transformation and examples of practical application , below is a more detailed description of the present invention . nevertheless , this is no means to limit the form of practical application ; it should be understood that the intention is to include the concept and the extent of technology in all of the transformation , equivalents to alternatives . in describing the present invention , if any detailed description about the prior art is considered to deteriorate the fundamental principles of the present invention , the description will be omitted . telomere is known as a repetitive sequence of genetic material found at the ends of chromosomes that prevent chromosomes from damage or merging onto other chromosomes . the length of the telomere is shortened at each cell division , and after a certain number of cell division , the telomere length is extremely shortened to the extent in which the cell stops dividing and dies . on the other hand , the elongation of telomeres is known to extend the life span of a cell . for an example , cancer cells excrete an enzyme called telomerase , which prevents shortening of telomeres , thus resulting in proliferation of cancer cells . a recent , tumor necrosis factor ( tumor necrosis factor ; tnf ), monocytokine and cytokine which derived from macrophage may cause of cachexia . as above mentioned that tnf is probably the most common cause of cachexia due to produce inflammatory related cytokines . therefore , it is directly observed in cancer patients and weight loss patients caused by severe infections . hence , scientific research has been studied the various mechanism of tnf . thus , it was reported that tumor necrosis factor ( tumor necrosis factor ; tnf ) called cachectin which is play a major role in cancer cachexia . the mechanism activity of cachectin is also same as cytokine such as interleukin ( il ), il - 6 , lif and ifn ( kr 2001 - 0012613 a ). in addition , j . walsmith et al ., 2002 reported that weight loss occurred is not caused by anorexia alone , chronic inflammation is also lead to weight loss . furthermore , another study found that weight loss occurred in adjuvant induced arthritis in mouse , as well as they seem to be loss of muscle mass and muscle fiber . this research showed the express of gene such as tnf - α ( tumor necrosis factor - α ) and il - 1β ( interleukin - 1β ) can be induced in the skeletal muscle . importantly , the expression of tnf - α and il - 1β in the skeletal muscle lead to a negative correlation with skeletal muscle fiber . hence , in the prior arts , showed that the prevent for loss of skeletal muscle mass more effectively in adjuvant induced arthritis in mouse by blocking both of tnf - α and il - 1 at the same time rather than only blocked tnf - α . the combination of tnf - α and il - 1β leads to muscle wasting . therefore , tnf - α not only affected to cachexia but , il - 1β can be presented evidence for occurs cachexia . cachexia occurs in chronic disease caused by loss of adipose tissues and the desire to eat , which is a unique disease with characteristics that lead to reduced body mass due to the loss of non - fat mass . anorexia nervosa causes the cachexia can be observed in cancer , chf , copd , ckd and the aging . this can be related with inflammatory factors such as tnf - α , il - 6 , il - 2 , and il - 1β . those inflammatory markers are able to regulate the feedback mechanism of hypothalamus , also it is contribute to the progress of cachexia . also , leptin is a hormone which acts as secreted by adipose tissue , reduced the food consumption by acting on the hypothalamus , stimulated energy consumption leads to weight loss . cachexia caused by ckd ( chronic kidney disease )- and chf ( congestive heart failure ) which indicated the high levels of leptin ( diana r . engineer et al ., 2012 ). in r . roubenoff et al ., 1997 , the chronic inflammatory represented in the rheumatoid arthritis ( ra ) which is related with loss of body cell mass . these symptoms are called as inflammatory cachexia which represented with hypermetabolism and degradation of protein in rheumatoid arthritis without malabsorption . in other words , according to the experiment , it is presumed that adjuvant arthritis leads to weight loss and joint swell . also , this study found that reduced cell mass leads to weight loss and was higher than the rate of weight loss . as a result , weight loss caused by inflammatory which directly closed to the reduced consumption of food intake and increased the production of tnf - α and il - 1 in splenocytes ( r . roubenoff et al ., 1997 ). also , the research of michael j . tisdale , 2009 found that muscle atrophy which represented in cachexia , its blocked by il - 6 receptor antibody in overexpression interleukin - 6 in il - 6 transgenic mice . according to this study , severe symptoms of cachexia and polyps are represented in mouse , which exhibited high amounts of il - 6 but , apcmin +/ il6 −/− mouse is not exhibiting any loss and low amounts of polyps . also , overexpression interleukin - 6 in transgenic mice showed the loss of skeletal muscle and polyps formed , but conversely , cannot find any loss of skeletal muscle in mouse without tumors . also , this research provided that muscle protein degradation by increased il - 6 which leads to activated the decomposed pathway of non - lisosome ( for example , proteasome ) protein and lisosome ( cathepsin ) protein ( michael j . tisdale , 2009 ). cachexia is a weakness syndrome which can be referred to various disease such as aids , cancer , after hip fracture , chronic heart failure , cold ( chronic obstructive pulmonary disease ) and copd ( chronic obstructive pulmonary disease ) comprising chronic obstructive pulmonary disease , liver cirrhosis , renal failure , rheumatoid arthritis comprising autoimmune disease , systemic lupus , sepsis , tuberculosis , cystic fibrosis , crohn &# 39 ; s disease and severe infections . as well as weakness observed in aging . in an exemplary embodiment of the present disclosure , a peptide of seq id no 1 , a peptide which is a fragment of the peptide of seq id no 1 or a peptide having 80 % or more sequence identity with the peptides includes a peptide derived from telomerase , specifically human ( homo sapiens ) telomerase . the peptides disclosed herein may include peptides comprising an amino acid sequence at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % of sequence homology with the peptide of seq id no 1 or a fragment thereof . moreover , the peptides disclosed in the present invention may include peptides having differences from seq id no : 1 or a fragment thereof in at least one amino acids , at least 2 amino acids , at least 3 amino acids , at least 4 amino acids , at least 5 transformed amino acids , at least 6 transformed amino acids , or at least 7 amino acids . in one embodiment of the present invention , changes in amino acids include modifications of peptide &# 39 ; s physical and chemical characteristics . for example , amino acid modification can be performed for improving thermal stability of the peptide , altering substrate specificity , and changing the optimal ph . the term “ amino acid ” herein includes not only the 22 standard amino acids that are naturally integrated into a peptide but also the d - isomers and modified amino acids . therefore , in a specific embodiment of the present invention , a peptide herein includes a peptide having d - amino acids . on the other hand , a peptide may include non - standard amino acids such as those that have been post - translationally modified . examples of post - translational modification include phosphorylation , glycosylation , acylation ( including acetylation , myristorylation , plamitoylation ), alkylation , carboxylation , hydroxylation , glycation , biotinylation , ubiquitinylation , modification in chemical properties ( e . g . β - removing deimidation , deamidation ) and structural modification ( e . g . formation of disulfide bridge ). also , changes of amino acids include the changes of amino acids that occur due to chemical reaction during the combination process with cross - linkers for formation of a peptide conjugate , such as changes in an amino group , carboxyl group or side chain . a peptide disclosed herein may be a wild - type peptide that has been identified and isolated from natural sources . on the other hand , when compared to seq id no : 1 or its fragments , the peptides disclosed herein may be artificial variants that comprise one or more amino acids substituted , deleted and / or inserted . amino acid alteration in wild - type polypeptides — not only in artificial variants — comprises protein folding and / or conservative substitutions of amino acids that do not influence activities significantly . examples of conservative substitutions may be within the groups of basic amino acids ( arginine , lysine and histidine ), acidic amino acids ( glutamic acid and aspartic acid ), polar amino acids ( glutamine and asparagines ), hydrophobic amino acids ( leucine , isoleucine , valine and methionine ), aromatic amino acids ( phenylalanine , tryptophan and tyrosine ), and small amino acids ( glycine , alanine , serine , and threonine ). the amino acid substitutions that do not generally alter the specific activities are known in the art . most common occurring alterations are ala / ser , val / ile , asp / glu , thr / ser , ala / gly , ala / thr , ser / asn , ala / val , ser / gly , tyr / phe , ala / pro , lys / arg , asp / asn , leu / ile , leu / val , ala / glu , asp / gly , and the opposite alterations thereof . other examples of conservative substitutions are shown in the following table 1 . the substantial modification of the biological properties of peptides are performed by selecting significantly different substitution in the following efficacies : ( a ) the efficacy in maintaining the structure of the polypeptide backbone in the area of substitution , such as sheet or helical three - dimensional structures , ( b ) the efficacy in maintaining electrical charge or hydrophobicity of the molecule in a target area , or ( c ) the efficacy of maintaining the bulk of the side chain . natural residues are divided into groups by general side chain properties as the following : non - conservative substitutions may be performed by exchanging a member of the above classes with that of different classes . any cysteine residues that are not related to maintaining the proper three - dimensional structure of the peptide can typically be substituted with serine , thus increasing the oxidative stability of the molecule and preventing improper cross - linkage . conversely , improvement of stability can be achieved by adding cysteine bond ( s ) to the peptide . another type of amino acid variants of peptides are those having a changed pattern of peptide glycosylation . the term “ change ” herein means deletion of at least one carbohydrate residues that are found in a peptide and / or addition of at least one glycosylated residues that do not exist within a peptide . glycosylation in peptides are typically n - linked or o - linked . the term “ n - linked ” herein refers to that carbohydrate residues are attached to the side chain of asparagine residues . as tripeptide sequences , asparagine - x - serine and asparagine - x - threonine ( wherein the x is any amino acid except proline ) are a recognition sequence for attaching a carbohydrate residue enzymatically to the side chain of asparagine . therefore , with the presence of one of these tripeptide sequences in a polypeptide , the potential glycosylation sites are created . “ o - linked glycosylation ” means attaching one of sugar n - acetylgalactosamine , galactose , or xylose to hydroxyl amino acids . the hydroxyl amino acids are most typically serine or threonine , but 5 - hydroxyproline or 5 - hydroxylysine can be used . addition of glycosylation site to a peptide is conveniently performed by changing an amino acid sequence to contain the tripeptide sequence mentioned above ( for n - linked glycosylation sites ). these changes may be made by addition of at least one serine or threonine residues to the first peptide sequence , or by substitution with those residues ( for o - linked glycosylation sites ). the seq id no : 1 ( hereinafter ‘ pep 1 ’) as used herein is a telomerase - derived peptide comprised of 16 amino acids . also , in one aspect , the present invention is a peptide comprising amino acid sequence of seq id no : 1 , a peptide having above 80 % homology of amino acid sequence with above - mentioned sequence , or a fragment of the above - mentioned peptides has an advantage in that it has high feasibility due to its low toxicity within a cell . in one aspect , the present invention is a composition for preventing or treating cachexia comprising , as an active ingredient , a peptide comprising amino acid sequence of seq id no : 1 , a peptide having above 80 % homology with above - mentioned sequence , or a fragment of the above - mentioned peptides . in one aspect , the present invention is a method for preventing or treating cachexia comprising administration of a peptide comprising amino acid sequence of seq id no : 1 , a peptide having above 80 % homology with above - mentioned sequence , or a fragment of the above - mentioned peptides to a subject in need thereof . in one aspect , the present invention is a use of a peptide for preventing or treating cachexia comprising administration of a peptide comprising amino acid sequence of seq id no : 1 , a peptide having above 80 % homology with above - mentioned sequence , or a fragment of the above - mentioned peptides to a subject in need thereof . in one aspect , the present invention is a kit comprising a peptide comprising an amino acid sequence of seq id no : 1 , a peptide including an amino acid sequence having a sequence identity of 80 % or greater to the amino acid sequence , or a peptide fragment of the above - mentioned peptides ; and instructions at least one of administration dose , administration route , administration frequency , and indication of the peptide or composition . in one aspect , the fragment may consist of at least 3 amino acids . in other aspects , the fragment may consist of at least 4 amino acids , at least 5 amino acids , at least 6 amino acids , at least 7 amino acids , at least 8 amino acids , at least 9 amino acids , at least 10 amino acids , at least 11 amino acids , at least 12 amino acids , at least 13 amino acids , at least 14 amino acids , or at least 15 amino acids . in one aspect , the peptide may be derived from human telomerase . specifically , the peptide of seq id no : 1 means the peptide position in 611 - 626 of an entire human telomerase sequence ( 1132 amino acids , seq id no : 2 ). in one aspect , the peptide may be used for eliminating symptoms related to carchexia , or preventing or treating cachexia . in one aspect , the peptide may be administered in a single dose of from 0 . 001 to 1 ng / kg or from 0 . 01 to 0 . 4 ng / kg . in other aspect , the dose of administering may be at least 0 . 001 ng / kg , at least 0 . 005 ng / kg , at least 0 . 01 ng / kg , at least 0 . 02 ng / kg or at least 0 . 03 ng / kg . in other aspect , the dose of administering may be less than 1 ng / kg , less than 0 . 9 ng / kg , less than 0 . 8 ng / kg , less than 0 . 7 ng / kg , less than 0 . 6 ng / kg , less than 0 . 5 ng / kg , less than 0 . 4 ng / kg , less than 0 . 3 ng / kg , less than 0 . 2 ng / kg . in one aspect , the peptide may be administered in once at 1 - 5 days or in once at 1 . 5 - 2 . 5 days . in one aspect , the composition may contain a peptide of 0 . 05 - 5 nm concentrations . according to an embodiment of the present invention , the composition may contain 0 . 1 μg / mg to 1 mg / mg , specifically 1 μg / mg to 0 . 5 mg / mg , more specifically 10 μg / mg to 0 . 1 mg / mg of a peptide comprising an amino acid sequence of seq id no : 1 , a peptide comprising an amino acid sequence having a sequence identity of 80 % or greater to the amino acid sequence , or a peptide fragment thereof . when the peptide is contained in the above - mentioned range , all the safety and stability of the composition may be satisfied and cost - effectiveness may be achieved . according to an embodiment of the present invention , the composition may have applications to all animals including humans , dogs , chickens , pigs , cows , sheep , guinea pigs , and monkeys . according to an embodiment of the present invention , a pharmaceutical composition may be administered through oral , rectal , transdermal , intravenous , intramuscular , intraperitoneal , intramedullary , epidural , or subcutaneous means . forms of oral administration may be , but not limited to , tablets , pills , soft or hard capsules , granules , powders , solutions , or emulsions . forms of non - oral administration may be , but not limited to , injections , drips , lotions , ointments , gels , creams , suspensions , emulsions , suppositories , patches , or sprays . according to an embodiment of the present invention , the pharmaceutical composition , if necessary , may contain additives , such as diluents , excipients , lubricants , binders , disintegrants , buffers , dispersants , surfactants , coloring agents , aromatics , or sweeteners . according to an embodiment of the present invention , the pharmaceutical composition may be manufactured by conventional methods of the industry in the art . according to an embodiment of the present invention , the active ingredient of the pharmaceutical composition may vary according to the patient &# 39 ; s age , sex , weight , pathology state and severity , administration route , or prescriber &# 39 ; s judgment . dosage may be determined by one of ordinary skill in the art based on the above - mentioned factors , and the daily dose may be , but is not limited to , about 0 . 0000001 ng / kg / day to about 10000 ng / kg / day or about 0 . 00001 ng / kg / day to about 100 ng / kg / day , specifically about 0 . 0001 ng / kg / day to about 10 ng / kg / day , and more specifically about 0 . 01 ng / kg / day to about 0 . 4 ng / kg / day . according to an embodiment of the present invention , the pharmaceutical composition may be administered , but is not limited to , 1 to 3 times per 1 to 5 days . the terms used herein is intended to be used to describe the embodiments , not to limit the present invention . terms without numbers in front are not to limit the quantity but to show that there may be more than one thing of the term used . the terms “ comprising ”, “ having ”, “ including ” and “ containing ” shall be interpreted openly ( i . e . “ including but not limited to ”). mention of a numerical range is used instead of stating separate numbers within the range , so unless it is explicitly stated , the range should be construed as if all the numbers within the range are separately described herein . the end values of all the ranges are included in the range and can be combined independently . unless otherwise noted or clearly contradicting in context , all methods mentioned herein can be performed in a proper order . the use of any one embodiment and all embodiment , or exemplary language ( e . g ., “ such as ”, “ like ˜”), unless included in the claims , is used to more clearly describe the present invention , not to limit the scope of the present invention . any language herein outside of the claims should not be interpreted as a necessity of the present invention . unless defined otherwise , technical and scientific terms used herein have meanings ordinarily understood by a person skilled in the art that the present invention belongs to . the preferred embodiments of the present invention include the best mode known to the inventors to perform the present invention . variations in the preferred embodiments can become clear to those skilled in the art after reading the statements above . the present inventors hope that those skilled in the art can use the variations adequately and present invention be conducted in other ways than listed herein . thus , the present invention , as allowed by the patent law , includes equivalents , modifications and variations thereof , of the key points of the invention stated in the appended claims . in addition , all possible variations within any combination of the above - mentioned components are included in the present invention , unless explicitly stated otherwise or contradicting in context . although the present invention is described and shown by exemplary embodiments , those skilled in the art will understand well that there can be various changes in the form and details without departing from the spirit of the invention and range , defined by the claims below . hereinafter , the present disclosure will be described in detail through examples and test examples . however , the following examples and test examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples and test examples . the peptide of seq id no : 1 was synthesized according to the conventionally known method of solid phase peptide synthesis . more specifically , the peptide was synthesized by coupling each amino acid from c - terminus through fmoc solid phase peptide synthesis , spps , using asp48s ( peptron , inc ., daejeon republic of korea ). those peptides with their first amino acid at the c - terminus being attached to a resin were used as follows : all the amino acids to synthesize the peptide were protected by fmoc at the n - terminus , and the amino acid residues were protected by trt , boc , t - bu ( t - butylester ), pbf ( 2 , 2 , 4 , 6 , 7 - pentamethyl dihydro - benzofuran - 5 - sulfonyl ) that can be dissolved in an acid . examples include the followings : fmoc - ala - oh , fmoc - arg ( pbf )- oh , fmoc - glu ( otbu )- oh , fmoc - pro - oh , fmoc - leu - oh , fmoc - ile - oh , fmoc - phe - oh , fmoc - ser ( tbu )- oh , fmoc - thr ( tbu )- oh , fmoc - lys ( boc )- oh , fmoc - gln ( trt )- oh , fmoc - trp ( boc )- oh , fmoc - met - oh , fmoc - asn ( trt )- oh , fmoc - tyr ( tbu )- oh , fmoc - ahx - oh , trt - mercaptoacetic acid . hbtu [ 2 -( 1h - benzotriazole - 1 - yl )- 1 , 1 , 3 , 3 - tetamethylaminium hexafluorophosphate ]/ hobt [ n - hydroxybenzotriazole ]/ nmm [ 4 - methylmorpholine ] were used as the coupling reagents . piperidine in 20 % dmf was used to remove fmoc . in order to remove the protection from residues or to separate the synthesized peptides from resin , cleavage cocktail [ tfa ( trifluoroacetic acid )/ tis ( triisopropylsilane )/ edt ( ethanedithiol )/ h 2 o = 92 . 5 / 2 . 5 / 2 . 5 / 2 . 5 ] was used . the peptide synthesis was performed by using solid phase scaffold with the repetition of the following processes : starting with the amino acid protection , separate reaction of each amino acid , washing with solvents , and deprotection . each peptide was synthesized by using the solid phase scaffold combined to starting amino acid with the amino acid protection , reacting the corresponding amino acids separately , washing with a solvent and deprotected , and repeating the processes . upon the release from the resin , the synthesized peptides were purified by hplc , validated by mass spectrometry , and freeze - dried , and verified for synthesis by ms , and then freeze - dried . specific peptide synthesis process is described as the following based on the synthesis process of pep 1 which has seq id : no . 1 . the amino acid ( 8 equivalent ) protected with nh 2 - lys ( boc )- 2 - chloro - trityl resin , and coupling agent hbtu ( 8 equivalent )/ hobt ( 8 equivalent . )/ nmm ( 16 equivalent ) melted in dmf were mixed together , and incubated at room temperature ( rt ) for 2 hr . following the incubation , the reaction mixture was subjected to the sequential washes of dmf , meoh , and dmf . piperidine in 20 % dmf was added and incubated at rt for 5 minutes 2 times , then sequentially washed with dmf , meoh , and dmf . 3 ) making the basic framework of peptide , nh 2 - e ( otbu )- a - r ( pbf )- p - a - l - l - t ( tbu )- s ( tbu )- r ( pbf ) l - r ( pbf )- f - i - p - k ( boc )- 2 - chloro - trityl resin ) by repeating the above - mentioned reactions 1 ) and 2 ). 4 ) cleavage : cleavage cocktail was added to the completely synthesized peptide , thus separating the synthesized peptide from the resin . 5 ) pre - chilled diethyl ether was added into the obtained mixture , and then centrifugation was used to precipitate gathered peptide . 6 ) after purification by prep - hplc , the molecular weight was confirmed by lc / ms and lyophilized to produce in a powder form . pep 1 prepared by the method described in example 1 was used to perform an experiment of effectiveness to preventing or treating rheumatoid arthritis . investigation of the inhibitory effect of pep1 on tnf - α level in hepg2 cells pbmc ( peripheral blood mononuclear cell ) was separated from the blood samples ( 50 ml ) collected from healthy subjects using ficoll - paque ™ plus ( ge healthcare life sciences , piscataway , n . j ., usa ). pbmcs were then enriched in complete rpmi 1640 medium containing 20 % of human serum , followed by transferring to 100 mm polystyrene cell culture plate coated with human serum for 30 mins . after 2 hr incubation at 37 ° c . and 5 % co 2 , the monocytes were detached from the bottom of cell culture plate using cold pbs ( phosphate buffered saline ) ( gibco / life technologies , carlsbad , calif ., usa ), and 1 × 10 5 cells were cultured in each well of 96 - well plate in rpmi 1640 medium ( supplemented with penicillin - streptomycin ; 100 mg / ml , human serum ; 20 %) overnight . for luciferase analysis , hek293 / null ( human embryonic kidney ) cells and hek293 / trl stably expressing tlr2 ( toll - like receptor2 ) obtained from seoul national university school of dental medicine were used . one day before the luciferase experiment , 2 . 5 × 10 5 cells were seeded into each well of 12 - well plate and cultured overnight in dmem ( dulbecco &# 39 ; s modified eagle &# 39 ; s medium ) medium ( supplemented with blasticidin ; 10 μg / ml , fetal bovine serum ; 10 %)( invitrogen / life technologies , carlsbad , calif ., usa ) to investigate the effect of pep - 1 on tnf - α level in terms of protein expression level , elisa ( enzyme linked immunosorbent assay ) was performed . 1 × 10 5 pbmc - derived monocytes were cultured in 96 - well plate overnight . after then , lps ( lipopolysaccharide ; 10 ng / ml , sigma ) was treated for 2 hours , followed by 3 times washes with pbs . opti - mem medium ( invitrogen / life technologies , carlsbad , calif ., usa ) was then treated for an hour to induce the starvation , and 4 um of fitc ( fluorescein isothiocyanate ), fitc - tat , pep - 1 - fitc , and fitc - pep - 1 were treated for 2 hours before measuring the tnf - α level . after culturing , cell soup was collected , and the amount of tnf - α was measured using elisa kit ( r & amp ; d , minneapolis , minn ., usa ) as follows . tnf measurement uses sandwich elisa method . 100 μl of tnf - α primary antibody was added into each well of pre - coated 96 - well plate , and the plate was incubated at 4 ° c . overnight . on next day , the plate was washed 3 times with 0 . 5 % tween20 wash solution for 5 min each , and then 100 μl of each sample and standard solution was added and left at room temperature for 2 hrs . after washing the plate like above , 100 μl of hrp - conjugated secondary antibody was added into each well and left at room temperature for 2 hrs . again , plate was washed , and avidin / biotin was added for measuring the absorbance . tnf - α level of each sample was quantified using the standard graph calculated from the absorbance of standard solution . pbmc - derived monocytes were stimulated with endotoxin lps ( 10 ng / ml ) for 2 hrs , starved for 1 hr using opti - mem , and then 4 um of fitc , fitc - tat , pep 1 - fitc and fitc - pep 1 were treated for 2 hrs . after incubation , tnf - α level was measured with cell culture medium using elisa . as a result , in case of fitc and fitc - tat , tnf - α level increased due to lps ( 6 . 2 and 6 . 7 ng / ml , respectively ), but tnf - α level significantly decreased in case of pep - 1 - fitc and fitc - pep - 1 ( 0 . 17 and 0 . 25 ng / ml , respectively ) and the difference was statistically significant ( p & lt ; 0 . 01 ) ( fig1 ). analysis of peptides effect on cachexia marker leptin , il - β and il - 6 in thp1 cell line thp - 1 cell culture was performed to investigate the effects of pep 1 on cachexia markers . thp - 1 ( human acute monocytic leukemia derived cell line ) cell was purchased from atcc ( american type culture collection , manassas , va ., usa ), cell was maintained in rpmi - 1640 ( life technologies , carlsbad , calif ., usa ) supplemented with 10 % fbs ( life technologies ), 1 % penicillin / streptomycin and 2 - mercaptoethanol ( sigma - aldrich , st . louis , mo ., usa ), and cells were cultured in a 37 ° c ., 5 % co 2 humidified incubator . in general , thp - 1 cells were grown under suspension condition , and it was differentiated into adherent macrophages by treating cells with phorbol myristate acetate ( pma ) for 24 hr . thp - 1 cells ( 3 × 10 6 cells / plate , 95 ˜ confluency ) were seeded into well plate for incubator . after differentiation of thp - 1 cells , macrophage - like thp - 1 cells were washed two times using complete rpmi 1640 ( 5 min / wash ). then , cells were treated with 10 ng / ml lps and / or 20 μm pep 1 at 37 ° c . for 4 hr . total rna samples were isolated from peptide - treated thp - 1 cells by using trizol ( qiazol ) reagent and , and cdna was synthesized by reverse transcriptase pcr using reverse transcription pcr kit from promega following manufacturer &# 39 ; s protocol . then , cdna samples were used as a template for synthesized by real time qpcr . real time pcr primers for leptin , il - 1β and il - 6 shown in table 2 . real time qpcr was performed using cfx96 ( bio - rad ) instrument with sybr green system . the pcr cycling conditions were 95 ° c . for 10 min for activation of hotstart dna taq polymerase , followed by 50 cycles of 95 ° c . for 10 sec , 55 ° c . for 30 sec , and 72 ° c . for 30 sec . all samples were measured in triplicate and differences in gene expression were calculated using the 2 - cycle threshold method . all the data were normalized against β actin ( housekeeping gene ) and presented as means of +/− s . e . from at least three independent experiments . the result of the above experiment , thp - 1 cells are involved in the inflammatory responses induced by lps then the levels of leptin , il - 1β and il - 6 were decreased in thp - 1 cells after being treated with pep 1 ( see fig2 to fig4 ). in order to find effectiveness of the peptide according to the present invention to rheumatoid arthritis ( ra ), cia ( collagen induced arthritis ) mouse were used to confirmation . non - patent document disclosed in the present invention describes about cia animal model in detail . in reference to this , the present embodiment established the cia animal model as follows . in the first and second experiment mentioned below , lyophilized and formed in powder pep1 according to the example 1 was dissolved in 0 . 9 % saline solution and was used . after doing amendment of the purity ( purity : 97 . 3 %, content : 85 . 3 %) of pep1 , the solution for injection was made in each concentration with 0 . 9 % saline solution as an additive just before an administration . every dose was administered by the solution in an amount of 100 μl . the first induction was done to 38 mice at day 1 by using 5 - weeks male dba / 1j mouse ( orient bio inc ., korea ), and the mice were divided into the preventive group consisting of 16 mice administered by a peptide composition before cia inducement ( i . e . 8 mice of 1 nm , 100 μl ( around 0 . 2 ng dose ) and 8 mice of 10 nm , 100 μl ( around 2 ng dose )), the therapeutic group consisting of 16 mice administered by a peptide composition after cia inducement ( i . e . 8 mice of 1 nm , 100 μl and 8 mice of 10 nm , 100 μl ), and the pbs treatment group consisting of 6 mice . to the preventive group , the treatment was done by intradermal injection in each suitable concentration at day 2 , 4 , 6 , 21 , 23 , 25 , 35 , 37 and 39 . at day 19 , the second inducement was done to 38 mice , and to the therapeutic group the treatment by intradermal injection in each suitable concentration at day 21 , 23 , 25 , 35 , 37 and 30 from day 21 ( see fig1 ). the assessment of rheumatoid arthritis index was done at from the day of second inducement to the day 42 per every two days and the joint and serum was collected after sacrificing all mice at day 42 . like the first experiment , the first inducement was done to 38 mice at day 1 by using 5 - week mice , the mice were divided into the group consisting of 32 mice administered by a peptide composition before cia inducement ( 8 mice of 0 . 2 nm , 100 μl ( i . e . around 0 . 04 ng ); 8 mice of 1 nm , 100 μl ( around 0 . 2 ng dose ); 8 mice of 2 nm , 100 μl ( i . e . around 0 . 4 ng ); 8 mice of 5 nm , 100 μl ( i . e . around 1 ng )) and the pbs treatment group consisting of 6 mice . the second inducement was done at day 21 , and the treatment was done by intradermal injection in each suitable concentration to each group at day 23 , 25 and 27 ( see fig2 ). the assessment of rheumatoid arthritis index was done at from the day of second inducement to the day 42 per every two days and the joint and serum was collected after sacrificing all mice at day 42 . the preventive group ( pre - 1 nm and pre - 10 nm ) showed the decreasing arthritis effect until day 36 and after then the decreasing effect has gone . the therapeutic group ( thera - 1 nm and thera - 10 nm ) showed the decreasing arthritis effect in whole period but the 10 nm group showed the more effect than the 1 nm group . also , the preventive group and the therapeutic group showed increased body weights compared to the cia control group ( see fig5 to fig7 ). when the peptide treatment group of each concentration compared with the cia control group , the peptide treatment group appeared to decrease the arthritis index in whole period . also , when the body weights were measured , the increased body weight was confirmed at the low - level peptide treatment group compared to cia control group ( see fig9 ). as summarizing the result of the first and second experiments mentioned above , the administration of 1 nm peptide was more effective than the 1 nm peptide having lower level , and the therapeutic group treated after second inducement was more effective to inhibit rheumatoid arthritis than the preventive group . also , as a result of measuring the change of the body weight in each group , decrease of body weight in the therapeutic group of the 1 nm peptide treatment is lower than any other group and this is also the effect of inhibiting arthritis . hela cell line was purchased from atcc . the hela cell line was maintained in mem supplemented with 10 % fetal bovine serum ( invitrogen , usa ), earle &# 39 ; s salts , non - essential amino acids , sodium pyruvate , 100 ug / ml penicillin and 100 units / ml streptomycin , and then incubated at 37 ° c ., 5 % co 2 . the cells were seeded into 96 - well plates and added to each well for medium supplemented with 10 % fetal bovine serum ( invitrogen , usa ), 100 ug / ml penicillin and 100 units / ml streptomycin . the cells were cultured in 37 ° c ., 5 % co 2 for 12 h incubator . after incubated , plates washed by pbs , and added mem ( minimum essential medium ) for starvation during 1 h . the 20 um of pep 1 with 100 μl of the aqueous solution were added to each well , and then the cells were incubated at 37 ° c . for 24 . after incubated , the cell viability and toxicity were evaluated using an mtt assay . the result are shown in fig1 . choi , sang gyu , “ recent advances in cancer cachexia ”, j . korean oncol . nurs ., 1 , 20 - 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