Patent Abstract:
diastereomeric peptides with a net positive charge greater than + 1 , and cyclic derivatives thereof , are provided , having at least 13 amino acid residues , comprising histidine and one or more hydrophobic amino acid residues , optionally esterified or amidated at the c - terminus and / or acylated at the n - terminus . the peptides may contain other amino acid residues including non - natural amino acids . the peptides are particularly useful in the treatment of cancer .

Detailed Description:
the diastereomeric peptides of at least 13 amino acid residues of the present invention are characterized by comprising one or more histidine residues , thus differing from the 15 - mer peptides disclosed in wo 02 / 040529 . they have not been disclosed in the above - mentioned wo 98 / 37090 and wo 02 / 040529 , and exhibit an enhanced or similar activity for the treatment of cancer in comparison to the closest diastereomer ( peptide 4 ) disclosed in wo 02 / 040529 . the diastereomeric peptides of the invention are cytolytic agents of very low toxicity as evaluated herein in animal models . in the acute toxicity tests performed in mice , no mortality was observed with the peptides of the invention administered at concentrations considerably higher than those necessary for their anticancer activity , whereas 100 % mortality was observed for the 15 - mer peptide 4 of wo 02 / 040529 , herein designated peptide 1 ( seq id no : 1 ), administered at these high concentrations . in order to reduce the toxicity and to improve the cytolytic activity of the diastereomeric peptide , the effect of several important parameters such as length , amphipathic organization , the variety of positively charged amino acids , the location and number of d - amino acids , additional amino acid residues at the n - and c - termini , and / or addition of hydrophobic chains to the n - and / or c - terminus , and polarity of the diastereomeric peptides , on their potency , selectivity and spectrum of activity were examined . for this purpose , we synthesized and structurally and functionally characterized a series of linear and cyclic diastereomers , basically comprised of various ratios of leucine and histidine and optionally containing lysine and arginine residues and additional amino acid residues , preferably at the n - terminus and / or c - terminus . the peptides were then characterized with regard to their biological activity towards pathogenic cancerous cells and normal mammalian cells such as nih - 3t3 normal fibroblasts cell line , and their toxicity was tested in vivo . the potency and selectivity of the novel diastereomers of the invention is demonstrated herein in the anticancer assays . the diastereomers disclosed herein exhibit similar activity as that of peptide 1 disclosed in wo 02 / 040529 against several malignant cells and are more active against other cells . furthermore , they are active against the malignant cells at concentrations that are 2 - 8 lower than the concentrations at which they act against nih - 3t3 normal fibroblasts cells . in addition , they are significantly less toxic to mice in comparison with peptide 1 of wo 02 / 040529 . thus , the new diastereomeric peptides of the invention are useful as anticancer agents and can be used for treatment of solid tumors such as , but not limited to , breast , prostate , lung , kidney , and colon cancer as well as melanoma and basal and squamous cell carcinomas and non - solid tumors such as leukemias . the observed high potency of the positively charged diastereomeric peptides against a variety of malignant cells as shown in the examples herein indicates the existence of a common target for their action . this target is most probably the malignant cell membrane that has been shown to express higher levels of negatively charged phosphatidylserine than normal mammalian cells ( utsugi et al ., 1991 ). in one preferred embodiment , the present invention provides pharmaceutical compositions comprising a diastereomeric peptide of the invention for the treatment of cancer . it is contemplated that all peptides of the invention are useful for the treatment of malignant tumors as shown herein for peptides of seq id nos : 5 , 8 , 12 - 16 , 36 - 39 , 42 and 44 , designated in the examples herein as peptides 5 , 8 , 12 - 16 , 36 - 39 42 and 44 , respectively . in particular , peptides 12 , 13 and 37 - 39 , 42 and 44 were shown in experiments in vitro to be effective against prostate tumor . the high potency and the low in - vivo toxicity of the model diastereomers of the invention pave the way for their use also in topical applications against a wide variety of pathogenic organisms in topical infections including , but not limited to , treatment of acne , fungal infections of the scalp , fungal or bacterial infections related to surgical or traumatic wounds , chronic or poorly healing skin lesions ( especially in diabetics ), vaginal infection ( vaginitis ), eye and ear infections and burn wounds , infections of mouth and throat , and localized infections such as chronic pulmonary infections in cystic fibrosis , emphysema or asthma that can be treated with aerosol or other formulation for nasal or pulmonary application . the observed resistance of the diastereomers to proteolytic digestion may enable them to reach the digestive system in intact form and to eliminate there bacterial infections such as chronic gastric mucosal infestation by helicobacter pylori and intestinal bacterial infections . as used herein , the term “ topical ” means “ pertaining to a particular surface area ” and the topical agent applied to a certain area of said surface will affect only the area to which it is applied . therefore , any and all applications in which the peptides act locally and not through the blood circulation are encompassed by the present invention . for systemic administration , the peptide may be administered as such without any additional carrier or , in general , buffered aqueous compositions are employed . alternate compositions utilize liposome carriers . the solution is buffered at a desirable ph using conventional buffers such as hank &# 39 ; s solution , ringer &# 39 ; s solution , or phosphate buffer . other components which do not interfere with the activity of the peptide may also be included such as stabilizing amounts of proteins , for example , serum albumin , or low density - or high density - lipoprotein ( ldl and hdl , respectively ). systemic formulations can be administered by injection , such as intravenous ( i . v . ), intraperitoneal ( i . p . ), intramuscular , or subcutaneous ( s . c .) injection , or can be administered by transmembrane or transdermal techniques . for topical application , the active components can be formulated with a variety of cosmetically and / or pharmaceutically acceptable carriers . formulations appropriate for transdermal or transmembrane administration include sprays and suppositories containing skin penetrants , which can often be detergents . the term “ pharmaceutically acceptable carrier ” refers to a vehicle that delivers the active components to the intended target without being harmful to humans or other recipient organisms . as used herein , “ pharmaceutical ” will be understood to encompass both human and animal pharmaceuticals . useful carriers include , for example , water , acetone , ethanol , ethylene glycol , propylene glycol , butane - 1 , 3 - diol , isopropyl myristate , isopropyl palmitate , or mineral oil . the carrier may be in any form appropriate to the mode of delivery , for example , solutions , colloidal dispersions , emulsions ( oil - in - water or water - in - oil ), suspensions , creams , lotions , gels , foams , mousses , sprays and the like . methodology and components for formulation of pharmaceutical compositions are well known , and can be found , for example , in remington &# 39 ; s pharmaceutical sciences , eighteenth edition , a . r . gennaro , ed ., mack publishing co . easton pa ., 1990 . the formulation , in addition to the carrier and the anticancer components , also can comprise other optional materials that may be chosen depending on the carrier and / or the intended use of the formulation . additional components include , but are not limited to , antioxidants , chelating agents , emulsion stabilizers , e . g . carbomer , preservatives , e . g . methyl paraben , fragrances , humectants , e . g . glycerine , waterproofing agents , e . g . pvp / eicosene copolymer , water soluble film - formers , e . g . hydroxypropyl methylcellulose , oil - soluble film formers , cationic or anionic polymers , and the like . the diastereomers of the invention may also be used for food preservation , as food supplements in veterinary compositions , as alternative to antibiotics for animal nutrition , as anti - mycoplasma , antibacterial , and antifungal agents for tissue culture media , and as reagents for transformation / transfection of target cells with desired dna or rna molecules . the invention will now be described with reference to the following non - limiting examples . 4 - methyl benzhydrylamine resin ( bha ) and butyloxycarbonyl ( boc ) amino acids were purchased from calbiochem - novabiochem co . ( la jolla , calif ., usa ). other reagents used for peptide synthesis included trifluoroacetic acid ( tfa , sigma ), n , n - diisopropylethylamine ( diea , sigma ), dicyclohexylcarbodiimide ( dcc , fluka ), 1 - hydroxybenzotriazole ( 1 - hobt , pierce ), and dimethylformamide ( dmf , peptide synthesis grade , biolab , ill .). xtt reaction solution for cytotoxicity assay and matrigel were purchased from biological industries ( beit haemek , israel ). all other reagents were of analytical grade . buffers were prepared in double - distilled water . the cl1 human prostate carcinoma ( pc ) cell line is an androgen - independent ( ai ) subclone of lncap cell line , which was generated by culturing androgen - dependent ( ad ) lncap cells in charcoal - stripped , ad serum , as described ( patel et al . 2000 ). 22rv1 human pc cells are ai sub - clones of the ad prostatic adenocarcinoma cwr22 xenograft ( sramkoski et al . 1999 ). cl1 and 22rv1 ( atcc , usa ) were grown in rpmi - 1640 supplemented with 10 % fcs ( biological industries , beit haemek , israel ). pc3 and du145 are androgen - insensitive ( ai ) ( non - responsive ), invasive human prostate cancer cell lines . nih - 3t3 mouse fibroblast cell lines ( atcc , usa ) were grown in dmem supplemented with 10 % bs . murine lewis lung carcinoma ( llc ) cell lines were also grown in dmem medium supplemented with 10 % fetal calf serum and antibiotics under the same conditions as above . peptides were synthesized by a solid phase method on 4 - methyl benzhydrylamine resin ( bha ) ( 0 . 05 meq ) ( merrifield et . al ., 1982 ; shai et . al ., 1990 ). the resin - bound peptides were cleaved from the resin by hydrogen fluoride ( hf ) and after hf evaporation and washing with dry ether , the peptides were extracted with 50 % acetonitrile / water . hf cleavage of the peptides bound to bha resin resulted in c - terminus amidated peptides . each crude peptide contained one major peak , as revealed by rp - hplc ( reverse phase high - performance liquid chromatography ) that was 60 - 80 % pure peptide by weight . the synthesized peptides were further purified by rp - hplc on a c 18 ( supleco ) reverse phase bio - rad semi - preparative column ( 250 × 10 mm , 300 m pore size , 5 - μm particle size ), in 30 min , using a linear gradient of 30 - 50 % acetonitrile in water , both containing 0 . 1 % tfa ( v / v ), at a flow rate of 1 . 5 [ 1 . 8 ] ml / min . the purified peptides , which were shown to be homogeneous (˜ 95 %) by analytical hplc , were subjected to amino acid analysis and electrospray mass spectroscopy to confirm their composition and molecular weight . n - acylation was carried out using the same protocol used to attach protected amino acids for peptide synthesis . the cyclic peptides were synthesized by a solid - phase method as described in section ( iii ) above , with or without cysteine residues at both the n - and c - termini of the peptides . the cyclization without cystein is carried out by protecting the n - terminal , activating the c - terminal , then deprotecting the n - terminal and reacting the c - and n - terminal groups while still bound to the resin . when the peptide contains cystein residues at both the n - and c - termini , after hf cleavage and rp - hplc purification , the peptides are solubilized at low concentration in pbs ( ph 7 . 3 ), and cyclization is completed after 12 h . the cyclic peptides are further purified on rp - hplc and subjected to amino acid analysis to confirm their composition , and sds - page to confirm their monomeric state . the following 15 - mer peptide 1 and 13 - 17 - mer c - amidated diastereomeric peptides 2 - 44 ( seq id nos 2 - 44 ) composed of his , one or more hydrophobic amino acids selected from leu , ile , val , ala , thr and trp , or another non - natural hydrophobic amino acid , optionally the positively charged amino acids lys , his and / or arg and / or the n - cap amino acids gln and asn , and optionally further acylated at the n - terminus , containing from 3 to 9 d - amino acid residues , were synthesized as described in material and methods , sections ( iii ) and the cyclic peptides 34 and 35 are prepared as described in section ( iv ). peptide 1 is a 15 - mer diastereomer described in the above - mentioned wo 02 / 040529 and herein used for comparison purposes . the peptides will be represented hereinafter by numerals in bold and by a sequence identity number ( seq id no .). as representative examples , the analysis data of peptides 13 and 1 are given . peptide 13 was obtained as a white powder of ≧ 98 % purity as determined by hplc . amino acids content : his - 2 , leu - 9 and lys - 3 . 85 . molecular weight by mass spectra analysis : 1822 . 5 . peptide 1 was obtained as a white powder of ≧ 99 % purity and molecular weight 1804 . 5 . amino acids content : leu - 9 and lys - 5 . 80 . the anticancer activity of the diastereomers 1 , 5 and 11 - 16 , 36 - 39 , 42 and 44 was examined against human cl1 prostate cancer , murine llc ( lewis lung carcinoma ), du 145 and pc3 cell lines . the cell selectivity of the diastereomeric peptides was also studied by examining their effect on nih - 3t3 normal mouse fibroblasts cell line . a 96 - well plate ( falcon ) was used for the xtt proliferation assay . cancer cells were grown for 24 hours ( day 1 ) in rpmi - 1640 medium ( 5 × 10 3 , 7 × 10 3 , 1 × 10 4 and 7 × 10 3 cells / 100 μl for llc , cl1 , 22rvi , du 145 and pc3 , respectively ) supplemented with 10 % fetal calf serum and antibiotics , at 37 ° c ., in humidified atmosphere at 5 % co 2 and 95 % air , resulting in growth medium ph of 7 . 4 . nih - 3t3 fibroblast cells ( 1 × 10 4 cells / 100 μl ) were grown in dmem medium supplemented with 10 % bovine calf serum and antibiotics under the same conditions as described above for the cancerous cells . wells in the last two rows served as blanks ( medium only , for measuring the background color of the medium ) and 100 % survival controls ( cells and medium only without treatment ), respectively . in day 2 , the peptides were dissolved in sterile pbs to a concentration of 200 μm ( or 500 μm ). the medium in the assay wells was replaced with 100 μl serum free medium . for the assays at ph 6 , the medium was initially concentrated (× 5 or × 10 ) and then diluted with double distilled water and addition of sodium carbonate . before reaching the correct dilution , the medium was adjusted to ph 6 and then more water was added to reach the final dilution . the cells were grown in physiological ph , and the acidic ph was used only during the 24 hours incubation with the peptides . a sign for the correct acidity was a light yellow color of the medium . in line a of the plate , 160 μl of serum free medium was added , if the initial peptide concentration was 500 μm . peptide solutions , 100 μl ( or 40 μl ) were added to each assay well in line a , such that the final concentration of the peptide was 100 mm and the volume 200 μl . the medium in the wells was mixed with multichannel pipette 5 times and 100 μl of it were transferred to the next row of wells ( line b ), to give a peptide concentration of 50 μm . the double dilution of the peptides continued downstream the plate in the same manner . the plates were then incubated for 24 h . in day 3 , xtt reaction solution was prepared by adding to 100 μl aliquots of activation solution ( sodium 3 ′-[ 1 -( phenyl - aminocarbonyl )- 3 , 4 - tetrazolium ]- bis ( 4 - methoxy - 6 - nitro ) benzene sulfonic acid hydrate and n - methyl dibenzopyrazine methyl sulfate ; mixed in a proportion of 50 : 1 ) ( protected from light and kept in − 20 ° c . ), the substrate . 50 μl of xtt reaction solution were added to each well and the plates were incubated for 2 hours ( 37 ° c . and 5 % co 2 + 95 % air . in cases the incubation was not enough for the creation of the color , it was extended for up to 24 hours ). the optical density was read at wavelength of 450 nm in an elisa plate reader . cell viability was determined relative to the control and final results were recorded . the results were confirmed using replications in at least three independent experiments . the lc50 for each peptide was obtained from the curve of cell viability versus concentration of peptide and taken from the concentration at which cell viability was 50 %. the data shown in table 1 are for only one experiment , but representative of all replications . with regard to human cells , the results for peptides 5 , 8 and 12 - 16 show that the diastereomers 12 and 13 of the invention are similarly or significantly more potent than the known 15 - mer diastereomer 1 against the human cl1 cell line , at ph 6 . 0 , and peptide 12 was more active than peptide 1 against llc at both ph 6 and ph 7 . 4 . furthermore , peptides 8 , 12 , 13 , 14 , 16 , 36 , 42 and 44 , were active against cl1 and llc at ph 7 . 4 at concentrations , which are 2 - 8 fold lower than the concentration at which they act against nih - 3t3 normal mouse fibroblast cells . at ph 7 . 4 , peptide 14 was active against cl1 at concentrations 8 - fold lower than the concentrations at which it was active against nih - 3t3 cells , although it was half as active against cl1 and 22rvi as peptide 1 . the diastereomers 5 , 8 , 12 , 13 , 14 , 16 , 36 - 39 , 42 and 44 were significantly more potent against 22 rvi cells at ph 6 than peptide 1 , particularly peptides 39 and 44 were 8 and 16 times more active compared to 1 . in addition , peptides 5 , 14 and 16 were active against cl1 cells at concentrations at least two - fold lower than the concentrations at which they act against nih - 3t3 cells at the same ph . particularly high activity was observed for 38 , 39 , 42 and 44 at ph 6 against 22rvi and llc cells as compared to nih3t3 cells . peptides 10 and 11 were significantly less potent against du 145 cells at both ph 7 . 4 and 6 , and against pc3 cells at ph 7 . 4 compared to their activity against other cells . these results clearly reveal that the new diastereomeric peptides of the invention are more selective and more effective than the known diastereomer 1 . acute toxicity of peptides 1 , 13 and 16 was examined by intravenously injecting mice ( n = 3 ), each with one dose per day for 2 days of 0 . 5 ml solution containing peptides 1 , 13 or 16 at 3 , 9 , 15 , 20 and 30 mg / kg of body weight . no mortality was observed with all the peptides administered at 3 mg / kg and 9 mg / kg . at 15 and 20 mg / kg of body weight , 100 % mortality was observed only with prior art peptide 1 and no mortality was observed with peptides 13 and 16 of the invention . no mortality was observed with peptide 16 of the invention at 30 mg / kg . a week after injecting the peptides , blood samples were taken from the survived mice . all the differential and biochemistry tests were in the range of normal values ( i . e . neutrophils , lymphocytes , monocytes , eosinophils , basophiles , creatine phosphokinase , alkaline phosphatase , alanine aminotransferase , aspartate aminotransferase and creatinine ). thus , the peptides 13 and 16 of the invention are not toxic even when administered at concentrations considerably higher than those necessary for their anticancer activity . ( i ) inhibition of tumor growth in human prostate cancer xenografts . subcutaneouse ( s . c .) implantation of human pc cells in mice was done as previously described in gavish et al . ( gavish et al . 2002 ). briefly , 0 . 1 ml ai cl1 or 22rv1 human pc cells ( 5 × 10 6 cells ) in matrigel were inoculated s . c . into the dorsal side of five to six week - old nude male mice weighing 20 - 25 g ( harlen co ., israel ). two weeks after cell implantation , when the tumors diameter reached ≧ 5 mm ( denoted as day 1 ), the diastereomer 13 and its all l - amino acid analog peptide ( at 1 mg / kg , 0 . 1 mm ), or vehicle ( pbs , ph = 7 . 4 ) were injected intratumorally ( dosing volume of 2 . 5 ml / kg ) three times a week for a total of 9 doses . tumor size was measured by a caliper and recorded twice a week during a period of 28 days . mice were weighed and tumor weight ( mg ) was estimated by using the formula of length × width × depth × 0 . 52 in mm 3 , assuming the specific gravity to be 1 . at the end of the treatment , the mice were killed , and the tumors were removed , photographed , and weighed . the animal experimentation was conducted according to the rules of the institutional animal care and use committee . serum psa levels . four weeks after the first treatment , blood was withdrawn from the 22rv1 - inoculated mice in order to determine the level of prostate specific antigen ( psa ). the blood samples were taken directly to heparin containing tubes , centrifuged , and the supernatants were stored at − 20 ° c . the canag psa eia kit ( canag diagnostics ) was used to determine the total psa in the mice plasma ( gavish et al ., 2002 ). tumor weight and psa levels , represented as the mean ± se , were calculated from the raw data and then subjected to student &# 39 ; s t test . a value of p & lt ; 0 . 05 was considered as statistically significant . independently of the xenograft type , a significant reduction in tumor weight was observed with the mice treated with peptide 13 but not in mice treated with the analog l - diastereomer . in some mice , the tumor completely disappeared . furthermore , in the psa - secreting 22rv1 xenografts , the reduction in tumor weight was accompanied by a marked decrease in the psa serum levels . the treatment with peptide 13 showed an increase in the body weight of the animals compared with the vehicle - treated control group . in contrast , the l - diastereomer was inactive in both xenograt models . to check the reason why all l - amino acid analog was not active as opposed to the diastereomer peptide 13 , both peptides were mixed with matrigel matrix for one hour and the solution was analyzed by using rp - hplc and mass spectroscopy . upon interaction with the matrigel matrix , the all l - amino acid analog peptide was fully inactivated in contrast to peptide 13 , which preserved ˜ 50 % of its activity . since 22rv1 prostate tumor is metastatic , we analyzed the ability of the systemically administered peptide 13 to inhibit the formation of lung metastases derived from prostate cancer in cd1 nude mice that were pre - injected systemically with cells . during the experiment , the mice were monitored continuously for clinical signs of toxicity . it was observed that throughout the assay period the animals that had been treated with peptide 13 were in good condition and did not express any sign of weakness . at the end of the experiment , we found that the lung metastases were entirely abolished in the peptide 13 treated animals as compared to the untreated controls . moreover , the peptide 13 treated mice also showed a significant increase in the body weight compared with the vehicle treated control group . rfp - mda - mb - 231 breast cancer ( bc ) cells were injected ( 5 × 10 6 cells in 0 . 1 ml pbs ) into the left mammary fat pad of 8 - week - old female scid / ncr mice ( nci , usa ) as previously described ( dadiani et al ., 2004 ). one week after cell implantation , when the tumor diameter reached ˜ 5 mm , peptide 13 ( at 5 mg / kg , 0 . 14 mm ), or vehicle ( pbs , ph = 7 . 4 ) was injected systemically ( dosing volume of 22 ml / kg ) three times a week for a total of 9 doses for ten mice . mice were weighed and tumor volume was measured by a caliper ( expressed in weight units ( mg ) ( papo et al ., 2003 ) twice a week for a period of 45 days . monitoring of solid breast tumor and its derived metastases was done by in vivo fluorescence . tumor fluorescence intensity was monitored in real time by using in vivo optical imaging system ( ivis ) and was recorded once a week during the period of 38 days . a major reduction in tumor size was recorded from caliper measurements . the reduction in tumor size was accompanied by a marked lowering of the tumor fluorescence as recorded from in vivo optical imaging by ivis . however , since the accuracy and sensitivity of the fluorescence detection was much greater then caliper measurements , a lowering of tumor fluorescence was observed much sooner . the treatment with peptide 13 also resulted in an increase in the body weight of the animals compared with the vehicle - treated control group animals treated with peptide 13 were in good condition throughout the assay period and did not express any signs of weakness . ( iv ) inhibition of formation of lung and lymph node metastases derived from breast cancer since mda - mb - 231 breast tumor cells were metastatic , the ability of the systemically administered peptide 13 to inhibit the formation of metastases in the lymph nodes and lungs of scid / ncr mice was analyzed . during the experiment , the mice were monitored continuously for clinical signs of toxicity . monitoring of solid metastases derived for breast tumor was done by in vivo fluorescence using ivis as described above in ( iii ). at the end of the treatment ( day 38 ), the mice were killed , and the lungs and lymph nodes were removed and monitored for fluorescence of metastases derived from the breast cancer . for metastases quantification , the lungs and right lymph nodes were excised and fixed in 4 % buffered formaldehyde . paraffin - embedded 5 - μm sections were stained with h & amp ; e . the percentage of metastatic cell area of total section area was calculated using the image - pro plus 4 . 1 software . a significant reduction in lymph node metastasis fluorescence intensity was observed in the mice after treatment with peptide 13 . images of dissected lungs and lymph nodes from the untreated mice were also analyzed showing strong fluorescence relative to the treated ones . the dissected lungs and lymph nodes were analyzed by histology . the lungs and lymph nodes in the control untreated mice were significantly more populated by the cancer cells while the tumors in the 15 - mer treated mice were much less densely populated . metastasis quantification was done according to areas from three different sections of each organ ( p & lt ; 0 . 005 ). in order to reach their target , the diastereomers have to withstand proteolytic digestion of proteases , which may occur after their administration and during the time until they reach the target site . the susceptibility of the peptides 13 and 14 to proteolytic digestion by pepsin ( from porcine stomach mucosa , sigma ), trypsin ( from bovine pancreas , sigma ), and elastase ( from human leukocytes , sigma ) was assessed by reverse - phase hplc . as a negative control , the all l - amino acid analog peptides were used . equal amounts of the peptides were dissolved in pbs ( 35 mm phosphate buffer / 0 . 15 m nacl , ph 7 . 3 ) at a final concentration of 140 μm , to which 25 μm of protease were added . the samples were incubated under agitation for 30 min at 37 ° c . after the addition of the appropriate protease inhibitor to stop the reaction , aliquots were injected to c 18 column and the amounts of the intact peptides 13 and 14 and their all l analogs were evaluated using their absorbance at 215 nm . the diastereomers of the invention were significantly less susceptible to proteases digestion (˜ 50 % after 2 hr ) whereas the all l analogs were completely degraded after 30 min . liposomes serve as convenient delivery vehicles for biologically active molecules . hydrophilic drugs can be encapsulated in the internal aqueous compartment , whereas hydrophobic drugs may bind to or are incorporated in the lipid bilayers . in this experiment , liposomal diastereomeric peptides were prepared in order to further lower the peptide toxicity and increase their selectivity . liposomes composed of different ratios of phosphatidylcholine ( pc )/ phosphatidylglycerol ( pg ) ( 9 : 1 ; 4 : 1 ; 1 : 1 w / w ) or phosphatidylethanolamine ( pe )/ pg ( 9 : 1 ; 4 : 1 ; 1 : 1 w / w ) were prepared . briefly , dry lipid mixtures were dissolved in chcl 3 / meoh ( 2 / 1 , v / v ). the solvents were evaporated under nitrogen stream , and the lipid mixtures at the compositions described above were resuspended in pbs by vortex mixing . the lipid suspension was extruded through 3 different polycarbonate filters ( 1 μm , 0 . 2 μm and 0 . 1 μm pore size filters , 15 times each ). finally , the resulting suspensions of large unilamellar vesicles ( luv ) were added to different concentrations of a peptide of the invention to give lipid / peptide ratios of 50 : 1 ; 30 : 1 ; 10 : 1 w / w , respectively . the mixtures were sonicated for 2 minutes and the liposomes were stored at 4 ° c . until used . the anticancer activity of the resulting liposomal diastereomeric peptide preparations was examined as described in example 2 above . the lc 50 of liposomal peptides 5 and 12 - 16 in various lipid compositions and peptide / lipid ratios ( as described above ), or of liposomes at lipid compositions equivalent to the loaded liposomes or peptides alone , were determined using lc1 , llc and 22rvi cell lines . liposomal peptides exhibited lc 50 results similar to those of the peptide alone , indicating that the peptides of the invention entrapped within liposomes can maintain their anticancer activity . however , this activity is dependent on the liposomes &# 39 ; lipid composition and on the lipid / peptide ratio . based on the in vitro test results , the in vivo toxicity of the liposomal peptides preparations was examined , utilizing the liposomal composition which gave the best anticancer activity . groups of 5 cd1 male mice weighing 24 - 27 g ( 5 - week old ), bred in an animal isolator ( ivc racks ) under specific pathogen - free ( spf ) conditions at 24 ± 1 ° c ., were used . twelve mg / kg liposomal peptide or peptide alone dissolved in pbs in a dosing volume of 10 ml / kg , were administered by single i . v . bolus injection via the mice tail vein . in parallel , control groups received i . v . injections of equivalent liposomes alone or pbs in a dosing volume of 10 ml / kg . the liposomal peptides maintained their activity but were less toxic , thus leading to reduced mortality in mice . no incidence of mortality occurred following the i . v . injection of pbs or the liposomes alone . avrahami d . and shai y . a new group of antifungal and antibacterial lipopeptides derived from non - membrane active peptides conjugated to palmitic acid . j . biol . chem ., 279 : 12277 - 12285 , 2004 . avrahami d . and shai y . bestowing antifungal and antibacterial activities by lipophilic acid conjugation to d , l - amino acid - containing antimicrobial peptides : a plausible mode of action . biochem ., 42 : 14946 - 14956 , 2003 . baker , m . a ., maloy , w . l ., zasloff , m ., and jacob , l . s . anticancer efficacy of magainin 2 and analogue peptides . cancer res ., 53 : 3052 - 3057 , 1993 . biragyn , a ., surenhu , m ., yang , d ., ruffini , p . a ., haines , b . a ., klyushnenkova , e ., oppenheim , j . j ., and kwak , l . w . mediators of innate immunity that target immature , but not mature , dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens . j . immunol ., 167 : 6644 - 6653 , 2001 . boman , h . g . peptide antibiotics and their role in innate immunity . annu . rev . immunol ., 13 : 61 - 92 , 1995 . chan , s . c ., yau , w . l ., wang , w ., smith , d . k ., sheu , f . s ., and chen , h . m . microscopic observations of the different morphological changes caused by anti - bacterial peptides on klebsiella pneumoniae and hl - 60 leukemia cells . j . pept . sci ., 4 : 413 - 425 , 1998 . chen , y ., xu , x ., hong , s ., chen , j ., liu , n ., underhill , c . b ., creswell , k ., and zhang , l . rgd - tachyplesin inhibits tumor growth . cancer res ., 61 : 2434 - 2438 , 2001 . dadiani , m ., margalit , r ., sela , n ., and degani , h . high - resolution magnetic resonance imaging of disparities in the transcapillary transfer rates in orthotopically inoculated invasive breast tumors . cancer res . 64 : 3155 - 3161 , 2004 . draize j . h ., woodward , g . & amp ; 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