Patent Application: US-201415026819-A

Abstract:
the present invention concerns a hexapeptide and its medical and / or diagnosis uses , in particular for the diagnosis and / or treatment of cancer .

Description:
peptide ( also named in the description as minipeptide or hexapeptide - rmnsod - lp ) was synthesized step - wise in batch - mode on solid support with automatic synthesizer syro ( multisyntech ), using fmoc / tbu chemistry [ 12 ] starting from the c - terminal residue pre - loaded on ps - phb resin with an average of 0 . 57 mmol / g substitution ( rapp polymere ). synthesis on a 30 micromol scale proceeded through standard cycles of fmoc deprotection [ piperidine : dmf , 0 . 2 : 1 ( v / v )] and fmoc - aminoacid coupling steps [ resin : fmoc - aminoacid : hbtu : diea , 1 : 4 : 4 : 8 ] [ 13 ]. as pt binder , diamino - ethyl - glycine was used which , by virtue of the presence of 2 free amine groups is able to combine platinum ( ii ) ions as ptcl2 [ 16 ]. side chain de - protection with concomitant cleavage of peptide from solid support was achieved suspending the protected peptide - resin in a mixture tfa : water [ 95 : 5 ( v / v )] for three hours . the resin was removed by filtration under reduced pressure . precipitation was achieved collecting the filtrate on a bed of cold ether . after several ether washes , crude compound was suspended in water ( 0 . 1 % tfa , v / v ) and lyophilized . purity was assessed by analytical rp - hplc ( shimadzu ): vydac c18 column ( 4 . 6 × 150 mm ); eluent a : 0 . 1 % tfa in water ; eluent b : 0 . 1 % tfa in acetonitrile ; gradient : from 5 % b to 65 % b in 20 min ; flow 1 ml / min . ( r . t . ): 16 . 5 minutes . identity was assessed by mass spectrometry performed on a maldi - tof spectrometer ( applied biosystem ) [ mh += 870 ]. rp - hplc purification was performed on either vydac c18 column ( 22 × 250 mm ); eluent a : 0 . 1 % tfa in water ; eluent b : 0 . 1 % tfa in acetonitrile ; gradient : from 5 % b to 65 % b in 40 min ; flow 20 ml / min ). hexapeptide ability to penetrate into the cells . assessment by confocal microscopy the confocal analysis was performed by placing the cells of human breast cancer ( mcf - 7 , atcc htb - 22 ) on suitable slides ( mattek , ashland corporation ma , u . s .). the hexapaptide to the concentration of 0 . 125 μm was then added to the cells . peptide was synthesized step - wise in batch - mode on solid support with automatic synthesizer syro ( multisyntech ), using fmoc / tbu chemistry [ 1 ] starting from the c - terminal residue pre - loaded on ps - phb resin with an average of 0 . 57 mmol / g substitution ( rapp polymere ). synthesis on a 30 micromol scale proceeded through standard cycles of fmoc deprotection [ piperidine : dmf , 0 . 2 : 1 ( v / v )] and fmoc - aminoacid coupling steps [ resin : fmoc - aminoacid : hbtu : diea , 1 : 4 : 4 : 8 ] [ 2 ]. fmoc - ala - oh , fmoc - val - oh , fmoc - cys ( trt )- oh , fmoc - gly - oh , fmoc - thr ( tbu )- oh . fluorescein was coupled to peptidil - resin using 5 ( 6 )- fam : hbtu : diea in ratio 4 : 4 : 8 respect peptidyl - resin . side chain de - protection with concomitant cleavage of peptide from solid support was achieved suspending the protected peptide - resin in a mixture tfa : water [ 95 : 5 ( v / v )] for three hours . the resin was removed by filtration under reduced pressure . precipitation was achieved collecting the filtrate on a bed of cold ether . after several ether washes , crude compound was suspended in water ( 0 . 1 % tfa , v / v ) and lyophilized . vydac c18 column ( 4 . 6 × 150 mm ); eluent a : 0 . 1 % tfa in water ; eluent b : 0 . 1 % tfa in acetonitrile ; gradient : from 5 % b to 65 % b in 20 min ; flow 1 ml / min . ( r . t . ): 16 . 5 minutes identity was assessed by mass spectrometry performed on a maldi - tof spectrometer ( applied biosystem ) [ mh += 866 ]. the protocol used for the internalization of the pep - 6 fam includes two separate experiments . a first experiment was done using pep 6 - fam to 92 μm for 3 hr and the internalization was assessed . to these cells , already treated with pep6 - fam , primary ab anti - pep 24 1 : 20 overnight at 4 ° c . was added and , as secondary antibody goat anti rabbit fluorescent cf 488a ; and to 1 plate , already treated with pep6 fam , it was added the primary antibody anti rmnsod 1 : 20 overnight and the secondary cf555 rodamynated donkey anti sheep . for 45 min - 1 hr . in the second experiment , the pep 6 - fam was used at 92 μm to 1 hr and 3 hr with fixation in ethanol and paraformaldehyde , and for both times it was assessed the penetration alone , without the addition of the antibody and with the addition of primary antibody anti rmnsod with three different dilutions ( 1500 - 1 : 1000 - 1 : 2000 ) in 5 % ngs ( normal goat serum ) in pbs + 0 . 1 % triton overnight at 4 ° c . and , the secondary antibody anti rhodium cf 555 sheep 1 : 400 for 45 ′- 1 hr . the nuclei were subsequently stained with 10 μg for ml − 1 dapi ( 4 ′, 6 - diamidin - 2 - phenylindole , life ™ thencologies ). the slides were then put together and stored at 4 ° c . until the examination was achieved at the lsm 510 meta confocal microscope , zeiss ( 10 ). the analysis performed by confocal microscopy clearly showed that the fluorescent peptide is internalized in the cytoplasm of mcf - 7 cells ( fig1 a ). the image ( by transmission and fluorescence ) of mcf - 7 cells incubated with 0 . 125 μm of ftc - peptide for 3 hr ., indicates the internalization of the peptide ( exc . 488 nm argon laser / em . bp500 - 550 filter ). the image in fluorescence optical microscopy shows how the synthetic sequence of minipeptide combined to fluorescein ( 5 . 6 fam ) penetrates into the mcf - 7 breast cancer cells spreading in their cytoplasm ( fig1 b ). ability of the hexapeptide to transport into the cells a conjugated molecule . hexapeptide conjugated to cisplatin ( cisplatin - avcgtg ) samples of each cell line : pa - tu 8902 pancreatic adenocarcinoma ( dsmz no . acc179 ), a375 melanoma ( atcc crl 1619 ™) prostate cancer du145 ( atcc htb 81 ), huh - 7 hepatocarcinoma jcrb cell bank ( jcrb043 nibio , national institute of biomedical innovation ), a2780 ovarian cancer ( european collection of cell cultures , ecacc , salisbury , wiltshire , uk ), mrc - 5 human lung fibroblasts normal ( atcc ccl - 171 ™) breast cancer mcf - 7 ( atcc htb - 22 ™) used in the study were treated , independently , for 3 hours with a solution containing 92 μm of cisplatin alone ( cc , bristol - mayer co . ( newyork city , united states ) or with a solution of 92 μm of leader - hexapeptide rmnsod - lp - cc ( hex - lp - cc ) containing 11 . 1 μg of cisplatin . the cells were maintained in dmem culture medium added with 5 % fetal bovine serum ( fcs ) ( gibco ), and the controls were prepared by maintaining the cells in the same culture conditions but in the presence of a solution of 92 μm of the peptide only , therefore in the absence of cisplatin . the cells to be examined , after the incubation period , were harvested , washed twice in buffered solution ( pbs ) and treated with 50 μl of 35 % hno 3 for 16 hours . the platinum ( pt ) contained in the culture medium or in the cell pellet was determined by spectrophotometer at atomic absorption ( analyst 800 , perkin - elmer , norwalk , conn .) using the following criteria : temperature of pretreatment , 1300 ° c ., temperature of atomizing 2200 ° c . 0 . 015 μg of palladium were used ( palladium has been used for the modification of the matrix ) and 0 . 01 μg of mg ( no 3 ) 2 as a modifier of the matrix . the determinations were performed using a graphite furnace equipped with the correction system of the zeeman effect . for the determination of the metal , tubes of pyrolytic graphite coated with thga were used ( perkin - elmer ) with an integrated “ vov - type platform ” system . a standard solution of platinum in 2 . 5 % hno 3 ( spectrascan ) was used as a mother solution for the construction of a calibration curve with three points of reference . each determination was performed independently , in triplicate ( 11 ). the ability of the peptide to penetrate into tumor cells and to release cisplatin was assessed on 5 cell lines of human cancers ( breast , ovarian , hepatocellular carcinoma , renal cell carcinoma , melanoma ) by atomic absorption spectrophotometry . the results of the analysis are shown in table i . it is evident that the hexapeptide manages to transport directly inside the cancer cells an amount of platinum that is from 1 . 6 to 15 . 3 times higher than the amount of platinum that enters the cells when the cisplatin is used alone . after adding to the cells an amount of hexapeptide conjugated to cisplatin , the inventors have found that this synthetic construct had penetrated into tumor cells releasing an amount of platinum that percentage wise is significantly greater than the amount of platinum that reaches cancer cells when cisplatin is used alone . in terms of %, the uptake by tumor cells varies from cell to cell and ranges from 4 . 2 to 15 . 5 %. by contrast , the use of the same amount of cisplatin , alone , has shown that if it is not conjugated to the carrier peptide , the quantity of cisplatin that will be found within the cells will range from 0 , 7 % and 2 , 6 %. the solutions of cisplatin and peptide - conjugated cisplatin that were used have the same molarity which is equal to 92 μm and both contain 11 . 1 μg of cisplatin . immuno - histochemical assessment of the internalization of the hexapeptide into tumor cells of human origin — assessment performed qualitatively by optical microscope the tumor cells were incubated for 3 hours in the presence or absence of hexapeptide conjugated to cisplatin ( hex - lp - cc ), or cisplatin alone ( cc ) at concentrations of 92 μm . subsequently , a cytospin was used to obtain a smear of cells on slides . the cells were then placed in zamboni solution ( 4 % paraformaldehyde , 15 % picric acid ) for 60 min and then washed with a solution of 1 × pbs and incubated for 5 min with 3 % hydrogen peroxide to suppress the action of endogenous peroxidase . the immuno - coloring was performed using the dako lsa + system hrp kit ( dako , ca , usa ). the incubation lasted 30 minutes with primary antibody anti - peptide leader of 24 aa ( 1 : 200 ), produced in rabbits . this phase was followed by sequential method that requires a 30 - min incubation with biotinylated antibody and peroxidase - labeled streptavidin . to complete the reaction of differentiation , a substrate - chromogen solution was engaged . cell smears were colored with hematoxylin . a positive reaction is represented by a brownish coloration . a blue coloration indicates negativity . the immunocytochemical analysis was performed using a polyclonal antibody able to recognize the amino acid sequence of the peptide leader of rmnsod ( 7 ), ie of 24 aa . among which there are also those that form the hexapeptide . cells that have not undergone any treatment and used as a negative control , cells that were treated in the presence of hexapeptide conjugated to cisplatin , cells that have been treated with the antibody alone , cells that have received the entire molecule rmnsod bearing , as previously mentioned , the hexapeptide sequence included in the sequence of the leader peptide of 24 aa . after incubation with these molecules , the cells were washed and treated with the polyclonal antibody for three hours at rt , and then the cells were washed with a solution of pbs [ 17 , 18 ]. in control cells ( not treated and those treated with the antibody alone ), the reaction is negative . in cells treated with the hexapeptide ( alone ) or the entire protein that bears , within its leader sequence of 24 aa the six amino acids that compose the hexapeptide , the reaction was positive because the antibody has recognized the amino acids present in this sequence . the presence of the hexapeptide recognized by the antibody is represented by a brownish coloration , such as that represented in the monocytes ( fig2 b ). assessment of the ability of the hexapeptide conjugated to a chromophore group ( fluorescein ) to enter cells the test results are positive and confirms the previous data . that is , the hexapeptide , bounded ( conjugated ) with fluorescein before the execution of the immunocytochemistry examination , penetrates into the cells . therefore , the hexapeptide can be used to carry any molecule in the cells for diagnostic or therapeutic purpose , or to trigger an immune response . a peptide consisting of similar amino acids but with “ scrambled ” sequence ( actgvg , seq id no . 2 ) gave negative results , that is , it does not penetrate into the cells . the leader peptide of 24 aa . ( rmnsod - lp ), alone , or conjugated to cisplatin ( rmnsod - lp - cc ), the hexapeptide avcgtg ( seq id no . 1 ) alone ( hex - lp ), or conjugated to cisplatin ( hex - lp - cc ) and finally the cisplatin alone ( cc ) were added to the target cells ( mcf - 7 , a - 2780 ; huh - 7 ; mrc - 5 ; du - 145 ) at a concentration of 1 , 5 μm . the release of lactate dehydrogenase ( ldh ) was then measured quantitatively in the supernatant of the culture medium of the cells after 24 hr of incubation , using the cytotox 96 non riadioactive cytotoxic assay kit in compliance with the technical indications provided by promega . the experiments were performed in triplicate in an independent manner , using different preparations . ( table iii ) cisplatin alone ( cc ), the hexapeptide conjugated to cisplatin ( hex - cc ) and the hexapeptide alone ( hexapeptide ) were added to the target cells at a concentration of 1 . 5 μm and maintained for 48 hr . after the incubation the amount of lactate dehydrogenase ( ldh ) released from cells into the supernatant was measured . this quantity was expressed as a percentage of the total ldh ( released by cells in which cell lysis had been induced using a detergent ). approximately 3 % of ldh was found in the culture medium of not treated cells . the synthetic construct consisting of the hexapeptide conjugated to cisplatin , added to the tumor cells has penetrated into the cells , releasing an amount of cisplatin that has caused their immediate death . the assessment of the induced cytotoxicity was performed using the enzyme lactate dehydrogenase and expressed in terms of %. as can be seen from the results shown in table iii , the hexapeptide alone is practically devoid of toxicity , both on tumor and normal cells ( mrc - 5 ). in fact , the amount of ldh release ( ldh is an enzyme that reflects the degree of cell mortality ) is very low , ranging from 2 % to 5 %. on the contrary , when the hexapeptide conjugated to cisplatin is added to the cells , the cell mortality increases significantly ranging from 40 % to 85 %. cisplatin alone , instead causes a mortality that varies from a minimum of 25 % to a maximum of 48 %, that is twice or three times less than the one induced by the hexapaptide combined to cisplatin . the experiment clearly indicates that the hexapeptide transports and releases a double or triple amount of cytostatic directly into the cells . therefore , the therapeutic index of hexapeptide increased significantly . the peptide has greater efficacy and less side effects . human normal lymphocytes cells were collected from healthy donors and purified using ficoll hystopaque density gradient centrifugation . the cells were cultured at a density of 1 × 10 6 cells / ml in rpmi media supplemented with 1 % penstrep and 10 % fbs in a humidified atmosphere containing 5 % co 2 at 37 ° c . lymphocytes were incubated for five hours in the presence of fluorescinated hexapeptide 92 μm . a cytospin was used to smear cells onto slides . the smears were fixed in zamboni solution ( 4 % paraformaldehyde , 15 % picric acid ) for 60 minutes and then washed with 1 × pbs and incubated for five minutes with 3 % hydrogen peroxide to quench endogenous peroxidase activity . immunostaining was performed using the dako lsa + system hrp kit . rabbit anti - rmnsod - lp ( 1 : 200 ), was incubated on slides for 30 minutes , followed by incubation with the biotinylated secondary antibody for 30 minutes and peroxidase - labeled streptavidin for 30 minutes . to complete the reaction , a substrate - chromogen solution was used . the smears were counterstained with hematoxylin . the treatments performed respectively with 92 μm avcgtg ( seq id no . 1 )- fitc for 3 hours on the three cell populations gave the following percentages of labeled cells : 95 % of lymphocytes , while the controls displayed no labeling ( fig3 ). overall , these results indicate that most cells internalized avcgtg ( seq id no . 1 )- fitc , as they displayed both mainly cytoplasmic and less nuclear immunoreactivity ( fig3 ). it is observed that the fluorescent hexapeptide of the invention constituted of 6 aa . penetrates in white blood cells ( lymphocytes and neutrophils ) of healthy subject . therefore , the peptide of the invention is particularly useful to target leucocyte cells . the peptide by being conjugated to a citostatic molecule is advantageous for the treatment of leukemia . citostatic molecules include idarubicin , etoposide or cytarabin . further the peptide of the invention may also be conjugated to an antibiotic molecule , allowing such molecules to directly target white blood cells where bacteria tend to hide to provoke resistant infectious diseases . the peptide may also be successfully conjugated to disease specific antigens in order to induce immune response . jurkat cells ( attc , uk ) were seeded at density of 150000 cells / well in rpmi media supplemented with 1 % penstrep and 10 % fbs in a humidified atmosphere containing 5 % co 2 at 37 °. the next day , the cells were incubated with 92 μm avcgtg ( seq id no . 1 )- fitc for 3 h in multiwell 24 . subsequently , a cytospin was used to smear cells onto slides . the smears were fixed with 4 % paraformaldehyde / pbs for 10 min and rinsed twice with 1 × pbs containing 0 . 1 % triton to remove traces of fixative . cell samples without avcgtg ( seq id no . 1 )- fitc were used for the background control . after blocking solution with 5 % ngs ( normal goat serum , vector ) 1 × pbs + 0 , 1 % triton x - 100 , was added for 30 minutes . after removing the blocking solution , cells were incubated with primary antibody hexapeptide , diluted 1 : 200 in blocking solution , overnight at 4 ° c . the following day , after washing with lx pbs and 0 . 1 % triton x - 100 for 10 minutes , secondary antibody ( alexa fluo 488a , santacruz ) diluted 1 : 400 with 1 × pbs and 0 . 1 % triton x - 100 for 2 h was added . the excess of antibody was removed with 3 washes with 1 × pbs . nuclei were stained with dapi ( 1 μg / ml ). the slides were mounted and stored in the dark at 4 ° c . and then observed by fluorescence microscope zeiss hb050 by using the software axion vision re1 . 4 . 8 . as shown in fig5 - 8 , the fluorescent hexapeptide of the invention constituted of 6 aa . penetrates in jurkat cells . jurkat cells are stabilized human cells deriving from infantile acute lymphatic leukemia . the peptide of the invention may be conjugated to a citostatic molecule and is advantageous for the treatment of leukemia . citostatic molecules include idarubicin , epirubicine or cytarabin . further , the peptide of the invention may also be conjugated to an antibiotic to target infected organs , leucocytes or bacteria . in particular , the peptide may be conjugated to specific or wide spectrum antibiotics such as β - lactam antibiotics . the peptide of the invention may be advantageously used to render active molecules that became inactive due to bacteria acquired resistance . three fresh ejaculate from bovine , obtained from two different bulls , were mixed and diluted in tris solution with a concentration of 100 × 10 6 sperm / ml , incubated at 37 ° c . for 1 hour in the presence of 1 mg / ml of fitc - rmnsod - lp ( hexapeptide ) and 1 mg / ml of propidium iodide . propidium iodide stain is used to identify viable sperm ( not red ) from the dead ones ( red ), therefor to prove that sperm are viable . propidium iodide is a dye that does not cross the cell membrane of a viable sperm , unless this is not broken as a result of sperm death . taking into consideration only viable sperm ( which do not have a red fluorescence ), they present a localization of the green fluorescence ( hexapeptide ) at the level of the mid piece region of the tail , the same location of the mitochondria . the peptide also enters in the sperm with a non - intact membrane . these sperm must not to be taken into consideration in the penetration study because if the membrane is broken , its permeability is not selective . the hexapeptide enters even in the dead , but for normal diffusion , not for selective activity of intact membranes . the 100 % of sperm that do not show a red fluorescence ( viable sperm ), show a green fluorescence localized in the mid piece portion of the tail ( fig9 ). the percentage of positive sperm for propidium iodide varies from 20 % to 15 %. therefore the peptide of the invention may be advantageously used to target male gametes . in particular , the peptide of the invention may be linked or conjugated to proteins , nucleic acids , nanoparticles or bioconjugates for therapeutic and diagnostic use for human or animal health . 1 ) balmain a . 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