Patent Application: US-200913127346-A

Abstract:
the present invention relates to a multimeric molecule having the general formula a or b : and its use in the diagnosis and / or therapy of tumors .

Description:
tetrabranched nt ( 8 - 13 )- peg - k ( peg_fluorescein ) [ nt4 ( 8 - 13 )- fluo ] 1 , nt ( 8 - 13 )- peg - chlorambucil [ nt4 ( 8 - 13 )- clb ] 2 , tetrabranched nt ( 8 - 13 )- peg - 5 - fluorodeoxyuridine [ nt4 ( 8 - 13 )- 5 - fdu ] 3 , tetrabranched nt ( 8 - 13 )- peg - 6 - mercaptopurine [ nt4 ( 8 - 13 )- 6 - mp ] 4 , tetrabranched nt ( 8 - 13 )- peg - combretastatin [ nt4 ( 8 - 13 )- cbtst ] 5 , tetrabranched nt ( 8 - 13 )- peg - monastrol [ nt4 ( 8 - 13 )- mon ] 6 , tetrabranched nt ( 8 - 13 )- peg - tirapazamine [ nt4 ( 8 - 13 )- tpz ] 7 , tetrabranched nt ( 8 - 13 )- peg - dota [ nt4 ( 8 - 13 )- dota ] 8 and tetrabranched nt ( 8 - 13 )- peg - combretastatin ether [ nt4 ( 8 - 13 )- o — cbtst ] 9 , and tetrabranched nt ( 8 - 13 )- peg - monastrol ether [ nt4 ( 8 - 13 )- o - mon ] 10 ( fig1 ), were synthesized using fmoc - lys ( dde )- oh as first and β - ala as second amino acid on novasyn tgr resin , except from [ nt4 ( 8 - 13 )- fluo ] 1 where the second aminoacid is fmoc - peg - oh instead of β - ala . the tetramer was then built as above but with boc - arg ( pbf )- oh as last amino acid of the neurotensin sequence , so that the last two coupling steps occurred selectively on the side chain arm . once the aminoacid sequence is completed the dde protective group is removed with hydrazine and the intermediate is coupled with peg . after fmoc removal from peg the compound is coupled to functional unit ( w ) carrying a free carboxyl group on the linker . nt4 ( 8 - 13 )- fluo 1 , nt4 ( 8 - 13 )- clb 2 , nt4 ( 8 - 13 )- tpz 7 , nt4 ( 8 - 13 )- dota 8 as well as nt4 ( 8 - 13 )- o — cbtst 9 and nt4 ( 8 - 13 )- o - mon 10 were linked to the branched carrier through an amide bond originating from the free carboxyl group present on the fluorophore or on the drug and the amine group of the peptide ( fig1 ). nt4 ( 8 - 13 )- 5 - fdu 3 , nt4 ( 8 - 13 )- cbtst 5 and nt4 ( 8 - 13 )- mon 6 , on the other hand , were conjugated to the carrier peptide through a bifunctional linker that gave an amide bond on the peptide side and an ester bond on the drug side . nt ( 8 - 13 ) 4 - 6 - mp was conjugated to the carrier peptide through a bifunctional linker that gave an amide bond on the peptide side and an thio - enoic bond on the 6 - mp side . the three conjugation arrangements gave rise to different drug - releasing patterns , i . e . nt4 ( 8 - 13 )- fluo 1 , nt4 ( 8 - 13 )- clb 2 , nt4 ( 8 - 13 )- tpz 7 , nt4 ( 8 - 13 )- dota 8 , nt4 ( 8 - 13 )- o — cbtst 9 and nt4 ( 8 - 13 )- o - mon 10 hardly release fluo , clb , tpz , dota , cbtst and mon . whereas nt4 ( 8 - 13 )- 5 - fdu 3 , nt4 ( 8 - 13 )- cbtst 5 , nt4 ( 8 - 13 )- mon 6 and nt ( 8 - 13 ) 4 - 6 - mp 4 easily release fdu , cbtst , mon and 6 - mp from the adduct . tetrabranched peptides carrying the sequence pyroelyenkprrpyil ( seq id no . 2 ) were synthesized as described above . unrelated branched peptides carrying the sequence acddhsva ( seq id no . 4 ) were synthesized as described above and used for control . the branched conjugated peptides 1 - 10 differ by the linker which is used for the coupling between the branched peptide and the functional unit . the linkers are here considered fast releasing or slow releasing for their ability to release the functional unit from the carrier peptide . 10 6 panc - 1 cells were incubated with nt conjugated branched peptides nt ( 8 - 13 ) 4 - 5fdu 3 , nt4 ( 8 - 13 )- cbtst 5 , nt4 ( 8 - 13 )- o — cbtst 9 , nt4 ( 8 - 13 )- mon 6 and nt4 ( 8 - 13 )- o - mon 10 ( 100 μm ) and with the unrelated branched peptide ( 100 μm ), at 37 ° c . for different time intervals . cells were centrifuged , washed and then lysed in water after freezing and thawing . supernatant and lysed cells were analysed by mass spectrometry after addition of nt ( 8 - 13 ) 4 as internal standard , using an ettan maldi - tof mass spectrometer in reflectron mode with an acceleration voltage of 20 kv . nt4 ( 8 - 13 )- o — cbtst 9 and nt4 ( 8 - 13 )- o - mon 10 decreased gradually in cell medium while increasing in cell lysate , where it appeared after 1 h , reaching maximum concentration after 48 hours of incubation . the unrelated tetra - branched peptide was found intact after 48 hours in the cell medium and never detected in the lysed cells . nt ( 8 - 13 ) 4 - 5fdu decreased gradually in cell lysate and cell medium . the unrelated tetrabranched peptide conjugated to 5fdu [( acddhsva ) 4 - 5fdu ] was never found in the cell lysate and remained intact in the medium for 4 hours . in fact , nt ( 8 - 13 ) 4 - 5fdu and ( acddhsva ) 4 - 5fdu are challenged by hydrolyses of 5 - fdu , which is released from the ester linker , therefore they show a shorter half - life . nt4 ( 8 - 13 )- cbtst 5 , nt4 ( 8 - 13 )- mon 6 released the cbsts and mon after 2 hours by hydrolysis of ester bond , while ether conjugated drugs 9 and 10 were found intact in the supernatant or in lysate . release of 6 - mp from nt ( 8 - 13 ) 4 - 6 - mp nt ( 8 - 13 ) 4 - 6 - mp was incubated at 37 ° c . in a phosphate buffer solution ( ph = 7 . 4 ) in the presence of 1 , 5 and 15 equivalents of gsh . the crude mixture was then injected in hplc at different time intervals to measured 6 - mp release . nt ( 8 - 13 ) 4 - 6 - mp released 86 % of 6 - mp after 135 min in the presence of 1 equivalent of gsh and 100 % release after 30 min with 5 equivalents of gsh . the functionalized branched peptides were then classify as fast releasing or slow / non releasing : nt4 ( 8 - 13 )- fluo 1 , nt4 ( 8 - 13 )- clb 2 , nt4 ( 8 - 13 )- tpz 7 , nt4 ( 8 - 13 )- dota 8 , nt4 ( 8 - 13 )- o — cbtst 9 and nt4 ( 8 - 13 )- o - mon 10 are slow releasing adducts while nt4 ( 8 - 13 )- 5 - fdu 3 , nt4 ( 8 - 13 )- cbtst 5 , nt4 ( 8 - 13 )- mon 6 and nt4 ( 8 - 13 )- 6 - mp 4 are fast releasing . in vitro activity of branched nt peptides conjugated to functional unit peptide binding and internalization , of tetra - branched nt ( 8 - 13 ) conjugated to biotin ( nt ( 8 - 13 ) 4 - peg - biotin ) was analysed by confocal microscopy in human colon adenocarcinoma ( ht29 ), human pancreas carcinoma ( panc - 1 ) and human prostate carcinoma ( pc3 ) cell lines 15 . it was found in the present invention that nt ( 1 - 13 ) 4 - fluorescein and nt ( 8 - 13 ) 4 - fluorescein specifically bind to the three cell lines , which express nt receptors ( fig2 ). cells were plated , grown for 24 hours , blocked for 30 min at − 37 ° c . with 3 % bsa in tbs and then incubated with the peptides ( 2 μm in tbs — 0 . 3 % bsa ) compared with a four - fold molar excess of monomeric analogues . protease inhibitors cocktail was added to the buffer in experiments with monomeric peptides . cells were grown in the medium for 1 , 2 or 4 hours at 37 ° c . and then were fixed with 4 % formalin and plasma membrane was stained with lectin - cy3 ( 0 . 5 μg / ml in tbs — 0 . 3 % bsa ) and nuclei with 4 , 6 - diamidino - 2 - phenylindole ( dapi ) ( 1 μg / ml in tbs — 1 % bsa ). images were taken by confocal laser microscope ( leica tcs sp5 ). internalization of conjugated peptides was completed in 1 hour . peptides were degraded inside the cells within 1 . 8 hours 15 . no difference in cell binding or internalization rate was detected between nt ( 1 - 13 ) and nt ( 8 - 13 ) tetra - branched peptides . monomeric nt ( 1 - 13 )- fluorescein ( m ) gave no signal . the ability of tetrabranched peptides conjugated to a functional unit to bind cancer cell lines through nt receptors , to be rapidly internalized in cells and to still be detectable after 4 hours , show their importance for therapeutic applications . it is well known that classical chemotherapeutics used in the clinical practice have different activity on different tumors . this is due to natural resistance of cancer cells to the drugs , caused by different mechanisms , including a decreased uptake or increased export of drugs by the cell , increased inactivation of drugs inside the cell or enhanced repair of the dna damage produced by dna - alkylating agents . previously reported cytotoxicity experiments , performed by the authors of the present invention on ht - 29 15 , demonstrated that conjugation of methotrexate ( mtx ) or of the photosensitizer , chlorine e6 , to tetra - branched . nt ( 8 - 13 ) produces pro - drugs like molecules . such molecules can no longer be transported across plasma membranes by the mechanism of the corresponding free drug and can only be ‘ activated ’ via peptide - receptor binding , thus profoundly decreasing non - specific drug toxicity . introduction of a novel , peptide receptor - mediated , mechanism of cell internalization of the drug might allow by - passing natural mechanism of cell resistance . the authors then chose several different molecules , commonly used in classical tumor chemotherapy - methotrexate ( mtx , chlorarnbucil ( clb ), 6 - mercaptopurine and 5 - fluoro - 2 ′- deoxyuridine ( 5 - fdu )— or emerging new drugs — combretastatin ( cbtst ) 16 , monastrol ( mon ) 17 , tirapazaminc ( tpz ) 18 . the molecules were tested either as free drugs or conjugated to tetra - branched on ht - 29 , panc - 1 and pc - 3 tumor cell lines ( fig3 and 4 ). in details , ht - 29 , panc - 1 or pc - 3 cells were plated at a density of 2 . 5 × 10 4 per well in 96 - well microplates . different concentrations of free or nt - conjugated drugs , from 0 . 15 to 30 μmol / l , were added 24 h after plating . cells were grown without changing the medium for days . growth inhibition was assessed by 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide . ec50 values were calculated by non - linear regression analysis using graphpad prism 3 . 02 software . the best ec50 values ( expressed in molar concentrations ) obtained were : 1 . 9e - 006 for nt ( 8 - 13 ) 4 - clb on ht29 , 3 . 3e - 007 for nt ( 8 - 13 ) 4 - 5fdu on ht29 , 1 . 4e - 7 for nt ( 8 - 13 )- tpz on . pc3 , 43e - 007 for nt ( 8 - 13 ) 4 - cbtst on panc - 1 and 1 . 1e - 007 for nt ( 1 - 13 ) 4 - 5fdu on ht29 . the cellular toxicity of all the drug - conjugated nt4 was tested on the three cell lines and compared with the cytotoxicity of corresponding free drugs and with that of an unrelated tetra - branched peptide ( u4 ), identically conjugated to the same drug . 5 - fdu , monastrol and cbtst were fast released from the adducts . cytotoxicity of fast releasing drug - conjugated tetra - branched peptides was then tested in ht - 29 , panc - 1 and pc - 3 in experiments where cells were exposed to a 1 hour pulse of free or nt - conjugated drug , washed and incubated for 6 days or , alternatively incubated for 6 days with the peptides , without additional washing . the additional washing was performed in order to avoid free drug to diffuse inside the cells during the following six days . conjugation to tetrabranched nt ( 8 - 13 ) profoundly modified drug activity , which might result from the combination of both cell and drug features , including : i ) cell sensitivity to the drug ; ii ) drug mechanism of action ; iii ) mechanisms of cell resistance to the drug ; iv ) efficiency of membrane transport of the conjugated - drug . as expected , activities of the free drugs are very different from one another and from cell line to cell line ( fig3 and 4 , second panel from the left ). in principle , the conjugation to tetrabranched nt may produce as a result : an increase in drug specificity , an increase in drug activity , an increase in both specificity and activity or no improvement of the free drug . in some cases , like mtx and clb in pc - 3 , conjugation to branched nt can by - pass natural cell resistance to the drug ( fig3 ) switching the cells from completely non - sensitive to full responsive . an undoubted advantage of the branched peptide carrier is its target specificity , demonstrated by the lack of activity on the three cell lines of any drug ; when coupled to an unrelated branched peptide ( see all third panels from left fig3 and 4 ). the tetrabranched nt ( 8 - 13 )- peg - 6 - mercaptopurine [ nt4 ( 8 - 13 )- 6 - mp ] 4 and tetrabranched nt ( 8 - 13 )- peg - monastrol [ nt4 ( 8 - 13 )- mon ]] 6 gained no improvement when compared to the parent free drugs . results with fast releasing tetra - branched nt are very interesting , since both in the case of 5 - fdu and cbtst , activity of the drug is clearly increased by conjugation to branched nt4 ( fig4 panels f and g ). comparison of results obtained with cbtst in slow and fast releasing molecules , indicate that fast releasing molecules can be even more active than slow - releasing compounds . interestingly , cell that are not affected by a drug ; such as pc - 3 by mtx , clb and 5 - fdu , can become sensitive to it , when conjugated to branched nt ( fig4 panels a , b and f ). changing the mechanism of membrane transport , by switching to a peptide receptor - mediated mechanism , can deeply modify drug transport from outside to inside the cells . moreover , conjugation to branched peptides might as well impair mechanisms of drug export from inside to outside the cell , entrapping the conjugated drug into the target cell . this is extremely important for the therapy of tumors that over - express nt receptors and 1 . 0 do not respond to classical chemotherapy . analysis of human tumor samples from surgical resections using fluorophore - conjugated nt4 in order to validate the nt branched peptides of the present invention as possible targeting agents for therapy of either colon or pancreas adenocarcinoma , binding of tetra - branched nt peptides to human tumor surgical samples in comparison to healthy tissues , was analysed and quantified . surgical resections of 16 colon and 12 pancreas tumors were collected . tumor samples were compared to healthy tissues from the same patient obtained 5 cm away from the tumor edge . serial sections of the same biopsy were analysed both by hematoxylin / eosin ( h & amp ; e ) light microscopy and by fluorescent confocal microscopy . in details , samples were embedded in tissue tck and stored in liquid nitrogen . 10 μm thick sections , obtained with a 2800 frigocut n ( reichert - jung , depew , n . y . ), were dried at 37 ° c . for 30 min , fixed with 4 % formalin for 15 min at room temperature and incubated in glycine 0 . 1m for 12 hours at 4 ° c . blocking with fbs for 30 min at 37 ° c . was followed by 30 min incubation at room temperature either with nt ( 1 - 13 ) 4 - fluorescein ( 1 μg / ml in tbs — 0 . 3 % bsa ). each step was followed by washing with tbs . finally , sections were incubated for 0 . 5 min with 4 , 6 - diamidino - 2 - phenylindole ( dapi ) ( 1 μg / ml in tbs — 1 % bsa ). each step was followed by washing with tbs . controls were performed using an unrelated fluorescein - conjugated tetra - branched peptide . analogue monomeric peptides were assayed for comparison . peptide binding was analyzed by confocal laser microscope ( leica tcs sp5 ) with 488 nm absorption and 500 - 540 emission wavelength for fluorescein and 405 absorption and 420 - 460 emission for dapi . all images were processed using the imagej software ( nih ). resulting electronic data were reported as pixel distribution in the green color range of the rgb system . this enabled translation of the immunofluorescence signals of tumor and healthy tissues into numbers representing the mean of green staining in the range of the rgb system , for each sample ( fig5 panel b ). when treated with nt ( 1 - 13 ) 4 - fluorescein tumor tissues from both colon and pancreas adenocarcinoma showed remarkably higher fluorescence emission compared to normal tissues from the same patients ( fig5 panel a ). binding of nt ( 1 - 13 ) 4 to any tissue sample was identical to that of nt ( 8 - 13 ) 4 to the same sample . 14 out of the 16 colon cancers were histologically characterized as adenocarcinomas and 2 as adenomas . the latter had k / h values corresponding to the lowest range of the k / h ranking . for pancreas samples , 11 out of the 12 samples were adenocarcinomas and one was a lymphoma , ie it had a very different cell origin . this sample had the lowest k / h of all tested surgical samples . no correlation was found between staging of the tumors , either colon or pancreas , and receptor expression ( k / h value ). this means that even at early stages of the disease the difference between k and h tissues is statistically relevant . this is a very important point for a tumor marker that is not uniquely expressed by tumor cells but rather overexpressed by them . nt receptors might then be used as targets for early treatments with branched peptides ( table 1 , fig6 ) legend : nos : not otherwise specified . grading = tumor grading on the basis of cytology observations . tnm ( international staging of tumors ): t = tumor size ; n = number of lymph nodes involved , m = number of metastasis . statistical analysis was performed to evaluate significance of difference in peptide binding between healthy and tumor tissues from all collected surgical samples , except for the lymphoma because of its completely different cell origin , with respect to colon and pancreas carcinomas . as shown in the box - plots ( fig6 ), a remarkable difference in signal was observed for both pancreas and colon cancers , with respect to their healthy counterparts . for the comparison between healthy and tumor samples , the level of significance was p & lt ; 0 . 01 for pancreas and p & lt ; 0 . 02 for colon for two - sided testing . this result means that these peptides can discriminate between healthy and cancer samples , therefore they can be used as tumor markers . this result is very promising for possible therapy of colon carcinoma and pancreas exocrine carcinoma by means of nt - branched peptides . imaging of tumor biopsies might enable pre - treatment estimation of the efficacy of a nt - based target therapy , which might be evaluated on the basis of measured differences of branched peptide binding in tumor versus healthy tissue , in each patient . moreover , such a clear discriminating signal between healthy and tumor tissues indicates that branched nt might play a remarkable role in the specific diagnosis of colon and pancreas carcinoma . in vivo activity of branched nt peptides conjugated to functional unit . nude mice bearing ht29 tumors in the right flank were injected with six doses of nt ( 1 - 13 )- 5fdu . compound nt ( 1 - 13 )- 5fdu was chosen for the in vivo experiments among all , in the light of its in vitro cytotoxicity on ht29 , when compared to the analogue peptides coupled to mtx . in details , cd - 1 female nude mice ( charles river laboratories , inc . ), 5 to 6 weeks of age ( mean weight , 20 g ), were injected s . c . in the right flank with 1 × 10 6 ht29 cells . when tumors reached a diameter of 3 to 4 mm ( 5 days after tumor inoculation ) mice were randomly divided into groups ( four mice per group ) and repeatedly injected in the tail vein ( 0 , 30 , 70 , 140 , 190 , 240 hours post first injection ) with 500 μl of the following solutions in 0 . 9 % nacl : ( a ) 1 mg / ml nt ( 1 - 13 ) 4 - 5fdu ( 3 . 05 μmol / kg ); ( b ) 30 μg / ml 5fdu ( 3 . 05 μmol / kg ); and ( c ) 0 . 9 % nacl . tumor volumes were measured daily with a caliper using the following formula : volume = length * width2 * π / 6 . 21 days after tumor inoculation , ie 6 days after the last treatment , mice were sacrificed , tumors , removed and weighted . experiments were done following the local ethical committee approval for animal use in cancer research . the procedures related to animal use conform to all regulations protecting animals used for research purposes , including ukcccr ( 1998 ) united kingdom co - ordinating committee on cancer research guidelines for the welfare of animals in experimental neoplasia . 2nd ed br j cancer 77 : 1 - 10 . statistical analysis was done using student &# 39 ; s t test ( p & lt ; 0 . 05 ). after the last treatment ( 240 h ), the average tumor volume of the mice treated with nt ( 1 - 13 )- 5fdu was around 50 % that of animals treated with free 5fdu or with saline ( fig7 a ). tumor weight , measured 5 days after the last treatment ( 360 h ), was 40 % less in mice treated with the drug conjugated peptide than in the control groups ( fig7 c ). free 5 - fdu given six times , in a dose of 3 μg / kg , in time - frame of 10 days , gave only 10 % inhibition of tumor growth . 3 . langer m and beck - sickinger a g . curr med chem anti - canc agents . 2001 ; 1 ( 1 ): 71 - 93 . 4 . garcia - garayoa e , allemann - tannahill l , blauenstein p , willmann m , carrel - remy n , tourwe d , iterbeke k , conrath p , schubiger p a . nucl med biol . 2001 ; 28 ( 1 ): 75 - 84 . 5 . hillairet de boisferon m , raguin o , thiercelin c , dussaillant m , rostene w , barbet j , pelegrin a , gruaz - 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