Patent Application: US-24838199-A

Abstract:
the invention relates to the use of peptides individually or in combination , for treating and / or preventing angiogenesis . it also relates to the use of peptide analogs or a combination of peptides referred to as muj - 7 as anticancer drugs in restricting the tumor growth and spread by inhibiting tumor angiogenesis . muj - 7 , in addition inhibits metastasis through its antiangiogenic activity in all cancers . the invention also relates to a pharmaceutical composition containing either individual peptides or in combination , and methods of treatment of human beings and animals for curing and / or preventing angiogenesis .

Description:
we have observed that vip ( vasoactive intestinal peptide ), somatostatin , substance p , and bombesin are secreted by at least some human tumor and cancer cells and that there are binding sites for these peptides on these cells . specifically , out of a number of peptide growth regulators studied by indirect immunofluorescence , the four peptides ( i . e ., vasoactive intestinal peptide ( vip ), somatostatin , substance p , and bombesin ) were shown to bind , to tumor cells . ( herein , the terms “ peptide growth regulators ”, and “ peptides ” each refer to vip , somatostatin , substance p , and bombesin ). it may be that there is an autocrine mechanism for cell proliferation where the peptides are secreted by tumor cells and transduce a signal through specific receptors on the same cell type leading to cell proliferation . as will be described in more detail below , the effects of the analogs of somatostatin , vip , bombesin , substance p on the tumor cell growth and survival were studied using different assay systems . the amino - acid sequences of the seven analogs ( vip 1 , vip 2 , vip 3 , som 1 , som 1 , bom 2 and sp 1 ) are disclosed in u . s . application ser . no . 08 / 727 , 679 . as will be explained in more detail below , the combination of these seven analogs is known as muj - 7 . the analogs were synthesized manually and using a conventional peptide synthesizer . an example of a combination within the scope of the invention comprises som 2 , vip 1 , vip 2 , vip 3 , som 1 , bom 1 , and sp 1 . a combination hereinafter referred to as muj - 7 , was prepared using the following seven peptide analogs : ( 1 ) vip 1 , ( the vip antagonist ) having a molecular weight of approximately 3464 . 9 and a concentration of approximately 10 − 7 m ; ( 2 ) vip 2 . ( the receptor binding inhibitor ) having a molecular weight of approximately 1027 . 55 and a concentration of approximately 10 − 8 m ; ( 3 ) vip 3 , ( the vip receptor antagonist ) having a molecular weight of approximately 3342 . 09 and a concentration of approximately 10 − 8 m ; ( 4 ) som 1 ( the somatostatin analog ( ctop ) having a molecular weight of approximately 1061 . 59 and a concentration of approximately 10 − 9 m ; ( 5 ) som 2 , ( the analog of somatostatin ) having a molecular weight of approximately 1637 . 0 and a concentration of approximately 10 − 8 m ; ( 6 ) bom 1 ( the bombesin antagonist ) having a molecular weight of approximately 983 . 55 and a concentration of approximately 10 − 8 m ; and ( 7 ) sp 1 ( the substance p antagonist ) having a molecular weight of approximately 1515 . 83 and a concentration of approximately 10 − 8 m . the preceding sentence sets forth the preferred concentrations of the seven analogs comprising muj - expected that muj - 7 would be effective if the concentration of each of the seven analogs ranged from approximately 10 − 6 m to approximately 10 − 12 m . muj - 7 may be prepared in the following way . a stock solution of each of the seven peptide analogs is prepared with a ph of approximately 7 . 0 to approximately 7 . 4 . although sterile phosphate buffered saline was used to prepare the stock solutions for the testing described below , other diluents may be used such as rpmi 1649 , buffered saline , isotonic nacl , ringer &# 39 ; s solution , water ( for injection ) distilled water , polyethylene glycol ( near or in water ), 2 % tween in water , dimethylsulfoxide to 50 % in water , propylene glycol ( neat or in water ), balanced salt solution , glycerol , and other conventional fluids that are suitable for intravenous administration to obtain a ph in the range of approximately 7 . 0 to approximately 7 . 4 for each stock solution , the ph can be adjusted by using 1 n hcl for lowering the ph or 1 n naoh for raising the ph , although the conventional agents for adjusting the ph can be used , the concentration of the peptide analog in each stock solution is approximately 10 − 3 m . aliquots of the seven peptides analogs are mixed together such that the muj - 7 formulation contained approximately equal weights of each of the seven peptide analogs . in muj - 7 , approximately , the concentration of vip 1 , is 10 − 7 m ; the concentration of vip 2 is 10 − 8 m ; the concentration of vip 3 is 10 − 8 m ; the concentration of som1 is 10 − 9 m ; the concentration of som 2 is 10 − 8 m ; the concentration of bom1 is 10 − 8 m ; and the concentration of sp 1 is 10 − 8 m . in one exemplary embodiment , the ph of the muj - 7 solution may range from about 7 . 0 to 7 . 4 . to obtain a ph in this range , the ph can be adjusted by using 1 n ncl for lowering the ph or 1 n naoh for raising the ph , although other conventional agents for adjusting the ph can be used . in addition , a number of novel peptides also exhibited an antiangiogenic effect . these peptides are : muj - 7 was tested against primary tumor cells of human colon adenocarcinoma , and each of the peptide analogs comprising muj - 7 was tested individually against human colon adenocarcinoma cells . a three day mtt cytotoxicity assay was performed as described in u . s . patent application ser . no . 08 / 727 , 679 . the percent killing achieved by individual peptides was in the range of 54 to 79 % while percent killing achieved by muj - 7 was 94 %. five different subcombinations of seven peptide analogs comprising muj - 7 were tested against human colon adenocarcinoma cells . each subcombination was tested by performing a one day mtt cytotoxicity assay as described in u . s . patent application ser . no . 08 / 727 , 679 . the percent killing achieved by the subcombinations was in the range of 64 . 7 % to 94 . 9 %. ecv . 304 cells collected at exponential growth phase were resuspended in medium ( 3 . 3 × 10 6 cells / ml in rpmi 1640 containing 10 % fbs ). 150 μl of medium was added to the wells of a 96 - well tissue culture plate ( nunc , denmark ) followed by 30 μl of cell suspension . the plate was left in incubator ( 37 ° c ., 5 % co 2 ) overnight . each of the seven peptides of the combination muj - 7 were added at three molar concentrations of 10 − 7 m , 10 − 8 m and 10 − 9 m . 20 μl of muj - 7 at three concentrations of n / 10 , n and 10n was added to marked wells of the 96 - well plate . the value of n for each of the individual peptides was 10 − 8 m for vip 2 , bom 1 , sp 1 , vip 3 , and som 2 and 10 − 7 m for vip 1 , and 10 − 9 m for som 1 . each concentration was plated in triplicate . 20 μl of medium alone was added to control wells while wells without cells served as blanks . a total volume of 200 μl was ensured in each well and the plate was left in an incubator ( 37 ° c ., 5 % co 2 ). after 72 hours of incubation an mtt assay was performed and percentage inhibition in proliferation of treated cells was calculated with respect to control cells . results are given in table i . ecv304 cells collected at exponential growth phase were resuspended in medium ( 3 . 3 × 10 6 cells / ml in rpmi 1640 containing 10 % fbs ). 150 μl of medium was added to the wells of a 96 - well tissue culture plate ( nunc , denmark ) followed by 30 μl of cell suspension . the plate was left in incubator ( 37 ° c ., 5 % co 2 ) overnight . novel peptides shown below were incubated with cells at concentration range of 10 − 7 to 10 − 9 m ( except dt71 at concentration range 10 − 8 to 10 − 10 m ). each concentration was plated in triplicates . 20 μl of medium alone was added to control wells while wells without cells served as blanks . a total volume of 200 μl was ensured in each well and plate was left in incubator ( 37 ° c ., 5 % co 2 ). after 72 hours of incubation an mtt assay was performed and percentage inhibition in proliferation of treated cells was calculated with respect to control cells . results are given in table ii . polycarbonate filter transwell inserts ( 24 well size ) with 8 μm pores ( nunc , denmark ) were used for the migration assay . ecv304 ( 10 4 cells / 200 ul dmem containing 0 . 1 % bsa ) was added to the upper chamber . the lower chamber contained 600 ul of dmem with 0 . 1 % bsa . individual peptides of the composition muj - 7 at six different concentrations each of n / 100 , n / 10 , n , 10n , 100n and 1000n were added directly to the upper well and the plate incubated at 37 ° c . for 24 hours . the cells migrated to the lower chamber were viewed randomly at five different phase - contrast microscopic fields and the total number of cells counted using video pro 32 image analysis system . percent inhibition in migration was determined with reference to the control . similarly , five different concentrations of n / 100 , n / 10 , n , 10n , 100n and 1000n of muj - 7 were added directly to the upper well and the plate incubated at 37 ° c . for 24 hours . the cells migrated to the lower chamber were counted as described above . the molarity representing n for each peptide is given in table no . 3 . where the value of n for each peptide is 10 − 8 m for vip 2 , bom 1 , sp 1 , vip 3 , and som 2 and 10 − 7 m for vip 1 , and 10 − 9 m for som 1 . polycarbonate filter trasnwell inserts ( 24 well size ) with 8 μm pores ( nuno , denmark ) were used for the migration assay . ecv304 ( 10 4 cells / 200 ul dmem containing 0 . 1 % bsa ) was added to the upper chamber . the lower chamber contained 600 ul of dmem with 0 . 1 % bsa . individual peptides at optimum concentration ( as indicated in table 4 ) were added directly to the upper well and the plate incubatedat 37 ° c . for 24 hours . the sequence of each of the peptides added are described above . the cells migrated to the lower chamber were viewed randomly at five different phase - contrast microscopic fields and the total number of cells counted using video pro 32 image analysis system . percent inhibition in migration was determined with reference to control . polycarbonate filter transwell inserts ( 24 well size ) with 8 μm pores ( nunc , denmark ) were used for the migration assay as described in the previous example . ecv304 ( 10 4 cells / 200 ul dmem containing 0 . 1 % bsa ) was added to the upper chamber . the lower chamber contained 600 ul of dmem with 0 . 1 % bsa . ten different subcombinations of muj - 7 listed in table 5 were tested for inhibition of migration of endothelial cells across the filter as described previously . none of the ten subcombinations caused percent inhibition of migration greater than muj - 7 and was in the range of 11 . 3 to 69 . 3 % ( table 6 ). matrigel ( 350 ul ) was placed into each well of a 24 - well culture plate at 4 ° c . and was allowed to polymerize by incubation at 37 ° c . for 30 min . ecv304 ( 1 . 5 × 10 4 ) were seeded on the matrigel in 500 ul dmem supplemented with 10 % fbs . muj - 7 at four different concentrations of 0 . 25 ×, 0 . 5 ×, ×, & amp ; 5 . 0 × were added and . the plate incubated at 37 ° c . for 24 hour . “×” denotes normal concentration of muj - 7 where molar concentration of individual peptides was 10 − 8 m for vip 2 , bom 1 , sp 1 , vip 3 and som 2 and 10 − 7 m for vip 1 and 10 − 9 m for som 1 . tube like structures formed , the area of which was individually counted at five different phase - contrast microscopic fields using video pro 32 image analysis system . percent inhibition in tube area was determined with reference to untreated cells . matrigel ( 350 ul ) was placed into each well of a 24 - well culture plate at 4 ° c . and was allowed to polymerize by incubation at 37 ° c . for 30 min . ecv304 ( 1 . 5 × 10 4 ) were seeded on the matrigel in 500 ul dmem supplemented with 10 % fbs . new analogs of peptides as shown in fig1 were added at optimum concentration as denoted in table 8 , and the plate incubated at 37 ° c . for 24 hours . tube like structures formed , the area of which was individually counted at five different phase - contrast microscopic fields using video pro 32 image analysis system . percent inhibition in tube length was determined with reference to untreated cells . matrigel ( 350 ul ) was placed into each well of a 24 - well culture plate at 4 ° c . and was allowed to polymerize by incubation at 37 ° c . for 30 min . ecv304 ( 1 . 5 × 10 4 ) were seeded on the matrigel in each of the 24 wells . 500 ul dmem supplemented with 10 % fbs and was added to 8 wells . these wells served as controls . culture supernatants of primary tumor cells of human colon adenocacinoma from a confluent culture in log phase of growth was filtered through a 0 . 22 u filter and its ph adjusted to 7 . 4 with sodium bicarbonate . 500 ul of this supernatant was added to another 8 wells , containing ecv304 ( 1 . 5 × 10 4 ) cells seeded on matrigel . to the remaining 8 wells , muj - 7 was added and incubated at 37 ° c . for 24 hours . five different phase - contrast microscopic fields ( 4 ×) were reviewed and total tube length of the tube - like - structures ( tls ) measured using video pro 32 image analysis system . percent inhibition of tls was calculated with reference to controls . in controls , although sprouting was visible at day 1 , intussusception was still missing . however , endothelial cells treated ptc culture supernatant showed enhanced sprouting as well as intussusception leading to completion of tube formation . this clearly suggests pro - angiogenic activity of factors in culture supernatant of primary tumor cells of human colon adenocarcinoma . treating ptc culture supernatant treated endothelial cells with muj - 7 resulted in complete inhibition of intussusception and a significant decrease in sprouting . this indicates the ability of muj - 7 to inhibit two key steps of a new blood vessel formation , i . e ., sprouting and angiogenesis . a qualitative representation of the effect is given in table 9 . matrigel ( 350 ul ) was placed into each well of a 24 - well culture plate at 4 ° c . and was allowed to polymerize by incubation at 37 ° c . for 30 min . ecv304 ( 1 . 5 × 10 4 ) were seeded on the matrigel in 500 ul dmem supplemented with 10 % fbs . individual peptides of the composition muj - 7 at three different concentrations each of n / 10 , n , and 10n were added and the plate incubated at 37 ° c . for 24 hours . tube like structures formed , the length of which were individually counted at five different phase - contrast microscopic fields using video pro 32 image analysis system . percent inhibition in tube length was determined with reference to control . similarly , three different concentrations of n / 10 , n , and 10n of muj - 7 were added to the well and the plate incubated at 37 ° c . for 24 hours . percent inhibition in tube length was determined with reference to control as described previously ( table 10 ). where the value of n for each peptide is 10 − 8 m for vip 2 , bom 1 , sp 1 , vip 3 , and som 2 and 10 − 7 m for vip 1 , and 10 − 9 m for som 1 . matrigel ( 350 ul ) was placed into each well of a 24 - well culture plate at 4 ° c . and was allowed to polymerize by incubation at 37 ° c . for 30 min . ecv304 ( 1 . 5 × 10 4 ) were seeded on the matrigel in each of the 24 wells . 500 ul dmem supplemented with 10 % fbs was added to 8 wells . these wells served as controls . culture supenatants of primary tumor cells of human colon adenocarcinoma from a confluent culture in log phase of growth was filtered through a 0 . 22 u filter and its ph adjusted to 7 . 4 with sodium bicarbonate . 500 ul of this supematant was added to another 8 wells contains ecv304 ( 1 . 5 × 10 4 ) cells seeded on matrigel . to the remains 8 wells , muj - 7 was added and incubated at 37 ° c . for 24 hours . five different phase - contrast microscopic fields ( 4 ×) were viewed and total tube length of the tube - like - structures ( tls ) measured using video pro 32 image analysis system . percent inhibition of tls was calculated with reference to controls . in controls , 76 . 9 % of tls were in the range of 50 - 69 μm and remaining were between 69 - 107 μm . there were no structures greater than 107 μm . in ptc stimulated wells , the percentage of tls in range of 50 - 69 μm reduced to 50 . 0 % while 27 . 3 % were in the range of 69 - 107 μm . it is interesting to note that there were approximately 18 . 0 % tls in the range of 107 - 183 μm and 4 . 55 % tls in 221 - 240 μm . hence there was a significant increase in the length of tls in ptc supernatant stimulated cells as compared to the unstimulated controls . this clearly suggests pro - angiogenic activity of factors in culture supematant of primary tumor cells of human colon adenocarcinoma . in the third group of wells , which were treated with muj - 7 , 81 . 25 % tls were in the range of 50 - 69 μm and 18 . 75 % in the range of 69 - 88 μm ( table 6 ). this reversal of tls activity to control level on treatment with muj - 7 clearly suggests anti angiogenic activity of muj - 7 . 10 . o &# 39 ; reilly , m s , holmgren l , shing y , chen c , rosenthal r a , moses m , lane w s , cao y , sage e h , folkm n j . arigiostatin : a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma . cell , 19 , 315 ( 1994 ). 11 . o &# 39 ; reilly , m s , holmgren l , chen c , folkman j . angiostatin induces and sustains dormancy of human primary tumors in mice . nature med , 2 , 689 ( 1996 ). 12 . folkman , j . tumor angiogenesis . in : holland j r , frei e , bast r , kule d , morton d , weichselbaum r , eds . cancer medicine , ed 4 . baltimore : williams & amp ; wilkins ( 1996 ). 13 . modzelewski r a , davies p , watkins s c , auerbach r , chang m - j , johnson c s . isolation and identification of fresh tumor - derived endothelial cells from a murine rif - 1 fibrosarcoma . cancer res 54 , 336 ( 1994 ). 14 holmgren l , o &# 39 ; reilly , m s , folkman j . dormancy of micrometastases : balanced proliferation and apoptosis in the presence of angiogenesis suppression . nature med 1 , 149 ( 1995 ). 15 . teicher b a , holden s a , gulshan a , alvarez sotoinayer e , huang s d , chen y - n , brem h . potentiation of cytotoxic cancer therapies by tnp - 470 alone and with other antiangiogenic agents . int j cancer 57 , 920 ( 1994 ). 16 . dawson d w , pearse s f , zhong r , silverstein r l , frazier w a , bouck n p cd36 mediates the invitro inhibitory effects of thrombospondin - 1 on endothelial cells . j cell bio ; 138 ( 3 ): 707 - 17 ( 1997 ). 17 . anand - apte b , bao l , smith r , iwata k , olsen b r zetter b , apte s s . a review of tissue inhibitor of metalloproteinases - 3 ( timp - 3 ) and experimental analysis of its effect on primary tumor growth . biochem cell biol ; 74 ( 6 ): 853 - 62 ( 1996 ). 18 . yeung , a k . human cytostatin , a novel anti - angiogenic cytokine . diss abstr int ( b ); 55 ( 12 ): 5300 ( 1995 ). 19 . wu z , o &# 39 ; reilly m s , folkman j , shing y . suppression of turmor growth with recombinant murine angiostatin . biochem biophys res commun ; 236 ( 3 ); 651 - 4 ( 1997 ). 20 . minchinton a i , fryer k h , wendt k r , clow k a , hayes m m . the effect of thalidomide on experimental tumors and metastases . 21 . dameso r , del tacca m . the effects of the somatostatin analog octreotide on angiogenesis in vitro . metabolism 45 ( 8 ): 49 - 50 ( 1996 ). 22 . woltering e a , barrie r , o &# 39 ; dorisio t m , arce d , ure t , cramer a , holmes d , robertson j , fassler j . somatostatin analogues inhibit angiogenesis in the chick chorioallantoic membrane . j surg res 50 ( 3 ): 245 - 251 ( 1991 ). 23 . wiedermann c j , auer b , sitte b , reinisch n , schratzberger p , kahler c m induction of endothelial cell differentiation into capillary - like structures by substance p . eur j pharmacol 298 ( 3 ): 335 - 338 ( 1996 ). 24 . ziche m , morbidelli l , masini e , amerini s , granger h j , maggi c a , geppetti p , ledda f . nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance p . j clin invest 94 ( 5 ): 2036 - 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