Patent Application: US-76641201-A

Abstract:
the present invention provides peptides having potent anti - angiogenic activity and endothelial cell proliferation inhibition activity . the peptides can be administered as pharmaceutical compositions for prevention or treatment of undesired angiogenesis , for instance for prevention of tumor metastasis or inhibition of primary tumor growth .

Description:
small peptides were designed according to the amino acid sequences of the kringle domains of human plasminogen or from the amino acid sequences of endostatin , vegf , flt - 1 or kdr / flk - 1 using the “ proline bracket model ” described in references 14 and 15 . the peptides were synthesized using a peptide synthesizer ( applied biosystems , inc ., model 473a ). the peptides were then purified by typical hplc methods using a c18 reverse - phase column and eluting with an acetonitrile gradient . angiostatin - derived peptides of the present invention represent a portion of a kringle domain of plasminogen . the effective portion of a kringle domain is generally residues 30 to 38 or 39 of a kringle domain , numbered as in reference 13 . however , it is acceptable to add additional amino acids to this generally effective core peptide . thus , peptides representing residues 27 to 40 or 41 of a kringle domain would be expected to be effective . preferred peptides of the invention are derived from human or mouse plasminogen . the exemplified peptides of seq id nos : 1 - 3 , 11 and 12 represent residues 29 - 38 or 29 - 39 of human plasminogen kringle domains ( 13 ). the peptides of the invention preferably range from 8 to 20 amino acids long . peptides of the invention typically range from 7 - 20 , more typically from 10 - 15 amino acids in length . the peptides are often about 10 amino acids in length . the peptides are more preferably 8 , 9 or 10 residues long , most preferably 10 residues long . the peptides of the invention may contain a single cysteine residue . in such a case , it is desirable that the cysteine residue be derivatized to block disulfide bond formation . most preferably , peptides of the invention lack cysteine residues altogether . the peptides also preferably contain a pair of proline residues . each proline residue of the pair is preferably the residue penultimate to a terminus of a peptide , but either or both of the proline residues can be a terminal residue . imino acids similar to proline , in that their side chains form a ring containing the peptide bonding nitrogen and a - carbon , and that will “ break ” an α - helix or β - sheet secondary structure , can be substituted for one or more of the proline residues . exemplary peptides are shown as seq id nos : 1 - 50 . the more preferred peptides are those of seq id nos : 1 - 3 , 11 , 12 , 29 - 33 , 35 - 38 , 40 , 41 , and 44 - 50 . the peptides of the invention are active in inhibiting angiogenesis in the in vitro bovine aorta endothelial ( bae ) cell proliferation assay with an ic 50 of 20 μm or less , preferably 5 μm or less , more preferably 1 μm or less and most preferably below 0 . 1 μm . much preferred peptides of the invention are active in the in vitro bae cell proliferation assay with an ic 50 of about 20 pm . alternatively , preferred peptides of the invention will exhibit inhibition of angiogenesis in a chick chorioallantoic membrane assay of at least 30 % ( i . e . inhibition is observed under at least 3 of 10 coverslips applied ) at a dose of 50 μg / coverslip . more preferably at least 50 % inhibition is observed at a dose of 25 μg / coverslip . most preferably , inhibition in the range of 50 - 80 % is observed for a dose ranging from 10 - 25 μg / coverslip . also , the peptides of the invention encompass peptides that have been derivatized , for example to improve transport across membranes by attachment of hydrophobic groups or that are carboxy - terminal amidated to improve resistance to serum proteases . methods for accomplishing such derivatizations are known in the art . formulation of the peptides for administration is standard in the art . it is anticipated that optimization of formulation , dosages and schedule of administration is also standard in the art . reference 16 , especially at parts 4 , 5 and 7 , provides guidance in these matters . administration of the peptides for anti - tumor treatment can be by any route , preferably oral , intravenous , intradermal , subcutaneous or aerosol . it is expected that subcutaneous administration is especially effective when anti - metastatic activity is desired ( see , reference 6 ). the dosage of the peptides will depend upon the therapeutic index of the compound . it is noted that angiostatin has been administered to mice in amounts of 100 mg / kg / day without observable toxicity . inhibition of metastasis by angiostatin is observed at doses of about 1 mg / kg / day and inhibition of primary tumor growth is observed at about 10 mg / kg / day ( 6 ). thus , it is expected that effective doses of the peptides of the present invention will be about 0 . 2 μg / kg / day to about 2 mg / kg / day for inhibition of metastasis . preferred dosages are in the 2 - 200 μg / kg / day range , most preferred dosages are in the 20 - 50 μg / kg / day range . it is expected that the dosage ranges will be about 10 - fold higher for inhibition of primary tumor growth . the peptides were tested in an in vitro bovine aorta endothelial cell ( bae ) proliferation assay for their ability to inhibit bae cell proliferation . cells were plated into 24 - well culture plates at 1 . 25 × 10 4 cells / well in complete sfm - endothelial medium ( gibco , usa ). after 24 h , the medium was removed and various amounts of peptides were added into the wells in 0 . 5 ml of sfm - endothelial medium without bfgf . after 30 minutes incubation , bfgf was added into each well at a final concentration of 1 ng / ml together with another 0 . 5 ml of sfm - endothelial medium . seventy - two hours later , cells were trypsinized and counted using a hemocytometer . commercial e . coli recombinant human angiostatin ( 1 μm ) was used as a control in the experiments . peptides angio - 2 , angio - 3 and angio - 4 , containing sequences from kringles 2 , 3 and 4 of human plasminogen ( seq id nos : 2 , 3 , and 11 ), all inhibited bae cell proliferation in a dose - dependent manner ( fig1 , 2 and 3 ). the ic 50 of the three peptides are about 2 mm , 20 nm and 200 um respectively . it is noted that peptide angio - 3 inhibited bae cell proliferation with an ic 50 in a range similar to the reported nanomolar range of angiostatin protein ( 5 ). in contrast , a randomly scrambled version of angio - 3 ( angio - 3s ) in which the same amino acids present in angio - 3 were put in a random order , completely lost angiogenesis inhibition ability ( fig1 ). this indicates that the antiangiogenic activity of the peptides are sequence dependent . no obvious cell cytotoxicity was observed by peptide angio - 3 at 20 μm with bae cells analyzed by trypan blue staining and microscopic cellular morphology analysis . the bae inhibition assay provides a method for determining the anti - angiogenic activity of peptides . for example , peptides angio - 3a , - 3b , - 3c , - 3d , - 3e and - 3f ( seq id nos : 4 - 9 ) are peptides in which the six amino acids surrounded by the two prolines are individually mutated into alanine . the contribution of each of the six amino acids between the proline residues to the biological activity of angio - 3 can be determined by testing these peptides . since peptides angio - 2 , angio - 3 and angio - 4 all inhibited bae cell proliferation , they were also tested in the chick embryo chorioallantoic membrane ( cam ) in vivo angiogenesis assay ( 7 ). in this assay , the peptides are coated onto 2 - 3 mm glass squares cut from microscope cover slips . all three peptides inhibited chick cam angiogenesis ( fig4 , 5 and 6 ). peptide angio - 3 reduced microvessel density at the very low dose of 12 pg / cover slip ( the lowest dose tested , comparing to a pbs control ). as a positive control , recombinant angiostatin at the dose of 100 ug / cover slip was tested in the same experiment . the amount of inhibition ( judging from the extent of reduction in microvessel density ) from 100 ug recombinant angiostatin is equivalent to 1 . 2 ug of angio - 3 peptide . based on this , we estimated that angio - 3 is at least as potent as angiostatin in inhibiting chick cam angiogenesis . at high doses , e . g ., 1 mg / cover slip , the blood vessels under the cover slip containing all three peptides died and circulation was stopped . the color of the blood changed into dark red instead of the bright red of the circulating blood . the above data demonstrated that angio - 2 , angio - 3 and angio - 4 , small peptides derived from the sequence of human plasminogen kringle domains 2 , 3 and 4 function as potent angiogenesis inhibitors . importantly , peptide angio - 3 is more potent in inhibiting bae cell proliferation compared to angiostatin protein and at least as potent as recombinant angiostatin in inhibiting chick cam angiogenesis . cao et al . ( 12 ) show that integrity of complete kringle domains is required for anti - angiogenic activity of angiostatin and that a tandem pair of properly folded domains is necessary . cao et al . ( 13 ) also show that , at least with respect to the k5 domain , appropriate disulfide bond formation is necessary for maintaining anti - angiogenic activity . thus , it is surprising that the exemplified small peptides of the invention , which ( 1 ) do not form complete kringle domains and ( 2 ) do not contain any cysteine residues and therefore do not form disulfide bonds , have such high activity . peptides were tested in an in vitro bovine aorta endothelial cell ( bae ) proliferation assay for their ability to inhibit bae cell proliferation . cells were plated into 24 - well culture plates at the number of 1 . 25 × 10 4 / well in complete sfm - endothelial media ( gibco , usa ). after 24 hours , the medium was removed and various amounts of peptides were added into the wells in 0 . 5 ml of sfm - endothelial medium without bfgf . after 30 minutes incubation , bfgf was added into each well at a final concentration of 1 ng / ml together with another 0 . 5 ml of sfm - endothelial medium . seventy - two hours later , cells were trypsinized and counted using a hemocytometer . peptides endo - 2 , endo - 3 and endo - 4 all inhibited bae cell proliferation in a dose - dependent manner while peptide endo - 1 has only a slight inhibition effect at high doses ( fig7 ). the ic 50 of the endo - 2 , - 3 and - 4 peptides is estimated to be 0 . 4 um , 0 . 3 um and 2 um respectively . all 4 endostatin - derived peptides were also tested in the chick embryo chorioallantoic membrane ( cam ) in vivo angiogenesis assay . these peptides reduced microvessel density with various efficacy comparing to a pbs control ( fig8 ). peptides designed from mammalian vegf sequences ( mvegf ) and zebrafish vegf ( fvegf ) showed dose - dependent inhibition of cam angiogenesis ( fig9 , 10 ). scrambled mutant peptide where the same amino acids in mvegf were placed in a random order completely destroyed the angiogenesis inhibition activity of mvegf peptide . two small peptides , hflt1 and hflt2 , were designed from the second immunoglobulin domain ( ig domain 2 ) of the extracellular region of flt - 1 . the peptide hflt2 showed very potent angiogenesis inhibition activity on cam ( fig1 and 11 ). surprisingly , a truncated version of hflt2 peptide , hflt2 - 11 , in which the n - terminal two amino acids have been deleted , showed a even more potent antiangiogenic activity comparing to hflt2 ( fig1 ). however , a single amino acid mutated version of the same peptide , hflt2 - t , in which the second amino acid proline was replaced with threonine , has a much weaker antiangiogenic activity ( fig1 ). peptide hflt2 - 9 in which four amino acids were removed from the n - terminal of hflt2 , showed similar level of antiangiogenic activity as the original peptide ( fig1 ). further truncation at the n - terminal of the peptide resulted in non - functional peptide ( hflt2 - 7 and hflt2 - 5 ). the above data demonstrated that small peptides derived from the sequence of human endostatin , vegf and flt - 1 can function as potent angiogenesis inhibitors . inhibition of endothelial cell proliferation and retardation of tumor growth in mice human hepatoma cell line hepg2 was cultured and 1 million cells were injected subcutaneously into the right back of each 6 weeks old balb / c nude mouse . visible tumors appear from about 2 weeks after tumor cell injection . mice with tumors were then randomly grouped and injected with the peptide at 300 μg / mouse every 12 h ( 16 - 19 g average weight ). no obvious toxicity was observed at this dose . tumor sizes were measured with a caliper every three days and volumes calculated using the formula ( width ) 2 × length × 0 . 52 . statistical analyses were done using student t - test using the sigmastat software ( spss , usa ). tumors are angiogenesis dependent . to test if peptide angio - 3 can inhibit tumor growth in vivo , human hepatoma hepg2 cells were injected subcutaneously into nude mice to induce tumor formation . tumor nodules appear in about 15 - 18 days after tumor cell injection . peptide was then injected at a distant site from the tumor into tumor bearing mice at 15 mg / kg every 12 h for up to a month . as shown in fig1 , injection of peptide angio - 3 inhibited hepg2 tumor growth by about 40 % at the time of experimental termination . experiments were terminated when the control group tumors started to show necrosis . statistical analysis indicates that the tumor size difference between the treatment and control groups is significant at p & lt ; 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