Patent Application: US-201113581457-A

Abstract:
disclosed are compositions and methods useful for delivering targeted therapies for pulmonary diseases , fibrotic disorders and cancer . the compositions and methods are based on peptide sequences that selectively bind to and home to diseased tissue and enable targeted therapies to effect a beneficial therapeutic result . the disclosed targeting is useful for delivering therapeutic and detectable agents to diseased tissue in an animal .

Description:
the term “ bioactive agent ” refers to a substance which is used in connection with an application that is therapeutic or diagnostic in nature , such as in methods for diagnosing the presence or absence of a disease in a patient and / or in methods for treating a disease in a patient . as to compatible bioactive agents , those skilled in the art will appreciate that any therapeutic or diagnostic agent may be incorporated in the stabilized dispersions of the present invention . for example , the bioactive agent may be selected from the group consisting of antiallergics , bronchodilators , vasodilators , antihypertensive agents , bronchoconstrictors , pulmonary lung surfactants , analgesics , antibiotics , leukotriene inhibitors or antagonists , anticholinergics , mast cell inhibitors , antihistamines , anti - inflammatories , anti - neoplastics , anesthetics , anti - tuberculars , imaging agents , cardiovascular agents , enzymes , steroids , genetic material , viral vectors , antisense agents , small molecule drugs , proteins , peptides and combinations thereof . particularly preferred bioactive agents comprise compounds which are to be administered systemically ( i . e . to the systemic circulation of a patient ) such as small molecule drugs , imaging agents , peptides , proteins or polynucleotides . as will be disclosed in more detail below , the bioactive agent may be incorporated , blended in , coated on or otherwise associated with the targeting peptide disclosed herein . particularly preferred bioactive agents for use in accordance with the invention include anti - allergies , peptides and proteins , bronchodilators , anti - inflammatory agents and anti - cancer compounds for use in the treatment of disorders involving diseased tissue reflecting altered heparan sulfate variants specific to said disease . yet another associated advantage of the present invention is the effective delivery of bioactive agents administered or combined with a targeting peptide . the phrase “ substantially identical ” means that a relevant sequence is at least 70 %, 75 %, 80 %, 85 %, 90 %, 92 %, 95 % 96 %, 97 %, 98 %, or 99 % identical to a given sequence . by way of example , such sequences may be allelic variants , sequences derived from various species , or they may be derived from the given sequence by truncation , deletion , amino acid substitution or addition . percent identity between two sequences is determined by standard alignment algorithms such as clustalx when the two sequences are in best alignment according to the alignment algorithm . a polypeptide “ variant ” as referred to herein means a polypeptide substantially homologous to a native polypeptide , but which has an amino acid sequence different from that encoded by any of the nucleic acid sequences of the invention because of one or more deletions , insertions or substitutions . variants can comprise conservatively substituted sequences , meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics . see zubay , biochemistry , addison - wesley pub . co ., ( 1983 ). it is a well - established principle of protein and peptide chemistry that certain amino acids substitutions , entitled “ conservative ” amino acid substitutions , can frequently be made in a protein or a peptide without altering either the conformation or the function of the protein or peptide . such changes include substituting any of alanine ( a ), isoleucine ( i ), valine ( v ), and leucine ( l ) for any other of these amino acids ; aspartic acid ( d ) for glutamic acid ( e ) and vice versa ; glutamine ( q ) for asparagine ( n ) and vice versa ; serine ( s ) for threonine ( t ) and vice versa ; and arginine ( r ) for lysine ( k ) and vice versa . in addition to the known functional variants , there are derivatives of the peptides disclosed herein which can also function in the disclosed methods and compositions . protein and peptide variants and derivatives are well understood by those of skill in the art and in can involve amino acid sequence modifications . for example , amino acid sequence modifications typically fall into one or more of three classes : substitutional , insertional or deletional variants . insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues . insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions , for example , on the order of one to four residues . deletions are characterized by the removal of one or more amino acid residues from the protein or peptide sequence . typically , no more than about from 2 to 6 residues are deleted at any one site within the protein or peptide molecule . these variants can be prepared by site - specific mutagenesis of nucleotides in the dna encoding the protein or peptide , thereby producing dna encoding the variant , and thereafter expressing the dna in recombinant cell culture , or via solid state peptide synthesis . techniques for making substitution mutations at predetermined sites in dna having a known sequence are well known . amino acid substitutions are typically of single residues , but can occur at a number of different locations at once ; insertions usually will be on the order of about from 1 to 10 amino acid residues ; and deletions will range about from 1 to 10 residues . deletions or insertions preferably are made in adjacent pairs , i . e . a deletion of 2 residues or insertion of 2 residues . substitutions , deletions , insertions or any combination thereof can be combined to arrive at a final construct . the mutations generally should not place the sequence out of reading frame ( unless a truncated peptide is intended ) and preferably will not create complementary regions that could produce secondary mrna structure . substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place . substantial changes in function or immunological identity are made by selecting substitutions that are less conservative , i . e ., selecting residues that differ more significantly in their effect on maintaining ( a ) the structure of the polypeptide backbone in the area of the substitution , for example as a sheet or helical conformation , ( b ) the charge or hydrophobicity of the molecule at the target site or ( c ) the bulk of the side chain . the substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which ( a ) a hydrophilic residue , e . g . seryl or threonyl , is substituted for ( or by ) a hydrophobic residue , e . g . leucyl , isoleucyl , phenylalanyl , valyl or alanyl ; ( b ) a cysteine or praline is substituted for ( or by ) any other residue ; ( c ) a residue having an electropositive side chain , e . g ., lysyl , arginyl , or histidyl , is substituted for ( or by ) an electronegative residue , e . g ., glutamyl or aspartyl ; or ( d ) a residue having a bulky side chain , e . g ., phenylalanine , is substituted for ( or by ) one not having a side chain , e . g ., glycine , in this case , ( e ) by increasing the number of sites for sulfation and / or glycosylation . similarly , the term “ conformational homology ” may be used herein to define a sequence which maintains a similar arrangement of amino acids from a conformational perspective to seq id no : 1 or seq id no : 2 . as used herein , the term “ peptide ” is used broadly to mean peptides , proteins , fragments of proteins and the like . the peptide may be animal , bacterial , viral or synthetic in origin . the term “ peptidomimetic ,” as used herein , means a peptide - like molecule that has the activity of the peptide upon which it is structurally based . such peptidomimetics include chemically modified peptides , peptide - like molecules containing non - naturally occurring amino acids , and peptoids and have an activity such as selective homing activity of the peptide upon which the peptidomimetic is derived ( see , for example , goodman and ro , peptidomimetics for drug design , in “ burger &# 39 ; s medicinal chemistry and drug discovery ” vol . 1 ( ed . m . e . wolff ; john wiley & amp ; sons 1995 ), pages 803 - 861 ). a variety of peptidomimetics are known in the art including , for example , peptide - like molecules which contain a constrained amino acid , a non - peptide component that mimics peptide secondary structure , or an amide bond isostere . a peptidomimetic that contains a constrained , non - naturally occurring amino acid can include , for example , an . alpha .- methylated amino acid ; . alpha .,. alpha .- dialkylglycine or . alpha .- aminocycloalkane carboxylic acid ; an n . sup . alpha .— c . sup . alpha . cyclized amino acid ; an n : sup . alpha .- methylated amino acid ; a . beta .- or . gamma .- amino cycloalkane carboxylic acid ; an . alpha .,. beta .- unsaturated amino acid ; a . beta .,. beta .- dimethyl or . beta .- methyl amino acid ; a . beta .- substituted - 2 , 3 - methano amino acid ; an n — c . sup . epsilon . or c . sup . alpha .— c . sup . delta . cyclized amino acid ; a substituted proline or another amino acid mimetic . a peptidomimetic which mimics peptide secondary structure can contain , for example , a non - peptidic . beta .- turn mimic ; . gamma .- turn mimic ; mimic of . beta .- sheet structure ; or mimic of helical structure , each of which is well known in the art . a peptidomimetic also can be a peptide - like molecule which contains , for example , an amide bond isostere such as a retro - inverso modification ; reduced amide bond ; methylenethioether or methylene - sulfoxide bond ; methylene ether bond ; ethylene bond ; thioamide bond ; trans - olefin or fluoroolefin bond ; 1 , 5 - disubstituted tetrazole ring ; ketomethylene or fluoroketomethylene bond or another amide isostere . one skilled in the art understands that these and other peptidomimetics are encompassed within the meaning of the term “ peptidomimetic ” as used herein . as used herein , the term “ dendrimer ” shall mean repeatedly branched and roughly spherical molecules . a dendrimer is typically symmetric around a core and usually adopts a spherical three - dimensional morphology . dendrimers generally contain three major portions : a core , an inner shell and an outer shell . dendrimers can be synthesized to have different and varying functionality in each of the major portions in order to control such variables as solubility , thermal stability and attachment of compounds suitable for particular applications . carsknkdc ( seq id no : 1 ) ( car ) peptide has been previously been shown to target wound healing ( jarvinen and ruoslahti , 2007 ). car peptide has also been linked to decorin for targeted anti - tgf - β scar minimization in skin wounds ( jarvinen and ruoslahti , 2010 ). here we describe the novel homing of car peptides to hypertensive pulmonary vasculature , acutely injured pulmonary tissue , and fibrotic pulmonary tissue . additionally , we disclose a novel means of achieving targeted therapy with car via simultaneous administration of car peptide along with another therapeutic . these findings provide the means to diagnose and deliver targeted therapies for pulmonary diseases such as pulmonary hypertension , interstitial lung disease , acute lung injury ( ali ), acute respiratory distress syndrome ( ards ), sepsis , septic shock , sarcoidosis of the lung , pulmonary manifestations of connective tissue diseases , including systemic lupus erythematosus , rheumatoid arthritis , scleroderma , and polymyositis , dermatomyositis , bronchiectasis , asbestosis , berylliosis , silicosis , histiocytosis x , pneumotitis , smoker &# 39 ; s lung , bronchiolitis obliterans , the prevention of lung scarring due to tuberculosis and pulmonary fibrosis , other fibrotic diseases such as myocardial infarction , endomyocardial fibrosis , mediastinal fibrosis , myelofibrosis , retroperitoneal fibrosis , progressive massive fibrosis , pneumoconiosis , nephrogenic systemic fibrosis , keloid , arthrofibrosis , adhesive capsulitis , radiation fibrosis , fibrocystic breast condition , liver cirrhosis , hepatitis , liver fibrosis , nonalcoholic fatty liver disease , nonalcoholic steatohepatitis , sarcoidosis of the lymph nodes , or other organs ; inflammatory bowel disease , crohn &# 39 ; s disease , ulcerative colitis , primary biliary cirrhosis , pancreatitis , interstitial cystitis , chronic obstructive pulmonary disease , atherosclerosis , ischemic heart disease , vasculitis , neoplastic / metastatic / oncological diseases ( including cancer ), pneumoconiosis , autoimmune diseases , angiogenic diseases , wound healing , infections , trauma injuries and systemic connective tissue diseases including systemic lupus erythematosus , rheumatoid arthritis , scleroderma , polyrnyositis , and dermatomyositis . these diseases can be treated by simultaneously administering car peptide with the bioactive agent to be targeted to the site of disease . we define simultaneous administration , or co - administration , as administration of car followed by administration of the therapeutic to be targeted within 1 hour of car administration . for example , if the disease is pulmonary hypertension and the desired goal is targeted pulmonary arterial vasodilation , an effective dose of car peptide can be co - administered with a minimal dose of systemic vasodilator to achieve targeted pulmonary vasodilation and a significant decrease in pulmonary pressure with minimal systemic hypotension . similarly , car peptide can be co - administered with other medications to increase therapeutic bioavailability , boost therapeutic efficacy , and minimize side effects . car may be administered in a linear or cyclical form , or in any conformation deemed physiologically appropriate as a means of conveying treatment . in addition to targeted vasodilation , we can also deliver targeted anti - coagulation . for example , in a disease like acute lung injury , which is often marked by pulmonary intra - alveolar coagulation , targeted anti - coagulation can be delivered to the affected pulmonary area by co - administering an effective dose of car with an anti - coagulant such as tissue factor pathway inhibitor ( tfpi ) or site - inactivated factor vila ( welty - wolf et al ., 2001 ) in a minimal dose to achieve targeted pulmonary anticoagulation with minimal changes in clotting ability over the areas of the body not undergoing thrombosis . selective pulmonary anti - coagulation can also be utilized to treat other pulmonary diseases marked by pulmonary thrombosis such as pulmonary hypertension , lung transplant rejection and others . in a disease like chronic obstructive pulmonary disease , which is often marked by shortness of breath , car peptide can be co - administered to boost the effective concentration and potency of drugs to relax airway smooth muscles such as long lasting β - 2 agonists such as salmeterol or formoterol ( richter , et al ., 2002 ). many pulmonary diseases are often marked by a decrease in glutathione ( gsh ), a powerful antioxidant ( morris and bernard , 1994 ). car peptide can be co - administered with n - acetylcysteine ( nac ), a glutathione precursor , in diseases like pulmonary fibrosis , pah , ali , and other pulmonary disorders to boost gsh production and scavenge reactive oxidants often found in pulmonary diseases . gsh may also serve to dampen the inflammatory immune response by binding to triggering receptor expressed on myeloid cells 1 ( trem1 ) and diminishing monocyte / macrophage - and neutrophil - mediated inflammatory responses . co - administration of car with nac can serve to lessen the severe inflammatory immune response that often characterizes severe pulmonary and fibrotic diseases like ali , pulmonary hypertension , autoimmune diseases and many other conditions . the levels of antioxidants such as superoxide dismutase ( sod ) ( rosenfeld , et al ., 1996 ), or synthetic superoxide dismutase mimetics like euk - 8 ( gonzalez et al ., 1996 ) can be increased through co - administration of car . treatments for pulmonary diseases like pulmonary fibrosis , pah and ali can also be improved by co - administering car with tgf - β inhibitors like decorin . decorin , which has been previously enhanced through direct conjugation with car ( janinen and ruoslahti , 2010 ), can also be co - administered with car to achieve the benefits of targeting without direct conjugation between the car and decorin molecules . in pulmonary hypertension , pulmonary fibrosis and other pulmonary diseases , the benefits of endothelin ( et - 1 ) receptor antagonists ( kuklin et al ., 2004 ), prostacyclin derivatives ( olschewski et al ., 1999 ), phosphodiesterase type 5 inhibitors ( kanthapillai et al ., 2004 ) and ontological agents such as imatinib ( ghofrani et al ., 2005 ) ( aono et al ., 2005 ) can be increased for patients through the co - administration of car . other pulmonary and fibrotic disease treatments such as ketoconazole which inhibits thromboxane and leukotriene synthesis ( sinuff et al ., 1999 ) can be improved in its efficacy while minimizing side effects through co - administration with car . newer therapeutic approaches such as small interfering rna ( sirna ), and microrna ( mirna ) therapies ( wurdinger and costa , 2007 ) can also be improved and side effects minimized through the selective targeting of diseased tissue through the co - administration of car . the present invention provides for establishing therapeutic targets by identifying altered gene expression levels at the heparan sulfate receptor , therefore indicating a diseased source . the diseased source may then be approached therapeutically with customized targeted therapies comprising a targeting peptide and bioactive agents disclosed in the present invention . in addition to targeted therapies , car &# 39 ; s homing to diseased pulmonary and fibrotic tissues can be utilized for the purposes of diagnosis through the conjugation or co - administration of car with imaging agents . a rat model of monocrotaline ( mct )- induced pulmonary arterial hypertension was used for this study . briefly , male sprague - dawley rats ( 150 - 200 g , harlan laboratories , in ) were administered with a single subcutaneous injection of monocrotaline at 60 mg / kg ( sigma - aldrich , mo ), while control rats administered 0 . 9 % saline ( fig1 ). rats were randomly selected and studied for peptide distribution studies on 1 , 3 , 7 , 14 or 21 days after the treatment of monocrotaline . the following peptides were labeled with 5 - carboxyfluorescein ( 5fam ) and used for the lung targeting studies : car , 5fam - carsknkdc ; vcam1 , cvhspnkkcggsk - 5fam ; control , 5fam - cgggggggc . all peptides were synthesized by anaspec ( anaspec inc ., ca ). peptides were resolved in pbs at the concentrations of 0 . 5 mg / ml . mct - treated or untreated rats were injected with peptide solution at a dose of 3 . 3 mg / kg body weight via the tail vein . at two hours after the injection , rats were perfused with pbs containing 1 % bovine serum albumin under the deep anesthesia with isofluorane at a rate of 3 . 0 % and euthanized . tissues were fixed by systemic perfusion with 10 % buffered formalin via right ventricle . the lung was inflated by injection of 10 % formalin through the trachea . various organs were excised from the rat and fixed for additional twenty four hours and processed for immunohistochemistory . to determine the localization of the peptides , paraffin - embedded tissue sections were immunostained with either hematoxylin and eosin ( fig2 - 3 ) or rabbit anti - fluoroscein isothiocyanate ( fitc ) antibody ( invitrogen , ca ) followed by horseradish peroxidase - labeled anti - rabbit igg secondary antibody ( fig4 ). the peptide localization was then visualized by diaminobenzidine ( dab ). an automated staining system , discovery xt ( ventana , ariz .) was used . to quantify the targeting efficiency of the peptides to the lung , the immunostained sections were scanned by aperio scanscope xt and analyzed using imagescope software ( aperio technologies , ca ). adult male sprague - dawley rats weighing approximately 200 g are injected subcutaneously with su5416 ( 20 mg / kg ; sugen inc ), which is suspended in carboxymethylcellulose ( 0 . 5 % [ wt / vol ] carboxymethylcellulose sodium , 0 . 9 % [ wt / vol ] nacl , 0 . 4 % [ vol / vol ] polysorbate , 0 . 9 % [ vol / vol ] benzyl alcohol in deionized water ). the rats are then exposed to chronic hypoxia in a hypobaric chamber ( 10 % o 2 ) for 3 weeks and are returned to normoxia ( 21 % o 2 ) for an additional 2 to 10 weeks . rats are anesthetized with intramuscular pentobarbital sodium ( 30 mg / kg ). the rats are placed on controlled heating pads . hemodynamic measurements are performed in anesthetized animals under normoxic conditions . polyvinyl catheters ( pv - 1 , internal diameter : 0 . 28 mm ) are inserted into the right jugular vein for measurement of right ventricular systolic pressure ( rvsp ) and into the left jugular vein for drug administration . a microtip p - v catheter ( spr - 838 , millar instruments ) is inserted into the right carotid artery and advanced into the left ventrical ( lv ). the signals are continuously recorded by mpvs - 300 system with powerlab / 4sp , a / d converter ( ad instruments ), and a personal computer . rvsp , heart rate , maximal left ventriclar systolic pressure , left ventricular end - diastolic pressure ( lvedp ), mean arterial pressure ( map ), cardiac output , and stroke volume are measured . if the heart rate falls below 300 beats / min , the measurements are excluded from analysis . at the end of each hemodynamic study , the rat is sacrificed by an overdose of pentobarbital sodium , and organs are removed for various measurements and analyses . after baseline hemodynamic measurements , a simple mixture of car ( 1 mg / 300 g rat ), or control peptide carm , and fasudil ( 0 . 1 , 0 . 3 , 1 , or 3 mg / kg ) or each agent alone is injected intravenously , and all hemodynamic parameters are continuously monitored . organs ( lung , heart , liver , spleen , and kidney ) are collected after blood is flushed with 30 ml phosphate buffered saline ( pbs ). lungs are inflated via trachea with 10 % formalin at a constant pressure of 20 cm h 2 o . after 24 hour - fixation with 10 % formalin , all organs are embedded in paraffin , and sectioned at 5 mm thickness . after deparaffinization , tissue sections are pretreated with 3 % hydrogen peroxidase for 10 minutes and blocked with normal horse serum for 1 hour . they are then incubated for 1 hour with an anti - fluorescein antibody ( 1 : 200 ; invitrogen ) as a primary antibody . after washing with pbs , the sections were incubated with biotinylated secondary antibodies , washed with pbs , and incubated in abc regent for 5 minutes . diaminobenzidine was used as a substrate for the immunoperoxidase reaction . sections were lightly counterstained with hematoxylin , and analyzed light microscopically ( fig5 - 13 ). car ( but not carm ) was detected in high intensity in all layers of severely remodeled arteries from lung tissue . neither car nor carm was found in other organs except for the kidney . the bleomycin ( bl ) model is usually considered a model of pulmonary fibrosis , but its administration is also associated with features of acute lung injury ( ali ). bleomycin is an antineoplastic antibiotic drug isolated in 1966 from the actinomycete streptomyces verticillus . bleomycin forms a complex with oxygen and metals such as fe2 +, leading to the production of oxygen radicals , dna breaks , and ultimately cell death . bleomycin can be inactivated by bleomycin hydrolase , a cysteine protease that shows variable levels of expression in the lungs . the susceptibility of the lungs to bleomycin - induced toxicity is largely dependent on the levels of expression of bleomycin hydrolase in the lungs ; species with high levels of expression , such as rabbits , are relatively resistant to bleomycin - induced toxicity , whereas species with low levels of expression , such as c57bl / 6 mice , are sensitive . in addition to species - related differences in bleomycin susceptibility , there are also differences in strain susceptibility , with c57bl / 6 mice being highly sensitive . a mouse model of bleomycin induced acute lung injury and pulmonary fibrosis was used for this study . briefly , 6 wt c57b1 / 6 male mice , 8 - 12 weeks were weighed and anesthetized , and given bleomycin ( bl ) intratracheally at 4 u / kg . at 3 days ( acute lung injury model ) and 14 days ( pulmonary fibrosis model ) after bl injection , peptides were injected via the tail vein . the following peptides were labeled with 5 - carboxyfluorescein ( 5fam ) and used for the lung targeting studies : car , 5fam - carsknkdc ; vcam1 , cvhspnkkcggsk - 5fam ; control , 5fam - cgggggggc . all peptides were synthesized by anaspec ( anaspec inc ., ca ). peptides were resolved in pbs at the concentrations of 0 . 5 mg / ml . bl - treated mice were injected with peptide solution at a dose of 3 . 3 mg / kg body weight via the tail vein . at two hours after the injection , mice were perfused with pbs containing 1 % bovine serum albumin under the deep anesthesia with isofluorane at a rate of 3 . 0 % and euthanized . tissues were fixed by systemic perfusion with 10 % buffered formalin via right ventricle . the lung was inflated by injection of 10 % formalin through the trachea . various organs were excised from the rat and fixed for additional twenty four hours and processed for immunohistochemistory . to determine the localization of the peptides , paraffin - embedded tissue sections were immunostained with rabbit anti - fluoroscein isothiocyanate ( fitc ) antibody ( invitrogen , ca ) followed by horseradish peroxidase - labeled anti - rabbit igg secondary antibody . the peptide localization was then visualized by diaminobenzidine ( dab ). an automated staining system . discovery xt ( ventana , ariz .) was used . to quantify the targeting efficiency of the peptides to the lung , the immunostained sections were scanned by aperio scanseope xt and analyzed using imagescope software ( aperio technologies , ca ) ( fig1 - 17 ). to measure the acute effects of fasudil with and without car administration on the right and left ventricular systolic pressure , blood pressure measurements were performed on catheterized su5416 / hypoxia / normoxia - exposed rats with pah ( fig1 - 19 ). surprisingly , co - administered car enhanced the blood pressure lowering effect of fasudil on rvsp with only a minor reduction in lvsp , as compared to fasudil alone . of additional importance , continuous infusion of car + fasudil resulted in a sustained , pulmonary - specific effect even after the cessation of the infusion ( fig2 ). an alternative analysis was conducted , observing the same pulmonary - specific effects when comparing pressure in the rvsp to systolic aortic pressure ( sap ). while the selective decrease in pulmonary pressure as measured in the rvsp is present , there is no increased car effect systemically when co - administered with fasudil ( fig2 ). severe occlusive pah rat model was used . animals were injected with su5416 ( 20 mg / kg ; sugen inc ), followed by 3 weeks hypoxia , then followed by 2 - 10 weeks normoxia . the peptide administered was a 7 amino acid variant to the car peptide used in previous examples . this variant ( cark ) consisted of the following sequence : carsknk ( seq id no : 2 ). in these experiments , cark was administered at a dose of 3 mg / kg and fasudil administered at 1 mg / kg . to measure the acute effects of fasudil with and cark administration on the right and left ventricular systolic pressure ( or systolic aortic pressure ), blood pressure measurements were performed on catheterized su5w / hypoxia / normoxia - exposed rats with pah . similar to car , cark co - administration enhanced the blood pressure lowering effect of fasudil on rvsp with only a minor reduction in sap ( fig2 ) and lvsp ( fig2 ), as compared to fasudil alone . interestingly , administration of 10 mg / kg of fasudil 30 minutes after cessation of cark infusion still resulted in a sustained , pulmonary - specific effect ( fig2 ). severe occlusive pah rat model was used . animals were injected with su5416 ( 20 mg / kg ; sugen inc ), followed by 3 weeks hypoxia , then followed by 2 - 10 weeks normoxia . the peptide administered was car , carsknkdc ( seq id no : 1 ). in this experiment , car was administered at a dose of 3 mg / kg and imatinib administered at 10 mg / kg . to measure the acute effects of imatinib with car administration on the right and left ventricular systolic pressure , blood pressure measurements were performed on catheterized su5w / hypoxia / normoxia - exposed rats with pah . similar to fasudil , car co - administration enhanced the blood pressure lowering effect of imatinib on rvsp with only a minor reduction in lvsp ( fig2 ). vi . altered levels of gene expression of enzymes involved in heparan sulfate proteoglycan biosynthesis found in a progressive porcine surgical shunt model of pah heparan sulfate biosynthetic enzymes are key components in generating a myriad of distinct heparan sulfate structures that carry out multiple biologic activities . to determine whether car or any variants utilized the heparan sulfate pathway , an analysis was first performed to identify differential gene expression in the pah model since car displayed both homing and selective therapeutic efficacy in models of pah . it was discovered that in the surgical shunt model of pah , a large increase in gene expression was identified in a select group of genes , all of which are related to the heparan sulfate biosynthetic pathway . the heparan sulfate 2 - o - sulfotransferase 1 ( hs2st1 ) gene , which encodes an enzyme responsible for catalyzing the transfer of sulfate to the c2 position of selected hexuronic acid residues within the maturing heparan sulfate , was found to be greatly increased over time in the pah pig model ( fig2 ). another gene which showed a selective increase in expression in the pah model was exostosin 1 ( ext1 ), a glycosyltransferase required for the biosynthesis of heparan sulfate ( fig2 ). specifically , ext1 encodes an endoplasmic reticulum - resident type ii transmembrane glycosyltranferase involved in the chain elongation step of heparan sulfate biosynthesis . other genes identified as exhibiting an increase in expression in the pah model were glycosyltransferase 8 domain containing 2 ( glt8d2 ) ( fig2 ), heparan sulfate n - deacetylase / n - sulfotransferase ( ndst1 ) ( fig2 ) and o - linked n - acetylglucosamine transferase ( ogt ) ( fig3 ). it is possible to modify the sequences disclosed in the present invention by truncation , i . e ., seq id no : 2 is a truncated variant of seq id no : 1 in which the terminal 2 amino acids of seq id no : 1 are deleted to produce seq id no : 2 . the conformation of peptide variants can be modeled using molecular and electronic structure modeling programs like molden . the molecular and electrostatic potential structure of seq id no : 1 or seq id no : 2 can be modeled , and compared to substitutional variants in which one or more amino acids have been substituted to predict if the variant will have a similar conformation with an expected similar function . seq id no : 2 was modeled in an energy minimized state and an electrostatic potential map was created to visualize its electrostatic surface . a library of single amino acid substitutional variants of seq id no : 2 were modeled by substituting each of the remaining 19 amino acids for the n terminus cysteine ( c ) ( fig3 ). the sequences from the library of single amino acid substitutional variants were as follows : aarsknk ( seq id no : 3 ), narsknk ( seq id no : 4 ), sarsknk ( seq id no : 5 ), tarsknk ( seq id no : 6 ), marsknk ( seq id no : 7 ), garsknk ( seq id no : 8 ), varsknk ( seq id no : 9 ), larsknk ( seq id no : 10 ), iarsknk ( seq id no : 11 ), harsknk ( seq id no : 12 ), farsknk ( seq id no : 13 ), warsknk ( seq id no : 14 ), yarsknk ( seq id no : 15 ), qarsknk ( seq id no : 16 ), earsknk ( seq id no : 17 ), darsknk ( seq id no : 18 ), rarsknk ( seq id no : 19 ), karsknk ( seq id no : 20 ), parsknk ( seq id no : 21 ). the electrostatic potential conformational structure was compared between seq id no : 2 and each substitutional variant ( fig3 - 50 ). some substitutional variants displayed nearly identical or very similar structures ( fig3 - 41 ) indicating a likelihood that substituting the amino acids a , s , m , v , h , t , n , g , l , or i for the n terminus cysteine ( c ) would result in the substitutional variants aarsknk ( seq id no : 3 ), sarsknk ( seq id no : 5 ), marsknk ( seq id no : 7 ), varsknk ( seq id no : 9 ), harsknk ( seq id no : 12 ), tarsknk ( seq id no : 6 ), narsknk ( seq id no : 4 ), garsknk ( seq id no : 8 ) and iarsknk ( seq id no : 11 ) having similar functional characteristics as seq id no : 2 . other substitutional variants showed greater differences in structure compared to cark ( fig4 - 50 ) with predicted different function . other substitutional variants can be modeled and predicted in a similar fashion for seq id no : 1 or seq id no : 2 by substituting any of the remaining 19 amino acids for a particular amino acid in either peptide with any resulting similar conformers predicted to have similar function . peptide dendrimers are radially branched macromolecules that contain a peptidyl branching core and / or peripheral peptide chains , and they can be divided into three categories . one category consists of “ grafted ” peptide dendrimers , having peptides only as surface functionalities . the second category is peptide dendrimers that composed entirely of amino acids . the third are dendrimers utilizing amino acids in the branching core and surface functional groups , but having non - peptide branching units . peptide dendrimers can be synthesized using either divergent or convergent approach , and the availability of solid - phase combinatorial methods enables large libraries of peptide dendrimers to be produced and screened for desired properties . dendrimer variants of seq id no : 1 or seq id no : 2 can be synthesized . for example , a cark dendrimer containing 8 cark residues on a polyamidoamine ( pamam ) core ( fig5 - 52 ) can be constructed . other cores such as poly ( ethylene glycol ) can also be used to create dendrimer variants . these dendrimer variants can have dozens , hundreds , or even thousands of car or cark peptide residues on the surface of the dendrimer to provide enhanced functional characteristics . these dendrimers can contain car or cark alone for disease selective homing , cell penetration and delivery of co - administered bioactive agents or contain a bioactive agent within the dendrimer for targeted delivery . aono y , nishioka y , inayama m , ugai m , kishi j , uehara h , izumi k , sone s . imatinib as a novel antifibrotic agent in bleomycin - 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