Patent Application: US-201213984522-A

Abstract:
the invention provides conjugates for imaging plaques , such as cardiovascular plaques , as well as associated pharmaceutical compositions . other aspects of the invention include methods for administering and imaging such conjugates and compositions , and using the imaging to characterise plaques . the conjugates of the invention distinguish between tropoelastin and elastin in plaques . the presence of tropoelastin can act as an indication that a plaque is liable to rupture or erode . such information allows assessment of disease progression and response to treatment .

Description:
tropoelastin - specific or lysyl oxidase - specific binding agent tropoelastin is a matrix protein , which is synthesized to form part of the walls of blood vessels . after expression of immature tropoelastin , it is covalently cross - linked by the enzyme lysyl - oxidase ( lox ) to structural mature elastin ( fig1 ), which provides tensile strength to the vessel wall . the present invention is therefore concerned with conjugates that are capable of differentiating between de novo synthesized tropoelastin and mature cross - linked elastin , especially in vivo , the former being associated with an increased risk of plaque instability and rupture , leading to ami and / or stroke and / or aortic aneurysm . the sequence of human tropoelastin , lysyl oxidase , and elastin are available on sequence databases along with the sequences of the corresponding polypeptides in animal models such as rabbits ( see also sequences section below ). tropoelastin from other species may also be used to design specific binding peptides or for screening antibody based binding agents . it may be advantageous to design peptides or antibodies that are capable of specifically binding to tropoelastin of more than one species , for example to enable the same conjugate to be used for imaging plaques in an animal model and in human patients . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide is capable of specifically binding tropoelastin . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide substantially does not bind to elastin . in a preferred embodiment , the tropoelastin - specific binding agent is capable of specifically binding tropoelastin in vivo and substantially does not bind to elastin in vivo . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide is specific for tropoelastin as compared to other intravascular components or proteins . in a preferred embodiment , the tropoelastin - specific binding agent is specific for tropoelastin as compared to other intravascular components or proteins in vivo . generally , the tropoelastin - specific binding agent may be a polypeptide or peptide that is capable of specifically binding to tropoelastin or may be an antibody molecule capable of specifically binding to tropoelastin . in a preferred embodiment , the tropoelastin - specific binding agent may be a polypeptide or peptide that is capable of specifically binding to tropoelastin in vivo or may be an antibody molecule capable of specifically binding to tropoelastin in vivo . equally , the lysyl - oxidase - specific binding agent may be a polypeptide or peptide that is capable of specifically binding to lysyl oxidase or may be an antibody molecule capable of specifically binding to lysyl oxidase . examples of tropoelastin - specific binding peptides include peptides having the amino acid sequence vvgspsaqdeaspls , egfepg or ypdhvqythy . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide consists of the sequence vvgspsaqdeaspls , egfepg or ypdhvqythy . the skilled person could readily design alternative peptide sequences using the known amino acid sequences of polypeptides known to bind to tropoelastin and / or lysyl oxidase , taking account of the need to avoid cross - reaction , for example , in the case of tropoelastin - specific binding agents , not to bind to a significant extent to mature elastin , especially in vivo . in the examples , the peptides used were chemically synthesized by peptide synthetics ( peptide protein research ltd ) after they had been designed . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide comprises a sequence of at least 4 , 6 , 8 , 10 , 12 or 14 amino acids from the amino acid sequence vvgspsaqdeaspls . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide is not more than 50 , not more than 30 , 20 , 18 , or 16 amino acids in length . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide comprises or consists of the amino acid sequence vvgspsaqdeaspls . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide comprises a sequence of at least 4 , 6 or 8 amino acids from the amino acid sequence ypdhvqythy . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide is not more than 50 , not more than 30 , 20 , 18 , 16 , 14 , 12 or 10 amino acids in length . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide comprises or consists of the amino acid sequence ypdhvqythy . in the present invention , the tropoelastin - specific binding agent may be a peptide or an antibody molecule capable of binding amino acid sequence vgvapg . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding agent may be a peptide comprising the amino acid sequence qdea . in some cases , in accordance with any one of the aspects of the present invention , the tropoelastin - specific binding peptide is not more than 50 , not more than 30 , 20 , 18 , 16 , 14 , 12 , or 10 amino acids in length . without wishing to be bound by any particular theory , the amino acid residues qdea on the tropoelastin - specific binding agent are thought to bind the tropoelastin hexapeptide vgvapg ( fig5 ). in the present invention , the tropoelastin - specific binding agent may be a peptide or an antibody molecule capable of specifically binding to tropoelastin , and preferably does not substantially bind to elastin and / or other components of the vascular system . in a preferred embodiment , the tropoelastin - specific binding agent may be a peptide or an antibody molecule capable of specifically binding to tropoelastin , and preferably capable of not substantially binding to elastin and / or other components of the vascular system in vivo . the tropoelastin - specific binding agent ( e . g . a peptide or an antibody molecule ) may have a dissociation constant for tropoelastin of less than 50 nm , less than 40 nm , less than 30 nm , less than 20 nm , less than 10 nm , or less than 1 nm . in contrast , preferably the tropoelastin - specific binding agent ( such as an anti - tropoelastin antibody or peptide ) may have a dissociation constant for elastin of more than 100 μmol / l . for example , the tropoelastin - specific binding agent ( such as an anti - tropoelastin antibody or peptide ) may have a dissociation constant for in vivo elastin ( e . g . elastin present in or derived from a mammalian , e . g . human , subject ) of more than 1 , 10 , 100 or 200 μmol / l . in the present invention , where the lysyl oxidase - specific binding agent is a peptide or an antibody molecule capable of specifically binding to lysyl oxidase , and not to other components of the vascular system , the peptide or anti - lysyl oxidase antibody may have a dissociation constant for lysyl oxidase of less than 50 nm , less than 40 nm , less than 30 nm , less than 20 nm , less than 10 nm , or less than 1 nm . binding kinetics and affinity ( expressed as the equilibrium dissociation constant k d ) of the tropoelastin specific peptide or anti - tropoelastin antibody molecules may be determined using standard techniques , such as surface plasmon resonance e . g . using biacore analysis . an anti - tropoelastin antibody molecule or anti - lysyl oxidase antibody molecules as described herein may be an immunoglobulin or fragment thereof , and may be natural or partly or wholly synthetically produced , for example a recombinant molecule . one example of an anti - tropoelastin antibody molecule can be purchased from calbiochem cat no . 324756 . anti - tropoelastin antibody molecules or anti - lysyl oxidase antibody molecules may include any polypeptide or protein comprising an antibody antigen - binding site , including fab , fab 2 , fab 3 , diabodies , triabodies , tetrabodies , minibodies and single - domain antibodies , as well as whole antibodies of any isotype or sub - class . antibody molecules and methods for their construction and use are described , in for example holliger & amp ; hudson , nature biotechnology 23 ( 9 ): 1126 - 1136 ( 2005 ). in some preferred embodiments , the anti - tropoelastin antibody molecule or anti - lysyl oxidase antibody molecules may be a whole antibody . for example an igg , iga , ige or igm or any of the isotype sub - classes , particularly igg1 and igg4 . the anti - tropoelastin antibody molecules may be monoclonal antibodies . anti - tropoelastin antibody molecules or anti - lysyl oxidase antibody molecules may be chimeric , humanised or human antibodies . anti - tropoelastin antibody molecules or anti - lysyl oxidase antibody molecules as described herein may be isolated , in the sense of being free from contaminants , such as antibodies able to bind other polypeptides and / or serum components . monoclonal antibodies are preferred for some purposes , though polyclonal antibodies may also be employed . anti - tropoelastin antibody molecules or anti - lysyl oxidase antibody molecules may be obtained using techniques , which are standard in the art . methods of producing antibodies include immunising a mammal ( e . g . mouse , rat , rabbit , horse , goat , sheep or monkey ) with the protein or a fragment thereof . antibodies may be obtained from immunised animals using any of a variety of techniques known in the art , and screened , preferably using binding of antibody to antigen of interest . for instance , western blotting techniques or immunoprecipitation may be used ( armitage et al ., 1992 , nature 357 : 80 - 82 ). isolation of antibodies and / or antibody - producing cells from an animal may be accompanied by a step of sacrificing the animal . as an alternative or supplement to immunising a mammal with a peptide , an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains , e . g . using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces ; for instance see wo92 / 01047 . the library may be naive , that is constructed from sequences obtained from an organism , which has not been immunised with any of the proteins ( or fragments ), or may be one constructed using sequences obtained from an organism , which has been exposed to the antigen of interest . in some embodiments , anti - tropoelastin antibody molecules or anti - lysyl oxidase antibody molecules may be produced by any convenient means , for example a method described above , and then screened for differential binding to tropoelastin relative to elastin ( and / or another component of the vessel wall ). suitable screening methods are well - known in the art and enable those skilled in the art to identify an antibody which displays increased binding to tropoelastin , relative to non - tropoelastin proteins such as elastin , or antibodies capable of binding to lysyl oxidase . after production and / or isolation , the biological activity of an anti - tropoelastin antibody molecule or anti - lysyl oxidase antibody molecules may be tested , for example using the binding experiments described above or in the production of a conjugate so that its properties as an imaging agent may be determined . antibody molecules normally comprise an antigen - binding domain comprising an immunoglobulin heavy chain variable domain ( vh ) and an immunoglobulin light chain variable domain ( vl ), although antigen binding domains comprising only a heavy chain variable domain ( vh ) are also possible ( e . g . camelid or shark antibodies ). the term also covers any polypeptide or protein comprising an antibody - binding domain . antibody fragments which comprise an antigen binding domain are such as fab , scfv , fv , dab , fd ; and diabodies . it is possible to take monoclonal and other antibodies and use techniques of recombinant dna technology to produce other antibodies or chimeric molecules , which retain the specificity of the original antibody . such techniques may involve introducing dna encoding in the immunoglobulin variable region , or the complementarity determining regions ( cdrs ), of an antibody to the constant regions , or constant regions plus framework regions , of a different immunoglobulin . see , for instance , ep 0 184 187 a , gb 2 , 188 , 638 a or ep 0 239 400 a . tropoelastin - specific antibodies and anti - lysyl oxidase antibody molecules are known in the art and are commercially available from sources such as calbiochem / abcam . alternatively , the skilled person could readily produce and screen candidate antibodies as discussed above . in addition to the tropoelastin - specific binding agent , the conjugates of the present invention include an imaging probe capable of detection by an imaging technique such as mri , pet or spect , or combinations thereof . examples of types of imaging probe include radionuclides , optical labels or paramagnetic labels . the present invention may also involve the use of further labelled probes that may be linked to or associated with the conjugates , for example to enable multi - modal imaging to be carried out . the possibility to incorporate optical probes as well as radionuclides and mri contrast agents provides the opportunity to combine modalities to enhance diagnosis and detection , for example the location of disease at the whole body level can be identified by whole body scanning with pet or spect . similarly , combined pet and mr imaging can provide the advantage of high sensitivity ( pet , spet ), quantification of signal ( pet ) and anatomical resolution ( mr ), and measurement of the microenvironment ( mr contrast enhancement ). one preferred class of conjugates of the present invention are mri agents that comprise a tropoelastin specific binding agent linked to a group capable of complexation to a mri active atom such as gadolinium . an alternative mri signal element may include iron oxides . a further possibility is the use of 19 f as a nmr or mri label and / or 18 f as a label , e . g . for pet or ct imaging . in one embodiment , the group capable of complexation to a mri active atom comprises dota . in some embodiments the group capable of complexation to a mri active atom is dota - lysine . the radionuclide probes used in accordance with the present invention may use a range of different radionuclides depending on the application for which the probes are intended . examples of radionuclides that may form part of the probes of the present invention include technetium , rhenium , copper , cobalt , gallium , yttrium , lutetium , indium , zirconium , carbon , iodine , fluorine and astatine isotopes such as tc - 99m , ga - 67 , in - 111 , i - 123 ( spect ), cu - 64 , cu - 60 , cu - 61 , cu - 62 , tc - 94m , ga - 68 , co - 55 , f - 18 , c - 11 , i - 124 , zr - 89 ( pet ), cu - 67 , re - 186 , re - 188 , y - 90 , lu - 177 , i - 131 ( radionuclide therapy ). the present invention may employ the radionuclides alone or in combinations . in general , technetium isotopes are employed for imaging purposes , rhenium isotopes for therapeutic purposes and copper and halogen isotopes for both imaging and therapy . examples of optical probes include fluorophores such as fluorescein , luminescent molecules and complexes such as lanthanide complexes . in some embodiments , the conjugates may comprise a linker or functional group to join the tropoelastin - specific binding agent and the imaging probe . the linker may be a short peptide sequence or may be a chemical linker . the use of peptide linker sequences will be between 6 and 25 amino acids in length , more preferably between 9 and 16 amino acids in length is known in the art . linked typically comprise reactive groups for linking to the binding agent and imaging probe , such as a free cysteine residue . in one aspect , the present invention provides conjugates for use in methods of imaging tropoelastin in the cardiovascular system of a subject , and in particular for imaging plaques . the method generally entails the steps of : ( a ) administering to the subject a composition comprising a conjugate for imaging cardiovascular plaques comprising a tropoelastin - specific binding agent and an imaging probe ; ( b ) allowing the imaging agent to bind to any tropoelastin present in plaques in the vascular system of the subject ; ( c ) detecting the imaging probe to determine the presence of the plaques . in order to come into contact with and bind tropoelastin in plaques , generally a composition comprising the conjugates will be for intravenous administration to the subject . after a suitable delay for binding to take place , the imaging probe may be detected using an imaging technique as described herein . the results of the detecting step may then be used to quantify the tropoelastin present in plaques , and may then be used to assess plaque burden and / or the likelihood of plaque rupture and / or monitor disease progression and / or response to therapy . the aim of this would be to determine a prognosis for a subject , in particular as regards the risk of having ami , a stroke and / or an aortic aneurysm , and / or to help determine therapeutic interventions intended to improve the condition of the subject . although the primary means of imaging using the conjugates employs mri , this may be used in conjunction with other nuclear medicine imaging techniques , such as single photon emission tomography ( spet ), an imaging technique that detects gamma rays emitted from a radionuclide to produce a three dimensional image of the distribution of the radionuclide in a sample or subject , and positron emission tomography ( pet ), an imaging technique that provides three - dimensional images by detecting pairs of gamma rays emitted indirectly by a positron - emitting radionuclide introduced into a sample or subject . by way of example spet studies can be carried out using 99m tc and pet studies using 94m tc . the skilled person , however , will be aware of other suitable spet and pet radionuclides that can be employed in the present invention . generally , the present invention may be employed for positron emission tomography ( pet ), single photon emission tomography ( spet ), optical ( oi ) and / or magnetic resonance imaging ( mri ) by appropriate selection of imaging probe . thus , the conjugates of the present invention may be used in methods of multi - modal imaging , that is where information or images are derived from two different techniques , either by the detection of the imaging probe capable of detection using two different techniques or by providing a second label at the site in the biological system where the nanoparticles become localised , most conveniently by linking or associating the second label with the conjugates as explained in detail above . multi - modal studies will be co - registered and may entail simultaneous imaging with two modalities or may need to take place in two steps , but generally employ the same sample so that spatial information obtained using the two techniques can be compared . examples of multi - modal imaging include pet / ct , spet / ct , pet / mr and spet / mr . by way of example , the following exemplary protocol may be used imaging according to the methods of the present invention . for visualization of contrast agent uptake in the coronary artery walls and large vessels such as the aorta , a navigator - gated , cardiac - triggered , fat - suppressed t1 - weighted 3d gradient echo inversion recovery targeted or whole heart sequence ( 3d ir tfe or 3d ir ssfp ) may be used . imaging parameters of a 3d ir tfe sequence may include field of view = 320 × 320 mm , matrix = 256 × 256 , acquired in - plane resolution = 1 . 25 × 1 . 25 mm , reconstructed slice thickness = 1 . 5 mm ( acquired : 3 mm ), acquisition window = 80 to 100 ms , repetition time / echo time = 5 . 8 ms / 1 . 9 ms , flip angle = 30 °, startup cycles = 5 , and number of slices = 20 but may differ for the whole heart and ssfp protocol . the patient - specific inversion time ( ti ) will be adjusted to null blood signal of blood using a look locker sequence . three different peptides ( vvgspsaqdeaspls , egfepg and ypdhvqythy ) were chosen for the tropoelastin - binding agent and conjugated with dota - lysine for gadolinium and pet / spect labelling . the proof of principle experiments described herein for the in vivo and ex vivo testing of the conjugates used mouse and rabbit models . binding studies with tropoelastin and tnf - alpha coated petri dishes will be performed to demonstrate specificity of the agents . furthermore , transmission electron microscopy of vessel specimens will be performed for elastin and macrophage visualization while x - ray spectra will be acquired for colocalization with gadolinium distribution in plaque laden vessel wall samples . animals will be euthanized immediately after mri . subsequently , the brachiocephalic artery and abdominal aorta will be excised and cut into 3 mm segments . sections will be cut into 3 μm slices for paraffin - embedded and 6 μm for oct - embedded sections . sections will be then stained with hematoxylin and eosin ( h & amp ; e ) for cellular infiltration , miller &# 39 ; s elastica van gieson ( evg ) for elastin and masson &# 39 ; s trichrome , and picrosirius red for plaque morphology and collagen deposition . in addition , immunostaining with specific antibodies for tropoelastin , tnf - alpha and lox will be performed . mass spectroscopy ( ms ) will be applied to quantify the molar concentration of gd in the investigated vessel specimens . mri will be performed in a mouse model of progressive atherosclerosis at 4 , 8 and 12 weeks post commencement of a high fat diet and in a model of angiotensin - ii ( ang - ii ) induced aortic aneurysm formation at 1 , 2 , 3 and 4 weeks post ang - ii releasing mini pump implantation . ten mice will be scanned at each time point either receiving the tropoelastin or tnf - alpha binding contrast agent ( ca ) resulting in a total of 60 and 80 mice , respectively . animals will undergo a pre and post contrast mri session at each time point and subsequently will be sacrificed for validation with histology , immunostaining , electron and mass spectroscopy . to demonstrate treatment effects , 10 mice will be scanned after 12 weeks of therapy with statins with the tropoelastin binding ca . to demonstrate the role of lox in tropoelastin synthesis , 10 mice will be scanned with the tropoelastin ca 12 weeks after commencement of lox inhibitor treatment . new zealand white rabbits will be fed a high cholesterol diet ( special diets services ) for 2 weeks and then undergo balloon injury of the abdominal aorta . subsequently , the high fat diet will be continued for another 6 weeks followed by 4 weeks of normal diet . plaques using this protocol have been shown to develop similar features compared with aha type ii - vi lesions ( excluding the presence of calcified lesions ). mri will be performed with the tropoelastin binding mr contrast agent prior to triggering of plaque rupture using histamine and russel &# 39 ; s viper venom ( rvv ). 48 h after induction of plaque rupture / erosion , mri will be repeated in order to detect the presence of intraluminal thrombi and to correlate thrombus location with pre - trigger tropoelastin - gd . a total of 16 rabbits will be scanned resulting in approximately 8 ( 50 %) rabbits with and without plaque rupture . immediately after the last scan , animals will be sacrificed for validation with histology , immunostaining , mass and electron spectroscopy . rabbit aortic segments were cryo - protected ( 30 % sucrose ), embedded in tissue freezing medium and stored at − 80 ° c . serial 10 μm thick cross - sections ( spanning 300 μm length ) were collected with 500 μm intervals . sections were used for masson &# 39 ; s trichrome for the detection the general plaque morphology , van gienson elastin staining for the detection of mature and immature elastin fibers and immunohistochemistry for the detection of tropoelastin fibers , lox , and macrophages . disrupted plaques were classified using the masson &# 39 ; s trichrome staining and included both ruptured and eroded , as defined for human plaques . non - disrupted plaques included those without an overlying thrombus . immunohistochemistry was performed by the avidin - biotin - peroxidase method ( vector laboratories , no . pk - 6102 ). anti - rabbit polyclonal antibodies for tropoelastin ( calbiochem , # 324756 ), lox ( imgenex , # img - 6442a ) and macrophages ( dako , clone ram11 , no . m0633 ) were used and the following steps were followed : 1 ) sections were incubated in 10 % formalin for 20 minutes at room temperature to adhere the tissue sections on the slides ; 2 ) sections were incubated in a citrate - based solution ( 10 mm citric acid , 0 . 05 % tween 20 , ph 6 . 0 ) ( vector laboratories , burlingame , calif ., no . h - 3300 ) at 100 ° c . for 20 min using a pressure cooker to retrieve the epitope ; 3 ) 1 % hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity ; 4 ) 10 % horse serum for 60 min to reduce nonspecific binding of the antiserum ; 5 ) primary antibodies for 2 h at room temperature . negative control sections were incubated with 10 % horse sera only ; 6 ) biotinylated horse anti - mouse immunglobulin g ( at a dilution of 1 : 200 ) for 1 hr at room temperature ; and 7 ) avidin - elite biotinylated horseradish peroxidase complex ( vectastain elite , vector laboratories , no . pk - 6102 ) at a dilution of 1 : 50 for 1 hr at room temperature . immunoreactive sites were visualized by incubation with 3 , 3 - diaminobenzidine ( dab substrate chromogen , vector laboratories , no . sk - 4100 ) at a dilution of 1 : 50 for 3 - 5 min . tris buffered saline ( ph 7 . 4 ) was used to dilute each solution and to wash the sections three times between each step . finally , tissue sections were counterstained with hematoxylin ( 1 min ). using an antibody that appears to bind to the immature ( tropo ) elastin and a rabbit model of controlled plaque disruption we found that : 1 . there is increase deposition of tropoelastin fibers during the progression of atherosclerosis as well as in vulnerable plaque . 2 . in the initial steps the tropoelastin fibers are scattered throughout the intima and in the later stages they increase in density and they are also found in the adventitia . 3 . the increase elastin content in vulnerable plaque may be used in molecular imaging for the in vivo detection of such lesions . 4 . in some cases , the tropoelastin fibers appear to co - localize with cd68 - positive macrophages indicating that macrophages maybe a source of elastin . 5 . however , there are also cases in which the macrophages do not co - localize with elastin fibers indicating that there might be a diversity of macrophage sub - populations with different local functionality . potential cleavage sites of the peptides vvgspsaqdeaspls and ypdhvqythy were investigated . only enzymes that are primarily present in the digestive system were found to cleave the peptides vvgspsaqdeaspls and ypdhvqythy . none of these enzymes were reported in blood or plaques and thus are unlikely to cleave the peptide vvgspsaqdeaspls or ypdhvqythy before it binds to the vessel wall / plaque specific target , tropoelastin . a protein blast was performed to screen for homologies . the amino acid sequences vvgspsaqdeaspls and ypdhvqythyk were only found in proteins described to interact with tropoelastin ( elastin - binding protein ( ebp ) and microfibril - associated glycoprotein - 1 ( magp - 1 ) respectively ) and not in other proteins . these results suggest that the chosen peptides are highly specific for the protein of interest , tropoelastin . in - vivo experiments in 12 weeks high - fat diet ( hfd ) fed apoe −/− mice injected with gadolinium labelled ( dota - gd )- vvgspsaqdeaspls showed a favourable biodistribution with preferential uptake in the plaque - laden brachiocephalic artery ( bca ) and aortic arch but not in the plaque - free carotid artery ( fig6 ), and rapid renal clearance allowing for imaging as early as 1 hour post contrast injection . in - vivo experiments in hfd fed apoe −/− mice with gadolinium - labelled k -( dota - gd )- ypdhvqythy showed promising results with uptake in the plaque laden brachiocephalic and aortic arch and no uptake in plaque free carotid artery ( fig7 ). the peptide also showed favorable biodistribution with rapid renal clearance and preferential uptake in the bca ( fig9 ). immunohistochemistry verified the presence of tropoelastin in the neointima and adventitia of the diseased bca , and the absence of tropoelastin in the media of both the plaque - laden ( diseased ) and plaque - free ( non - diseased ) bca vessel walls ( fig8 ). the bound relaxivity at 3t was measured as 20 . 88 mm − 1 s − 1 . all documents mentioned in this specification are incorporated herein by reference in their entirety . 1 . krettek et al ‘ elastogenesis in human arterial disease : a role for macrophages in disordered elastin synthesis ’ arterioscl . throm . vas . 23 ( 2003 ) 582 - 587 2 . xu et al ‘ hypercholesterolemia superimposed by experimental hypertension induces differential distribution of collagen and elastin ’ arterioscl . throm . vas . 20 ( 2000 ) 2566 - 2572 3 . akima et al ‘ soluble elastin decreases in the progress of atheroma formation in human aorta ’ circ . j . 73 ( 2009 ) 2154 - 2162 4 . kozel et al ‘ elastic fiber formation : a dynamic view of extracellular matrix assembly using timer reporters ’ j . cell . physiol . 207 ( 2006 ) 87 - 96 5 . starcher et al ‘ antibody raised to akaaakaaaka sequence on tropoelastin recognizes tropoelastin but not mature crosslinked elastin : a new tool in metabolic and structural studies of elastogenesis ’ connect . tissue res . 40 ( 1999 ) 273 - 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