Patent Application
20120034163
The present invention relates to non-invasive tools for detecting vulnerable atherosclerotic plaques.
Cardiovascular diseases, and in particular coronary events, are the leading cause of mortality worldwide. Coronary events are mainly caused by the rupture or erosion of a vulnerable coronary atherosclerotic plaque and subsequent thrombus formation. However, no effective non-invasive tool is available for the detection of vulnerable plaques, which would enable efficient prevention of coronary events.
Vulnerable plaques are characterized by a thin fibrous cap surrounding a large lipidic and necrotic core and by intense ongoing inflammation. The inflammatory process leading to the development of vulnerable atherosclerotic lesions is characterized by extensive recruitment of monocytes and lymphocytes into the arterial wall.
Several endothelial adhesion molecules are implicated in the process of leucocyte rolling, firm adhesion and transmigration, such as VCAM-1. VCAM-1 is an endothelial adhesion molecule involved in the adhesion of monocytes to the vascular endothelium which has been shown to be overexpressed at the surface of vulnerable coronary atherosclerotic plaques.
Peptides consisting of residues 75-84 and 84-75/75-84 of the major histocompatibility complex-1 (MHC-1) molecule B2702, corresponding respectively to B2702-p, RENLRIALRY (SEQ ID NO: 1) and B2702-rp, YRLAIRLNER RENLRIALRY (SEQ ID NO: 2) bind specifically to VCAM-1 (Ling et al. (2000) Transplantation 70:662-667). It has been shown that these peptides could be used as specific markers of the vulnerable coronary atheroma plaque, in particular, in non-invasive imaging methods. The results indeed indicated that B2702-p activity colocalized with areas of plaque development expressing VCAM-1 on ex vivo autoradiographic images of aortas, with significantly higher tracer activity in zones of lipid accumulation than in normal areas of the arteries (Broisat et al. (2007) Eur. J. Nucl. Med. Mol. Imaging 34:830-840). However, in vivo imaging was not optimal due to high basal levels of circulating B2702-p.
The present invention arises from the unexpected finding by the inventors that mutated derivatives of B2702-p displayed improved characteristics for the in vivo imaging of VCAM-1 expression.
The present invention thus relates to a polypeptide comprising or constituted of the sequence RILAR (SEQ ID NO: 3).
The present invention also relates to the polypeptide as defined above for use in imaging methods, in diagnostic or prognostic methods, or as a medicament.
The present invention also relates to the polypeptide as defined above for binding to VCAM-1.
The present invention also relates to an imaging method comprising the steps of:
a) administering a polypeptide as defined above to an individual;
b) detecting the binding or the absence of binding of the polypeptide in body areas of the individual;
c) visualizing the body areas of the individual wherein binding of the polypeptide can be detected.
The present invention also relates to a method of diagnosis and/or prognosis of a disease in an individual comprising the steps of:
a) administering a polypeptide as defined above to an individual,
b) detecting the binding or the absence of binding of the polypeptide in body areas of the individual;
c) visualizing the body areas of the individual wherein binding of the polypeptide can be detected,
d) deducing therefrom whether the individual suffers or will suffer of said disease.
The present invention also relates to the use of a polypeptide as defined above, for the in vitro detection of VCAM-1 in a sample.
In the context of the invention, the terms “polypeptide” and “peptide” are used indifferently and refer to native peptides (either proteolysis products or synthetically synthesized peptides) and further to peptidomimetics, such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, or more immunogenic. Such modifications include, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, including, but not limited to, CH2—NH, CH2—S, CH2—S═O, O═C—NH, CH2—O, CH2—CH2, S═C—NH, CH═CH or CF═CH, backbone modification and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified in Quantitative Drug Design, C A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).
According to the invention, a peptide consists of less than 50 amino acids, preferably less than 40 amino acids, more preferably less than 30 amino acids, still preferably less than 20 amino acids. More preferably, peptides according to the invention have a length of from about 5 to about 18 amino acids, from about 5 to about 17 amino acids, from about 5 to about 16 amino acids, from about 5 to about 15 amino acids, from about 5 to about 14 amino acids, from about 5 to about 13 amino acids. Most preferably, peptides according to the invention have a length of 12, 11, 10, 9 or 8 amino acids.
As used herein, the term “amino acid” is understood to include: the 20 naturally occurring amino acids i.e. alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; amino acids harbouring the post-translational modifications which can be found in vivo such as hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids.
As intended herein, the peptides of the invention can be substituted by one or more atoms or groups.
Preferably, the polypeptide according to the invention comprises or is constituted of the sequence RILARY (SEQ ID NO: 4). More preferably, the polypeptide according to the invention comprises or is constituted of the sequence LRILARY (SEQ ID NO: 5). Still preferably, the polypeptide according to the invention comprises or is constituted of the sequence NLRILAR (SEQ ID NO: 6). More preferably, the polypeptide according to the invention comprises or is constituted of the sequence ANLRILARY (SEQ ID NO: 7). More preferably, the polypeptide according to the invention comprises or is constituted of the sequence RANLRILARY (SEQ ID NO: 8). Most preferably, the polypeptide according to the invention is constituted of the sequence HGRANLRILARY (called EP43, SEQ ID NO: 9).
The invention also relates to a polypeptide comprising or consisting of the sequence selected from the group consisting of HGRENLRILARY (called EP35, SEQ ID NO: 10), HGRENLRILARA (called EP36, SEQ ID NO: 11), HGRENLRILAAY (called EP37, SEQ ID NO: 12), HGRENLRIAARY (called EP 38, SEQ ID NO: 13), HGRENLAILARY (called EP40, SEQ ID NO: 14), HGRENARILARY (called EP41, SEQ ID NO: 15), HGREALRILARY (called EP42, SEQ ID NO: 16), HGAENLRILARY (called EP44, SEQ ID NO: 17), HGRANLRILARA (called EP51, SEQ ID NO: 18), HGRANLRILAAY (called EP52, SEQ ID NO: 19), RENLRILARY (SEQ ID NO: 20), RENLRILARA (SEQ ID NO: 21), RENLRILAAY (SEQ ID NO: 22), RENLRIAARY (SEQ ID NO: 23), RENLAILARY (SEQ ID NO: 24), RENARILARY (SEQ ID NO: 25), REALRILARY (SEQ ID NO: 26), AENLRILARY (SEQ ID NO: 27), RANLRILARA (SEQ ID NO: 28) and RANLRILAAY (SEQ ID NO: 29).
In a particular embodiment, the polypeptide as defined above may be substituted with a detectable marker.
As used herein, a “detectable marker” refers to a compound which produces a signal that may be detected. When it is associated with a molecule, such as the polypeptide as defined above, it allows monitoring the fate of the molecule in the organism. Examples of detectable markers include radioisotopes, fluorophores such as Fluoresceine, Alexa, Cyanine, chemoluminescent compounds such as luminol, bioluminescent compounds such as luciferase or alkaline phosphatase, and contrast agents such as nanoparticles or Gadolinium. The choice of the suitable detectable marker, which depends on the detection system used, is well within the scope of the person skilled in the art.
Preferably, the detectable marker is a radioisotope. Examples of radioisotopes more particularly used in nuclear imaging include iodine 123 (123I), iodine 125 (125I), technetium 99m (99mTc), indium 111 (111In), fluorine 18 (18F), gallium 67 (67Ga), gallium 68 (68Ga) and any other radioisotope usable in human beings. Accordingly, the radioisotope is preferably selected from the group constituted of 123I, 125I, 99mTc, 111In, 18F, 67Ga, or 68Ga.
As used herein, the expression “polypeptide substituted with a detectable marker” means that a moiety of an amino acid residue of said polypeptide is substituted with a detectable marker. For example, an hydrogen atom may be substituted by an iodine atom in tyrosine residues.
In a preferred embodiment; the polypeptide according to the invention is constituted of HGRANLRILARY-123I or 99mTc-HGRANLRILARY.
In the context of the invention, “imaging methods” refer to methods that enable visualizing the inside of an organism or organs of an organism. Examples of imaging methods encompass invasive techniques such as angiography or coronarography, endocoronarian ecography, and non-invasive techniques such as Doppler velocimetry, angiography by magnetic resonance imaging or nuclear medicine such as scintigraphy. Preferably, the imaging method according to the invention is scintigraphy.
In a particular embodiment, the polypeptide as defined above is used in imaging methods for detecting atherosclerotic plaques.
In the context of the invention, an “atherosclerotic plaque” or “atheroma plaque” refers to a lesion of vessel walls. Preferably, an “atherosclerotic plaque” according to the invention comprises a lipid core and a fibrous cap, said cap being constituted by smooth muscle cells, collagens and an extracellular matrix and isolating the lipid core from the arterial lumen. Atherosclerotic plaques may be found for example in the aorta, the carotid, or in the coronary artery. When the plaque comprises a thin fibrous cap (about 65 to 150 μm thick) and a considerable lipid core, it is referred to as “vulnerable atherosclerotic plaque” or “vulnerable atheroma plaque” or “vulnerable plaque”. These instable plaques, which are prone to rupture, may be found in coronary arteries and in aorta and its branches.
Preferably, the polypeptide as defined above is used in imaging methods for detecting vulnerable atherosclerotic plaques. More preferably, the polypeptide as defined above is used in imaging methods for detecting aortic, carotidian, or coronary atherosclerotic plaques.
The present invention also relates to an imaging method comprising the steps of:
a) administering a polypeptide as defined above to an individual;
b) detecting the binding or the absence of binding of the polypeptide in body areas of the individual;
c) visualizing the body areas of the individuals wherein binding of the polypeptide can be detected.
In the context of the present invention, an “individual” denotes a human or non-human mammal, such as a rodent (rat, mouse, rabbit), a primate (chimpanzee), a feline (cat), a canine (dog). Preferably, the individual is human.
Any suitable method of administration, known from one skilled in the art may be used in step a). In particular, the polypeptide may be administered for example by the oral route, by inhalation, or by the parenteral route (in particular by intravenous injection). When the parenteral route is selected, the polypeptide may be in the form of injectable solutions and suspensions, conditioned in ampoules or flasks. The forms for parenteral delivery are conventionally obtained by mixing the polypeptide according to the invention with buffers, stabilizers, preservatives, solubilizing agents, isotonic agents and slurrying agents. According to known techniques, these mixtures can then be sterilized and conditioned in the form of intravenous injections. One of skill in the art may use organic phosphate salts-based buffers as buffer. Examples of slurrying agents include methylcellulose, acacia and sodium carboxymethylcellulose. Examples of stabilizers include sodium sulphite and sodium metasulphite, and examples of preservatives include sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol.
The amount of polypeptide administered naturally depends on the administration route, the size and/or weight of the individual, and the detection technique used.
As used herein, the expression “body area” refers to a determined region of the organism. It may be for example an organ, a part of an organ, or a tissue, such as a lung, the heart, the liver, the spleen, or a kidney, or a blood vessel such as an artery or a vein.
Any suitable system of detection and visualization, known from the one skilled in the art may be used in steps b) and c) defined above. In particular, steps b) and c) may be carried by scintigraphy. In this case, the polypeptide used is preferably substituted with a radioisotope selected from the group constituted of 123I, 125I, 99mTc, 111In, 18F, 67Ga or 68Ga. More preferably, the polypeptide used is constituted of HGRANLRILARY-123I or 99mTc-HGRANLRILARY.
In a preferred embodiment, the imaging method as defined above is used for visualizing atherosclerotic plaques of the individual. In particular, the imaging method as defined above may be used for visualizing aortic, carotidian, or coronary atherosclerotic plaques of the individual.
As used herein, a “diagnostic method” or “diagnosis” refers to a method for determining whether an individual suffers from a pathology.
As used herein, a “prognostic method” or “prognosis” refers to a method for determining whether an individual is likely to develop a pathology.
Preferably, the polypeptide as defined above is for use in the diagnosis of cardiovascular diseases.
In the context of the invention, a “cardiovascular disease” refers to a disease that involves the heart or blood vessels (arteries and veins). More particularly, a cardiovascular disease according to the invention denotes a disease, lesion or symptom associated with an atherogenesis process that affects the cardiovascular system. It includes especially the conditions in which an atheroma plaque develops as well as the complications due to the formation of an atheroma plaque (stenosis, ischemia) and/or due to its evolution toward an acute ischemic stroke (thrombosis, embolism, infarction, arterial rupture). Cardiovascular diseases include atherosclerosis, atheroma plaque, in particular vulnerable plaque, coronary heart disease, angina pectoris, thrombosis, stroke, myocardial infarction, vascular stenosis, and infarction. More preferably, the polypeptide as defined above is for use for diagnosing atherosclerosis or a coronary heart disease.
As used herein, “atherosclerosis” denotes a disease affecting arterial blood vessels. Atherosclerosis can be characterized by a chronic inflammatory response in the walls of arteries, mainly due to the accumulation of macrophages and promoted by low density lipoproteins without adequate removal of fats and cholesterol from macrophages by functional high density lipoproteins.
As used herein, a “coronary heart disease” denotes a progressive disease, due to a bad irrigation of the heart muscle, consecutive to the narrowing (stenosis) or calcification (sclerosis) of a coronary artery. The complete obstruction of a coronary artery leads to myocardial infarction.
In a particular embodiment, the polypeptide as defined above is for use for assessing the risk of occurrence of an acute ischemic stroke.
As used herein, the expression “risk of occurrence” refers to the probability that a subject develop a pathology.
As used herein, an “acute ischemic stroke” denotes the decrease of arterial blood uptake to a region of the organism. Its main local causes are thrombosis and embolism. When the atheroma plaque is located in the coronary artery, the acute ischemic stroke is in particular a myocardial infarction. When it is located in a carotid artery, it may lead to cerebral vascular accident. When it is located in a renal artery, it may lead to a renal artery embolism. When it is located in an artery of a limb, it may lead to an acute ischemic stroke of a limb.
Therefore, the polypeptide as defined above is preferably for use for assessing the risk of occurrence of an acute ischemic stroke selected from the group consisting of a myocardial infarction, a cerebral vascular accident, a renal artery embolism, and an acute ischemic stroke of a limb.
A method of treatment comprising the administration of a therapeutically effective amount of the polypeptide as defined above is also included in the present invention.
The polypeptide as defined above is advantageously coupled to a cytotoxic agent, so that the cytotoxic agent is contacted with a VCAM-1 expressing cell and selectively destroys this cell. The selection of a suitable cytotoxic agent is within the scope of one skilled in the art. Examples of cytotoxic agents include radioisotopes, toxins or chemotherapeutic agents.
The invention relates more particularly to the polypeptide as defined above for use for treating atherosclerosis and/or preventing an acute ischemic stroke, more preferably, for treating a vulnerable atherosclerotic plaque.
The present invention also relates to the polypeptide as defined above for binding to VCAM-1.
“VCAM-1” (Vascular Cell Adhesion Molecule 1) denotes a cellular adhesion protein, the transcription of which is induced in endothelial cells but which is also expressed in other cell types. VCAM-1 was identified and cloned by Osborn et al. in 1989 (Osborn et al. (1989) Cell 6:1203-11). VCAM-1 interacts with integrin α4β1, also called VLA-4 (Very Late Antigen 4), which is expressed constitutively in lymphocytes and monocytes. Like other adhesion molecules, such as ICAM-1, 2 and 3, VCAM-1 is involved in the adhesion of monocytes to the endothelium during atherosclerosis. VCAM-1 also interacts with integrin α4β7 to recruit lymphocytes in the intestine.
The polypeptide as defined above may also be used for the in vitro detection of VCAM-1 in a sample.
As used herein, a “sample” refers to a part of a bigger set. Preferably, a sample according to the invention is a substance of biological origin. Examples of biological samples include, but are not limited to, pieces of organs or of tissues such as kidney, liver, heart, lung, and the like, arteries veins and the like, blood and components thereof such as plasma, platelets, subpopulations of blood cells and the like.
The following examples and figures illustrate the invention without limiting its scope.
The objective of this study was to determine whether mutated derivatives of B2702-p (SEQ ID NO: 1) displayed improved characteristics for the in vivo imaging of VCAM-1 expression.
Material and Methods
Polypeptides and Tracers
Eleven derivatives of B2702-p were obtained by site-directed mutagenesis by selectively substituting each residue of the B2702-p sequence by an alanine residue.
The sequences of the eleven derivatives are listed in Table 1.
The polypeptides were labelled with 99mTc as described in Broisat et al. (2007) Eur. J. Nucl. Med. Mol. Imaging 34:830-840.
Animal Model and Experimental Protocol
An experimental model of ApoE−/− mice with left carotid artery ligation was used. Indeed, hypercholesterolemia associated with the vascular remodelling induced by carotid artery ligation induces the development of a focal atherosclerotic lesion at the site of ligation.
Left carotid artery ligation was performed under anesthesia and the animals were returned to their cages. Three weeks later, the animals were re-anesthetized and the radiotracers were injected in a lateral tail vein. Planar imaging was performed for 3 hours before the animals were euthanized. The major organs were excised, weighed and gamma-well counted for the determination of the tracers' biodistribution. Immunohistochemistry was also performed to evaluate VCAM-1 expression in the atherosclerotic lesion.
The experimental model was validated by assessing the presence of VCAM-1 in the atherosclerotic lesion using immunohistochemistry. The inventors observed a positive VCAM-1 staining in the atherosclerotic lesion as compared with a contralateral vessel.
In Vivo Planar Imaging
In vivo planar imaging was performed using a small-animal dedicated gamma-camera equipped with a parallel-hole collimator.
In Vivo Pinhole SPECT Imaging
In vivo pinhole SPECT imaging was performed using a small-animal dedicated gamma-camera equipped with a pinhole collimator for high-resolution imaging.
Results
Organ biodistributions of B2702-p and polypeptides EP35, EP36, EP37, EP38, EP40, EP41, EP42, EP43 and EP44 3 hours after injection are shown in Table 2.
The inventors observed that the elimination of the tracers was mainly renal and to a lower extent hepatic. The highest left carotid activity was observed following injection of EP43. The activity of the tracer in the atherosclerotic lesion was significantly higher than that observed in the contralateral, control carotid.
The inventors also analyzed the left-to-right carotid activity ratios (
The inventors demonstrated that EP43 left-to-right carotid activity and left carotid-to-blood activity ratios were both significantly higher than those of B2702-p. EP43 was the only derivative tested that displayed such improved characteristics with respect to B2702-p.
The inventors then analyzed the organ biodistributions (
In Vivo Planar Imaging
The inventors then performed in vivo whole-body planar imaging of mice injected with radiolabelled B2702-p and EP43 (
Images were obtained following injection of a similar dose of tracer. The high circulating blood activity precluded the non invasive imaging of B2702-p uptake in the carotid atherosclerotic lesion, whereas the improved characteristics of EP43 allowed the detection of VCAM-1 expression in the atherosclerotic lesion.
The inventors further quantified the above planar images (
The inventors also performed in vivo whole-body planar imaging of mice injected with radiolabelled EP51 and EP52 (
In Vivo Pinhole SPECT Imaging
High resolution pinhole SPECT imaging was also performed by the inventors following injection of EP43 to further confirm these results (
The inventors observed EP43 uptake at the level of the atherosclerotic lesion. In the other hand, no significant tracer activity could be detected in the right carotid area.
Image quantification was performed for all animals that underwent SPECT imaging of EP43 uptake (
Accordingly, the present inventors have shown that the new radiolabelled polypeptide EP53 allowed in vivo SPECT imaging of VCAM-1 expression in atherosclerotic lesions, indicating that EP43 is a tracer useful for non-invasive imaging of atherosclerotic lesions in patients.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09305016.9 | Jan 2009 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2010/050025 | 1/5/2010 | WO | 00 | 10/26/2011 |
