Patent Application
20110300071
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
Antiangiogenic therapy in conjunction with traditional chemotherapy and radiation represents a major step towards more selective and better-tolerated cancer treatments. However, there remains a need for imaging probes that permit sensitive detection and characterization of tumor angiogenesis and provide a means of following the progress of antiangiogenic tumor treatments (Dijkgraaf I, et al. Cancer Biother Radiopharm. 24:637-647, 2009).
Similarly, there is also a need for imaging probes that permit sensitive detection and characterization of atherosclerosis including atherosclerotic plaque, and provide a means of following the progress of treatments. Currently, most diagnostic modalities used for imaging atherosclerotic plaques assess the severity of the stenosis and/or plaque morphology. These tests include x-ray angiography, computed tomographic (CT) angiography, magnetic resonance imaging and intravascular ultrasound (Nissen, S. E., et al., Circulation 103: 604-616 2001; Saam T, et al., Radiology 244: 64-77 2007; Topol, et al., Circulation 92: 2333-2342 1995; Hong, C., et al., Radiology. 223: 474-480 2002; 2002; Sirol. M, Circulation 109: 2890-2896 2004). However, none of these modalities is able to provide information on the biology and metabolism of the plaque that may predict the rupture. (Fayad, Z. A., et al., Circ. Res. 89:305-316 2001; Fayad, Z. A., Neuroimaging Clin. N. Am. 12: 461-471, 2002.) Several radionuclide-based approaches for non-invasive, functional imaging of atherosclerosis have been developed and evaluated in animal models (Rosen, J. M., et al., J. Nucl. Med. 1990; 31:343-350; Vallabhajosula, S., et al., J. Nucl. Med. 29: 1237-1245, 1988; Rudd, J. H., et al., J. Nucl. Med. 49: 871-878, 2008; Langer. H. F., et al., J. Am. Coll. Cardiol. 52: 1-12, 2008; Vallabhajosula, S., et al., J. Nucl. Med. 38: 1788-1796, 1997; Tan, K. T., et al., Int. J. Cardiol. 127: 157-165, 2008; Ogawa, M., et al., J. Nucl. Med. 45: 1245-1250, 2004; Chang, M. Y., et al., Arterioscler Thromb. 12: 1088-1098, 1992; Kolodgie, F. D., et al., Circulation 108: 3134-3139, 2003; Lees, A. M., et al., Arteriosclerosis. 8: 461-470, 1988; Matter, C. M., et al., Circ. Res. 95: 1225-1233, 2004; Nahrendorf, M., et al., Circulation 117: 379-387, 2008; Prat, L., et al., Eur. J. Nucl. Med. 20: 1141-1145, 1993). Among the tracers for plaque imaging, those containing γ-emitters (technetium 99m, indium 111, iodine 123, etc.) suffer from the limited spatial resolution of single photon emission tomography (SPECT) (Davies, J. R., et al., J. Nucl. Med. 45: 1898-1907, 2004). In contrast, because of superior spatial resolution, positron emission tomography (PET) is more suitable for plaque imaging. (Langer. H. F., et al., J. Am. Coll. Cardiol. 52: 1-12, 2008; Davies, J. R., et al., J. Nuel. Med. 45: 1898-1907, 2004). To date, many PET radiotracers have been evaluated for imaging of atherosclerosis (Davies, J. R., et al., J. Nucl. Med. 45: 1898-1907, 2004). Among them, fluorine-18-fluorodeoxyglucose (FDG) is the most investigated (Rudd, J. H., et al., J. Nucl. Med. 49: 871-878, 2008; Ogawa, M., et al., J. Nucl. Med. 45: 1245-1250, 2004; Rudd, J. H., et al., Circulation 105: 2708-2711, 2002). Uptake of FDG in the aortic wall of patients with atherosclerosis has been attributed to infiltration of macrophages, smooth muscle cells, and lymphocytes within active atherosclerotic lesions (Tawakol, A., et al., J. Nucl. Cardiol. 12: 294-301, 2005). However, FDG accumulates in all metabolically active tissues as well as sites of inflammation and, therefore, its use for specific imaging of atherosclerotic plaques, and especially of vulnerable plaques, requires further evaluation (Laurberg, J. M., et al., Atherosclerosis 192: 275-282, 2007). The biology of atherosclerosis provides a number of other potential biomarkers for plaque imaging. For instance, degradation of the extracellular matrix and cell apoptosis are involved in plaque destabilization and can be imaged by using protease derivatives (cathepsin and matrix metalloproteinases) or radiotracers based on annexin-V (Jaffer, F. A., et al., J. Am. Coll. Cardiol. 47: 1328-1338, 2006). Also, the formation of plaque neovessels has been associated with interplaque hemorrage, cholesterol deposition and plaque growth, and therefore could be a marker of plaque vulnerability. Hence, angiogenesis markers such as, integrins, VEGF, and VCAM-1 are currently under evaluation for vulnerable plaque imaging with PET (Beer, A. J., et al., Cancer Metastasis Rev. 27: 631-644, 2008).
Natriuretic peptides (NPs) are a family of cardiac- and vascular-derived hormones that play a relevant role in cardiovascular homeostasis (Woodard G E, et al. Int rev Cell Mol. Biol. 268:59-93, 2008). Among the four family members, atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) have been demonstrated to suppress the signaling of vascular endothelial growth factor (VEGF), a key regulator of angiogenesis (Dijkgraaf I, et al. Cancer Biother. Radiopharm. 24: 637-647, 2009). Furthermore, ANP has been reported to attenuate the angiogenesis process (Kong, X., et al., Cancer Res. 68: 249-256, 2008; Vesely, D. L., J. Investig. Med. 53: 360-365, 2005). The NPs exert their biological effects through their interaction with NP receptors (NPRs) (Maack, T., et al., Science 238: 675-678, 1987). Among the NPRs, the clearance receptor (NPR-C) constitutes approximately 95% of the entire NPR population. In addition, NPR-C is the only NPR that recognizes all the NPs as well as NP fragments containing as few as five conserved amino acids (Arg-Ile-Asp-Arg-Ile) (Maack, T., Arq. Bras. Endocrinol. Metabol. 50: 198-207, 2006).
Molecular imaging, as an evolving technique, has played a major role in noninvasive, assessment of biologic processes in vivo and drug discovery over the past decade (Rosin R, et al. In: Schuster D P, Blackwell T S, eds. Molecular imaging of the lungs. New York: Taylor and Francis 2005:3-39; Dobrucki L W, et al. Nat Rev Cardiol 7:38-47, 2010; Sinusas A J, et al. Circ Cardiovasc Imaging. 1:244-256, 2008; Rudin M. Curr Opin Chem. Boil. 13:360-371, 2009).
In our previous study, we showed the 64Cu labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (ROTA)-C-type atrial natriuretic factor (CANF) conjugate (64Cu-DOTA-CANF) to be suitable as a tracer for PET imaging of NPR-C in the rabbit atherosclerosis model (Liu Y, et al. J Nucl Med. 51:85-91, 2010). Nevertheless, there remains a continuing need for imaging probes in disease processes.
Accordingly, the inventors herein have succeeded in devising new tracers which can be used for imaging distribution of natriuretic peptide receptors, including receptors which bind C-type atrial natriuretic factor (CANF). In some embodiments, these tracers can be used for imaging and monitoring angiogenesis during the course of anti-angiogenic treatment of cancer. In other embodiments, these tracers can be used for imaging and monitoring the presence and progression of atherosclerosis, including imaging of atherosclerotic plaque. In various embodiments, the tracers described herein can be used as probes for imaging angiogenesis or atherosclerosis using positron emission tomography (PET), scanning or magnetic resonance imaging (MRI) or other suitable imaging techniques.
Hence, in some embodiments, the present teachings disclose tracer molecules. A tracer of these embodiments comprises an amphiphilic comb-like nanostructure conjugated with a natriuretic peptide or fragment thereof and a signaling moiety. In some aspects, the oligopeptide can have the sequence of a C-type atrial natriuretic peptide or a fragment thereof. In various configurations, such oligopeptides can comprise the sequence Arg-Ile-Asp-Arg-Ile (SEQ ID NO:1). Thus, in some embodiments, the oligopeptide can be a fragment that is less than a full-length natriuretic peptide. A tracer comprising such oligopeptide fragments can be, in various configurations, a tracer which does not induce vasodilation or cause a drop in blood pressure in a subject following administration to the subject in an amount effective for imaging by positron emission tomography (PET) scanning. A tracer comprising such oligopeptide fragments can be, in various configurations, a tracer which does not induce vasodilation or cause a drop in blood pressure in a subject following administration to the subject in an amount effective for imaging by magnetic resonance imaging (MRI) scanning.
In other embodiments, the present teachings disclose imaging methods. In various aspects, these include methods of determining distribution of C-type atrial natiuretic peptide receptors in a subject. The methods include administering to a subject a tracer comprising a) an amphiphilic comb-like nanostructure conjugated with an oligopeptide comprising a fragment of a natriuretic peptide and b) a positron-emitting radionuclide. In various embodiments, the fragment can include the sequence Arg-Ile-Asp-Arg-Ile (SEQ ID NO:1). The method further includes subjecting the subject to positron emission tomography scanning. In various embodiments, the subject can be any mammal, including a human, such as a human in which angiogenesis is being monitored during an anti-angiogenic treatment for cancer, or a human in which means of imaging plaque is desired.
In yet another embodiment, the present teachings include methods of imaging angiogenesis in a subject. The methods include administering to a subject a tracer comprising a) an amphiphilic comb-like nanostructure conjugated with an oligopeptide comprising a fragment of a natriuretic peptide and b) a positron-emitting radionuclide. In various embodiments, the fragment can include the sequence Arg-Ile-Asp-Arg-Ile (SEQ ID NO:1). The method further includes subjecting the subject to positron emission tomography scanning. In various embodiments, the subject can be any mammal, including a human, such as a human in which angiogenesis is being monitored during an anti-angiogenic treatment for cancer.
In yet another embodiment, the present teachings include methods of imaging atherosclerotic plaque in a subject. The methods include administering to a subject a tracer comprising a) an amphiphilic comb-like nanostructure conjugated with an oligopeptide comprising a fragment of a natriuretic peptide and b) a positron-emitting radionuclide. In various embodiments, the fragment can include the sequence Arg-Ile-Asp-Arg-Ile (SEQ ID NO:1). The method further includes subjecting the subject to positron emission tomography scanning. In various embodiments, the subject can be any mammal, including a human, such as a human in which atherosclerosis is being monitored, such as during a stroke or heart attack.
In various embodiments of the present teachings, the oligopeptide can include at least 2 cysteine residues, which can comprise, in various configurations, at least one cystine (i.e., including a disulfide bridge). In some other configurations, the cysteines can be in reduced form (i.e., not including a disulfide bridge).
In some configurations, the oligopeptide comprised by the tracer can be no greater than about 20 amino acids in length. In some configurations, an oligopeptide comprised by a tracer can comprise the sequence Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys-NH2 (SEQ ID NO:2), in which the carboxy terminal cysteine is aminated. In some configurations, the cysteines of this sequence can comprise a disulfide linkage (a cystine).
In various configurations, an oligopeptide of the present teachings can be no greater than 25 amino acids, no greater than 24 amino acids, no greater than 23 amino, acids, no greater than 22 amino acids, no greater than 21 amino acids, no greater than 20 amino acids, no greater than 19 amino acids, no greater than 18 amino acids, no greater than 17 amino acids, no greater than 16 amino acids, no greater than 15 amino acids, no greater than 14 amino acids, no greater than 13 amino acids, no greater than 12 amino acids, no greater than 11 amino acids, or no greater than 10 amino acids in length. In some configurations, the cysteines of these oligopeptides can comprise a cysteine comprising a disulfide bridge, or can be in the reduced, free sulthydryl form. In addition, an oligopeptide of a tracer of the present teachings can further comprise a sequence unrelated to natriuretic peptide. Further, the tracer can include one or more non-peptidyl components such as a polymer such a polyethylene glycol.
Accordingly, in various aspects, the present teachings disclose a tracer that includes an amphiphilic comb-like nanostructure conjugated with an oligopeptide. A tracer can also include a signaling moiety. An oligopeptide moiety of these aspects can comprise a fragment of a natriuretic peptide, wherein the fragment comprises the sequence Arg-Ile-Asp-Arg-Ile (SEQ ID NO:1). An oligopeptide moiety can comprise a cysteine, and, in certain aspects, the oligopeptide can comprise the sequence Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys-NH2, (SEQ ID NO:2). In some configurations, the oligopeptide can be no greater than about 25 amino acids in length.
In some configurations, the oligopeptide moiety can be no greater than 20 amino acids in length, no greater than 19 amino acids in length, no greater than 18 amino acids in length, no greater than 17 amino acids in length, no greater than 16 amino acids in length, no greater than 15 amino acids in length, no greater than 14 amino acids in length, no greater than 13 amino acids in length, no greater than 12 amino acids in length, no greater than 11 amino acids in length, or no greater than 10 amino acids in length. In some configurations, the cysteine residues can comprise a cysteine. In some configurations, the oligopeptide moiety can be a fragment of a natriuretic peptide and consist of the sequence H-Arg-Ser-Ser-c[Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Cys]-NH2 (SEQ ID NO:3).
In some configurations, the tracer can comprise a signaling moiety that is a radionuclide such as a positron emitter. A positron-emitting radionuclide of these configurations can be, without limitation, carbon-11, nitrogen-13, oxygen-14, oxygen-15, fluorine-18, iron-52, copper-62, copper-64, zinc-62 zinc-63, gallium-68, arsenic-74, bromine-76, rubidium-82, yttrium-86, zirconium-89, technetium-94m, indium-110m, iodine-122, iodine-124, iodine-131, or cesium-137. In some configurations, a radionuclide can be selected from carbon-11, nitrogen-13, oxygen-15, fluorine-18, iron-52, copper-64, gallium-68, yttrium-86, bromine-76, zirconium-89, iodine-123 or iodine-124 or any combination thereof. In other configurations; a positron emitter can be selected from carbon-11, nitrogen-13, oxygen-15, fluorine-18 and copper-64 or any combination thereof. In some configurations, a radionuclide of the present teachings can be copper-64.
In various configurations, a radionuclide of the present teachings can be comprised by a carrier moiety, such as a chelating agent. In some configurations, a carrier moiety can be, without limitation, a dodecanetetraacetic acid such as 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetracetic acid (DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid).
In some configurations, the signaling moiety of a tracer of the present teachings can be a T I relaxation time-reducing agent, such as gadolinium, manganese or iron. In some configurations, the T1 relaxation time-reducing agent can be a gadolinium.
In some configurations, the signaling moiety of a tracer of the present teachings can be a T2 relaxation time-reducing agent. In some configurations, the T2 relaxation time-reducing agent can be a superparamagnetic iron oxide (SPIO) or an ultrasmall superparamagnetic iron oxide (USPIO).
The present inventors disclose a tracer and methods of using the tracer in molecular imaging. The tracer includes an amphiphilic comb-like nanostructure conjugated with an oligopeptide that is a natriuretic peptide or fragment thereof. In addition, the tracer includes a signaling moiety. The natriuretic peptide can be a CANF peptide so that the tracer comprises an amphiphilic comb-like nanostructure conjugated with a CANF peptide or fragment thereof.
In some embodiments, an amphiphilic comb-like nanostructure conjugated with a natriuretic peptide or fragment thereof can comprise CANF-comb copolymers. Such comb copolymers are based upon four building blocks: (a) polyethylene glycol (PEG) which is hydrophilic and can confer protein-resistance; (b) methyl methacryate which can serve as a hydrophobic backbone; (c) a chelator for a signaling moiety, such as, for example, 1,4,7,10-tetraazacyclododecane-1,4,7,10-teteraacetic acid (DOTA) for chelation of a positron emitter such as 64Cu; and (d) a targeting peptide such as CANF.
Synthesis of CANF-comb copolymers of the present teachings is described more fully in the Examples below. Briefly the synthesis involved the following. The DOTA methacryate was synthesized from bromomethyllacylate derivative and the tris-functionalized cyclen derivative. This allows direct incorporation of the complex containing the signaling moieties, such as 64Cu-DOTA into the interior of the nanoparticle after deprotection and 64Cu insertion. The CANF-PEG macromonomer was synthesized in two steps from a heterobifunctional PEG containing a hydroxyl and an azide chain end. The initial step involves introduction of the methacrylate functionality at the hydroxyl end of the heterobifunctional PEG through reaction with methacrylcyl chloride followed by attaching the acetylene-derivitized CANF using Cu(I) click chemistry (Lutz, et al. Angew Chem, Int. Ed. 46:1018-1025, 2007; Parrish, B. et al. J Am Chem Soc 127:7404-7410, 2005; Vestberg R, et al. J Polym Sci, Part A: Polym. Chem. 47:1237-1258, 2009). Using the components described above, the functionalized comb copolymers were synthesized by RAFT polymerization and assembled into comb-like nanoparticles as shown in
The tracers of the present teachings include an oligopeptide moiety which is a natriuretic peptide or fragment thereof (which does not contain the entire amino acid sequence of a full length natriuretic peptide). In various embodiments, the oligopeptide, moiety can comprise the sequence Arg-Ile-Asp-Arg-Ile (SEQ ID NO:1). In various configurations, the oligopeptide moiety can comprise, for example, no more than 25 amino acids of a full length natriuretic peptide, no more than 24 amino acids of a full length natriuretic peptide, no more than 23 amino acids of a full length natriuretic peptide, no more than 22 amino acids of a full length natriuretic peptide, no more than 21 amino acids of a full length natriuretic peptide, no more than 20 amino acids of a full length natriuretic peptide, no more than 19 amino acids of a full length natriuretic peptide, no more than 18 amino acids of a full length natriuretic peptide, no more than 17 amino acids of a full length natriuretic peptide, no more than 16 amino acids of a full length natriuretic peptide, no more than 15 amino acids of a full length natriuretic peptide, no more than 14 amino acids of a full length natriuretic peptide, no more than 13 amino acids of a full length natriuretic peptide, no more than 12 amino acids of a full length natriuretic peptide, no more than 11 amino acids of a full length natriuretic peptide, no more than 10 amino acids of a full length natriuretic peptide, no more than 9 amino acids of a full length natriuretic peptide, no more than 8 amino acids of a full length natriuretic peptide, no more than 7 amino acids of a full length natriuretic peptide, no more than 6 amino acids of a full length natriuretic peptide, or no more than 5 amino acids of a full length natriuretic peptide. The natriuretic peptide can be an atrial natriuretic peptide. In some embodiments, the peptide can be a C-type atrial natriuretic peptide. The natriuretic peptide can be a human atrial natriuretic peptide.
In various embodiments, a signaling moiety of the tracer can be any signaling moiety effective for providing a detectable signal using PET scanning. For PET, a signaling moiety can be any positron-emitting isotope known to skilled artisans. In various embodiments, a signaling moiety of a tracer of the present teachings can be any signaling moiety effective for providing a detectable signal using MRI. In these embodiments, the signaling moiety can be any T1 relaxation time-reducing agent, or any T2 relaxation time-reducing agent known to skilled artisans.
The present inventors disclose PET imaging of NPR-C receptor up-regulation associated with ischemia induced angiogenesis in mice. The NPR-C receptor presence was identified with the 64Cu-DOTA-CANF-Comb nanoprobe and PET/CT, as well as the immunohistochemistry. The imaging capability and superiority of the targeted 64Cu-DOTA-CANF-Comb nanoprobe over the 64Cu-DOTA-CANF peptide tracer were demonstrated.
The 64Cu-DOTA-CANF-Comb nanoprobe offers sensitive and targeted molecular imaging for NPR-C expression, for example in a rabbit atherosclerosis model. The superiority of the CANF-comb nanoprobe over the CANF-peptide is demonstrated in the examples below.
PET imaging illustrated significantly (p<0.05) higher standardized uptake values (SUV) of 64Cu-DOTA-CANF-Comb nanoprobe at the injured sites relative to the non-injured control site in a rabbit atherosclerosis models. Furthermore, the tracer uptake at the lesion of the targeted nanoprobe was much higher (p<0.05) than that of control nanoprobe. More importantly, in contrast to the previously published 64Cu-DOTA-CANF peptide tracer, the 64Cu-DOTA-CANF-Comb nanoprobe showed greatly increased (p<0.05) uptake and contrast ratio in targeting NPR-C receptor in atherosclerosis models. Western blot results showed the expression of NPR-C receptor. Both PET and IHC blocking studies confirmed receptor mediated tracer uptake.
A previous report had indicated the high sensitivity and specificity of 64Cu-DOTA-CANF for imaging NPR-C receptors in vivo (Liu, Y., et al. J Nucl Med. 51:85-91, 2010). However, its fast pharmacokinetics resulted in limited sensitivity and contrast in this murine angiogenesis model, thus making a CANF-modified nanoparticle a viable candidate for overcoming these difficulties. In related studies, the PMMA-core/PEG-shell amphiphilic nanoparticle showed in vivo behavior that could be accurately tailored by changing the molecular parameters of the starting functionalized copolymer (Welch, M. J., et al. J. Nucl. Med. 50:1743-1746, 2009). Scheme 1 (
The bio-distribution of the non-targeted control comb showed enhanced blood retention but presumably increased mononuclear phagocytic system (MPS) uptakes (
The HLI model has been used for many studies to identify various biomarkers (Limbourg, A., et al. Nat. Protoc. 4:1737-1746, 2009). The [15O] H2O PET imaging demoristrated the creation of ischemia and restoration of blood flow 7 days after. The PECAM staining of the previously ischemic thigh muscle showed a tightly packed bundle of newly formed capillaries, confirmed by H&E staining (
In summary, through modular construction of a DOTA-CANF-Comb nanoprobe with tailored physical and biological properties, we have demonstrated the usefulness of a multi-valent nanoprobe for targeting NPR-C receptors in the murine hindlimb ischemia model of angiogenesis. The blood retention, high specific activity, elevated targeting efficiency, and favorable uptake demonstrate the advantages of this amphiphilic DOTA-CANF-Comb nanoprobe.
The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999; and textbooks such as Fledrickson et al., Organic Chemistry 3rd edition, McGraw Hill, N.Y., 1970. Synthesis of tracers, including synthesis of oligopeptides, can be accomplished using routine methods well know to skilled artisans. In some cases, oligopeptides can be obtained from a commercial supplier, such as, for example, (Cys18)-Atrial Natriuretic Factor (4-18) amide (rat; Code H-3134) from Bachem (Torrence, Calif.) Pharmaceutical methods and compositions described herein, including methods for determination of effective amounts for imaging, and terminology used to describe such methods and compositions, are well known to skilled artisans and can be adapted from standard references such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C., et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003. As used in the present teachings and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise:
The following examples are intended to be illustrative of various embodiments of the present teachings and are not intended to be limiting of the scope of any claim.
The examples below illustrate the use of a positron labeled nanoprobe to image the natriuretic peptide clearance receptor in a hind limb ischemia model and demonstrate that nanoparticles can be very effective molecular imaging agents for positron emission tomography. In the studies reported below, a CANF fragment was conjugated to DOTA chelator and comb-like nanoparticle, respectively, to target the NPR-C receptor in murine hindlimb ischemia (HLI) model of angiogenesis.
This example illustrates the preparation of tracer comprising a CANF fragment conjugated to comb-like nanoparticle and DOTA chelator containing Cu as the signaling moiety.
Materials were purchased from Sigma-Aldrich (St. Louis) and used without further purification unless otherwise stated. The 64Cu (half-life=12.7 h, β+=17%, β−=40%) and [15O] H2O (half-life=2.07 min, β+=99.9%) were produced at the Washington University cyclotron facility according to methods well known in the art (McCarthy D W, et al. Nucl. Med. Biol. 24:35-43, 1997; Herrero P J, et al. Nucl. Med. 47:477-485, 2006). Functionalized polyethylene glycol) (PEG) derivatives were obtained from Intezyne Technologies (Tampa, Fla.). Tris-t-butylester-DOTA, 1,4,7,10-tetraazacyclododecane and DOTA-NHS were purchased from Macrocyclics (Dallas, Tex.). C-ANF (rat ANF(4-23), Des-Gln (Lees, A. M., et al., Arteriosclerosis 8:461-470, 1988)1, des-Ser (Matter, C. M., et al., Circ. Res. 95:1225-1233, 2004), des-Gly (Nahrendorf, M., et al., Circulation 117:379-387, 2008; Davies, J. R., et al., J. Nucl. Med. 45:1898-1907, 2004); des-Leu (, L., et al., Eur. J. Nucl. Med. 20:1141-1145, 1993) was purchased from Tianma Pharma (Suzhou, China). Centricon tubes were purchased from Millipore (Billerica, Mass.). HiTrap Desalting columns were from GE Healthcare Biosciences (Piscataway, N.J.). Zeba™ desalting spin columns were from Pierce (Rockford, Ill.). Dithiolester RAFT agent, DOTA methacrylate and N-succinimidyl 4-pentynoate were prepared by methods well known in the art (see for example, Liu, Y., J Nucl Med. 51:85-91, 2010; Pressly, ED., Biomacromolecules 8:3126-3134, 2007; Malkoch, M., Macromolecules 38:3663-3678, 2005; Perrier, S., Journal of Polymer Science, Part A: Polymer Chemistry 43:5347-5393, 2005; Shokeen, M., ACS Nano. 5:738-747, 2011).
Polymeric materials were characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy using either a Bruker 200 or 500 MHz spectrometer (Billerica Mass.) with the residual solvent signal as an internal reference. Gel permeation chromatography was performed in dimethylformamide on a Waters system equipped with four 5-μm Waters columns (300×7.7 mm) connected in series with increasing pore size (102, 103, 104, and 105 Å.) and Waters 410 differential refractometer index and 996 photodiode array detectors (Milford, Mass.). The molecular weights of the polymers were calculated relative to linear PMMA or PEG standards. Infrared spectra were recorded on a Perkin Elmer Spectrum 100 with a Universal ATR sampling accessory (Waltham, Mass.). Fast protein liquid chromatography was performed on GE ÄKTA system (Piscataway, N.J.) equipped with UV and Beckman 170 radio activity detectors (Fullerton) on a Superose 12 10/300 GL size exclusion column (10×300 mm, GE Healthcare Life Sciences, Piscataway, N.J.). An isocratic elution was performed at 0.8 mL/min by using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 150 mM NaCl mixture with neutral pH.
CANF and DOTA-NHS conjugation and purification were performed following standard procedures well known in the art (Liu, Y., J. Nucl. Med. 51:85-91, 2009; Rossini, R., J. Nucl. Med. 49:103-111, 2008; Sun, X., Biomacromolecules 6:2541-2554, 2005). Briefly, CANF and DOTA-NHS were mixed in 0.1 mmol/L Na2HPO4 (pH 7.5) at 4° C. overnight. The DOTA-conjugated CANF was purified by solid-phase extraction (C-18 Sep-Pak cartridges; Waters) and reversed-phase high-performance liquid chromatography (RP-HPLC), respectively. RP-HPLC was performed on a system equipped with a UV/VIS detector (Dionex) and a radioisotope detector (B-FC-3200; BioScan Inc., Washington, D.C.) on a C-18 analytic column (5 mm, 4.6-220 mm; Perkin Elmer). The linear gradient was from 100% H2O to 65% acetonitrile in 45 min at a flow rate of 1 mL/min and an ultraviolet absorbance at 210 nm. The conjugation efficiency was more than 95%, as determined by RP-HPLC. The presence of 1 DOTA per peptide was confirmed by liquid chromatography-electrospray ionization mass spectrometry on a 2695 separation and Micromass ZQ module (Waters).
CANF (59.3 mg, 0.037 mmol) was dissolved in 2 mL anhydrous DMF. 4-pentynoic anhydride (19.2 mg, 0.098 mmol) dissolved in 1.5 mL anhydrous DMF was added dropwise to the solution which was stirred for 2 days. Cold diethyl ether (15 mL) was added to the solution to triturate the product, which was subsequently dissolved in 2 mL of MilliQ water and freeze dried (yield 47.0 mg, 75%); Mw(ESI) 1674.73 {M+H+] (calc. 1674.80).
Synthesis of poly(ethylene glycol) CANF methacrylate (CANF-PEGMA)
N3-PEGMA (75.4 mg, 0.015 mmol) and Acetylene-CANF (42.8 mg, 0.025 mmol) Were dissolved in a solution of 1.0 g DMSO and 0.65 g MilliQ water followed by the additions of 50 μL 5 wt % aqueous CuSulfate (0.018 mmol) and 75 μL 5 wt % aqueous NaAscorbate (0.016 mmol), respectively. The mixture was allowed to stir for two days with repeat additions of CuSO4 (50 μL) and NaAscorbate (75 μL) solutions after one day. The product was purified by washing (10×) with MilliQ water in 15 mL centricon tubes (YM-5) and freeze-dried (yield 48 mg, 41%) (FT-IR, ν (cm−1): 3315, 2881, 1655, 1466, 1342, 1099, 962, 841. GPC Mn 6500, PDI 1.1 (PMMA standards, DMF).
The DOTA-CANF-Comb and non-targeted DOTA-Comb were synthesized as reported (Shokeen, M., et al., ACS Nano. 2011; 5:738-747, 2011) replacing the RGD-PEGMA with CANF-PEGMA. Mn 205 kDa, PDI 1.20 and Mn 220 kDa, PDI 1.25 for DOTA-CANF-Comb and control DOTA-Comb respectively, (GPC-DMF, PMMA standards).
The t-butyl protecting groups were removed by methods well known in the art (see for example Shokeen M, ACS Nano. 5:738-747, 2011; Pressly E D, Biomacromolecules. 8:3126-3134, 2007. Malkoch M, Macromolecules. 38:3663-3678, 2005). Typically the t-butyl groups of the DOTA functional groups of the copolymers were deprotected by dissolving in a 9:1 v/v mixture of dichloromethane/trifluoracetic acid (DCM/TFA) followed by solvent removal, redissolving in DCM/TFA and precipitation in hexane.
The deprotected polymers were then dissolved in DMSO (1 wt %), a rapid addition of an equal aliquot of water achieved assembly, and DMSO was removed by centrifugal filtration, resulting in particles of 22.0 nm and 20.4 nm (dynamic light scattering) for the targeting DOTA-CANF-Comb (zeta potential: −1.1±2 mV) and non-targeting DOTA-Comb (zeta potential: −35±2 mV) particles, respectively (see FIG. S1).
Copper-64 (t1/2=12.7 h, β+=17%, β−=40%) was produced on the Washington University Medical School CS-15 cyclotron by the 64Ni (p,n) 64Cu nuclear reaction at a specific activity of 1.85-7.40 GBq/γg at the end of bombardment. 19DOTA-CANF-Comb and control DOTA-Comb (5 μg, about 6 μmol) were labeled with 185 MBq 64Cu in 200 μL 0.1 M pH 5.5 ammonium acetate buffer at 80° C. for 1 h with a yield of 60.5±7.3% (n=15). The 64Cu-DOTA-CANF-Comb and 64Cu-DOTA-Comb were purified by 2 mL zeba spin desalting column after ethylene diamine tetraacetic acid (10 mM in 50 mM pH. 7.4 phosphate buffer) challenge. The radiochemical purity of the labeled nanoprobe was measured by radioactive thin layer chromatography (Washington D.C.).
This example illustrates the preparation of the Murine Hindlimb Ischemia (MHI) Model.
All animal studies were performed in compliance with guidelines set forth by the NIH Office of Laboratory Animal Welfare and approved by the Washington University Animal Studies Committee. Angiogenesis was induced in male C57BL/6 mice by placing two ligatures on a femoral artery above the saphenous branch and separated by 0.5 cm, followed by excision of the intervening segments. The contralateral femoral artery was exposed, but not ligated or excised as a sham control. The double ligation and vascular resection of a femoral arterial segment produced a severe ischemia of the affected hindlimb (HLI) verified by Doppler blood flow measurements (Perimed) in the distal thigh muscle and a significant increase in muscle blood flow (quantified as the ratio of ischemic to nonischemic hindlimb flow) after 7 days, consistent with lack the restoration of flow to the pre-surgery level and flow enhancement induced by angiogenesis (Almutairi, A., et al. Proc. Natl. Acad. Sci. USA. 106:685-690, 2009). Only animals showing this pattern of profound decrease in distal muscle blood flow at day 0 followed by a marked increase in muscle blood flow at day 7 were used in this study. Approximately 70% of mice undergoing HLI surgery showed the required pattern.
This example illustrates [15O] water PET estimation of blood flow change. PET using [15O] water offers direct physiological measurement of circulatory parameters for regional blood and vascular volume. In order to measure the blood flow changes caused by the surgical HLI and the resulting angiogenesis, blood flow was determined using [15O] H2O. In these experiments, the [15O] H2O (half-life=2.07 min, β+=99.9%) was produced at the Washington University cyclotron facility according to methods well known in the art (McCarthy, D. W., et al. Nucl. Med. Biol. 24:35-43, 1997; Herrero, P. J., et al. Nucl. Med. 47:477-485, 2006). About 22-37 MBq of [15O] water was intravenously (i.v.) injected into the same mice (n=4) after HLI surgery (day 0) and again 7 days later (day 7) (24). A 0-5 min dynamic scan was immediately obtained after the i.v. injection of [15O] H2O on an Inveon PET/CT system (Siemens Medical Solutions, Malvern, Pa.). The relative blood flow change was evaluated by standard uptake value (SUV) (Liu Y, et al Mol Pharm. 6:1891-1902, 2009).
After the induction of hind limb ischemia (HLI), blood flow was immediately decreased (
The following statistical analyses were used in some experiments reported herein. Group variation is described as mean±standard deviation. Group comparisons were made using 1-way ANOVA with a Bonferroni post-test. Individual group differences were determined with use of a 2-tailed Mann-Whitney test. The significance level in all tests was p<0.05. GraphPad Prism v. 5.02 was used for all statistical analyses.
This example illustrate bio-distribution studies using probes of the present teachings.
In these experiments, 64Cu-DOTA-CANF, 64Cu-DOTA-CANF-Comb, and Cu-DOTA-Comb were reconstituted in 0.9% sodium chloride (APP pharmaceuticals) for i.v. injection. Male C57BL/6 mice weighing 20-25 g (n=4) were anesthetized with inhaled isoflurane and about 370 kBq of labeled nanoparticles (0.8-1.2 μg/kg body weight) or DOTA-CANF peptide (0.8-1.1 μg/kg body weight) in 100 μL saline were injected via the tail vein. The mice were re-anesthetized before euthanizing them by cervical dislocation at each time point (1 h, 4 h, and 24 h) post injection (p.i.). Organs of interest were collected, weighed, and counted in a well gamma counter (Beckman 8000). Standards were prepared and measured along with the samples to calculate the percentage of the injected dose per gram of tissue (% ID/gram) (Liu, Y., et al. Mol Pharm. 6:1891-1902, 2009).
Biodistribution data of 64Cu-DOTA-CANF; Cu-DOTA-Comb, and 64Cu-DOTA-CANF-Comb are presented in
These data show a fast clearance profile of 64Cu-DOTA-CANF primarily through the kidney with minor accumulation in liver, lung and negligible uptake in other organs (
In contrast, the non-targeted 64Cu-DOTA-Comb displayed increased blood retention and slower clearance (25.4±3.04% ID/g at 1 h p.i., p<0.001, n=4) (
The targeted 64Cu-DOTA-CANF-Comb showed a superior bio-distribution profile with significantly improved circulatory retention (blood, lung, and heart) and reduced liver and renal clearance (
This example illustrates PET/CT imaging of 64Cu-DOTA-CANF in the HLI-induced angiogenesis model (
In these experiments, mice showing an increase in blood flow above, baseline level at 7 days after HLI surgery (n=6, 8, and 7 for DOTA-CANF, targeted DOTA-CANF-Comb nanoprobe, and non-targeted Comb, respectively) were anesthetized with isoflurane and injected i.v. with 3.7 MBq/100 μL of activity via the tail vein (8-11 μg/kg and 8-12 μg/kg of mouse body weight for the peptide and nanoprobes, respectively. The Molar ratio of 64Cu-DOTA-CANF-Comb to 64Cu-DOTA-CANF injected was 100:1.). For 64Cu-DOTA-CANF, a 0-60 min dynamic scan was performed on microPET Focus 120/220 (Siemens Medical Solutions) and the microCAT II (CTI-Imtek) scanners. The microPET images (corrected for attenuation, scatter, normalization and camera dead time) and microCT images were co-registered with fiducial markers attached to the animal bed and analyzed using AMIRA (Mercury Computer Systems, Chelmsford, Mass.). For 64Cu-DOTA-CANF-Comb and 64Cu-DOTA-Comb nanoprobes, the imaging sessions were carried out on an Inveon PET/CT system (Siemens Medical Solutions) and microPET Focus 220 at 1 h, 4 h (one 30-min frame, both) and 24 h p.i. (one 60-min frame). All the PET scanners were cross-calibrated periodically. The microPET images were analyzed with ASIPro (Almutairi, A., et al Proc Natl Acad Sci USA. 106:685-690, 2009). The tracer uptake values were not corrected for partial volume effects (Liu, Y., et al. J Nucl Med 51:85-91, 2010).
After the PET imaging, the animals were euthanized by exsanguination and the thigh containing the previously ischemic and nonischemic control muscles were perfusion fixed in situ with freshly prepared Michel's transport medium (American MasterTech Scientific Inc.) for histopathology and immunohistochemistry.
PET/CT imaging with 64Cu-DOTA-CANF at 7 days after HLI surgery showed tracer uptake in the distal thigh muscle, where ischemia had been induced previously, with weak signal deposited in the control, nonischemic limb (
This example illustrates PET/CT imaging of 64Cu-DOTA-CANF-Comb and 64Cu-DOTA-Comb in the HLI induced angiogenesis model obtained 7 days after ischemia, as shown in
With targeted 64Cu-DOTA-CANF-Comb nanoprobe, an increased accumulation at the lesion site of the ischemic limb was observed (
This example illustrates histopathology and immunohistochemistry.
As shown in
In these experiments, the perfusion-fixed tissue was stored overnight at 4° C. in Michel's Transport Medium before being frozen in OCT and step-sectioned (7 μm) at 100 μm intervals on a cryostat. Some of the sections at each step were stained with hematoxylin and eosin for identification of the morphology of the tissue.
Tissue sections at 100 μm intervals were also prepared for doubles immunofluorescent staining of NPR-C and either PECAM-1 for endothelial cells or alpha actin for vascular smooth muscle cells. The method for double immunolabeling followed the manufacturer's recommendations (Vector Laboratories). Briefly, the steps included avidin/biotin blocking; protein blocking with 5% normal horse serum for the first antibody (MEC 13.3 against CD31 [PECAM-1] or monoclonal anti-actin isotype IgG2a) and 5% normal goat serum for the second antibody (ANPC antibody [N-term] purified rabbit monoclonal); incubation for 30 min with the primary antibody diluted 0.04-0.5 mg/mL in buffer with normal serum added; incubation for 30 min with the biotinylated secondary antibody diluted 15 μg/mL in buffer with normal serum added; and incubation for 10 min with Fluorescin Avidin DCS (FITC, 10 μg/mL in buffer) for the first antibody (PECAM-1 or alpha actin) and Texas Red Avidin DCS (Rhodamine, 10 μg/mL in buffer) for the second antibody (NPR-C). Blocking of primary antibody binding to NPR-C were performed by pre-incubation of diluted antibody with the cognate peptide (0.5 mg/mL) overnight at 4° C. before immunohistochemistry staining. Slides were coverslipped and observed under the fluorescence microscope (Carl Zeiss) with appropriate filters.
This example, as shown in
Thigh muscle from the previously ischemic hindlimb showed areas of coagulation necrosis, but also an abundance of new capillaries, which in some sections appeared as tightly packed bundles identified by PECAM-1 staining of endothelial cells and by hematoxylin and eosin-staining (Couffinhal, T., et al. Am Pathol. 152: 1667-1679, 1998). Moreover, double immunostaining for PECAM-1 (
This example (
Competitive receptor blocking studies were performed in mice with HLI surgery (27±2.5 g) for 64Cu-DOTA-CANF by co-injection of unlabeled CANF peptide (CANF: 64Cu-DOTA-CANF=100:1 mole ratio, n=4) on day 7 after the surgery immediately followed by 0-60 min dynamic scans. For the 64Cu-DOTA-CANF-Comb, eight HLI mice (28±3.1 g) received co-injection of unlabeled DOTA-CANF-Comb nanoparticle and 64Cu-DOTA-CANF-Comb with 500:1 mole ratio on day 7 after the surgery and were scanned with PET/CT at 1 h, 4 h and 24 h p.i.
Competitive receptor blocking with co-injection of unlabeled DOTA-CANF resulted in a significant uptake decrease in the ischemic region (
This example illustrates materials and methods used in rabbit atherosclerotic-like lesion studies.
All animal studies were performed in compliance with guidelines set forth by the NIH Office of Laboratory Animal Welfare and approved by the Washington University Animal Studies Committee. Complex atherosclerotic-like arterial lesions containing a fibrous cap and a lipid-enriched core, similar to the structure of atheromatous plaques in human arteries, were induced in the right femoral artery of rabbits. Injury was induced by air desiccation and followed by angioplasty at a later time point as reported previously (Sarembock, I. J., et al., Circulation 80:1029-1040, 1989). Briefly, male New Zealand White rabbits were fed 0.25% cholesterol-enriched diet throughout the study and elevated serum cholesterol (>200 mg/dL) was confirmed at the time of vessel injury. The right femoral artery was exposed aseptically through a longitudinal skin incision and lidocaine was applied topically to prevent spasm. A 1-2 cm segment of the vessel was isolated between air-tight ligatures and small branches were ligated with suture. A 27-gauge needle was used to puncture the isolated segment proximally as a vent. A second 27-gauge needle was inserted distally into the segment and nitrogen gas was passed through the vessel at a flow rate of 80 mL/min for 8 min to dry and cause sloughing of the endothelium. The segment was then flushed with saline and the ligatures were released to restore blood flow, with gentle pressure applied to the puncture sites for a few minutes to maintain hemostasis. The skin incision was closed and the animal was recovered from anesthesia.
Four to six weeks after the air dessication-induced injury, the lesion site and extent of stenosis in the femoral artery were identified by an angiogram obtained with use of a 4F guide catheter introduced through a carotid arterial cutdown and advanced to the distal aorta. Heparin (100 U/kg, intravenous) was given to prevent clot formation in the catheters. A 0.014 in guidewire was then advanced across the lesion and the guide catheter was removed. A 2.0-2.5×20 mm coronary angioplasty balloon was advanced over the guidewire and the site of stenosis was dilated with three, 30 s balloon inflations of 6-8 atm with 1 min between inflations. After re-injuring the lesion site, patency of the femoral artery was confirmed by an angiogram through the angioplasty catheter before the catheter was removed. The carotid was ligated, the skin incision closed, and the animal was recovered from anesthesia.
The left femoral artery remained uninjured as a control.
The experimental design is schematized in
Receptor blocking studies were performed on three additional rabbits (4.1±0.34 kg). One-to-three weeks after the second injury, the animals were imaged with MRI and PET for a pre-blocking study to confirm the presence of atherosclerotic lesions and uptake of 64Cu-DOTA-C-ANF to NPR-C receptor on the plaque. One week later, besides the MRI imaging, the PET blocking studies were performed on the same rabbits scanned in pre-blocking studies by co-injection of 64Cu-DOTA-C-ANF with a blocking dose of unlabeled C-ANF peptide (1 mg, C-ANF: 64Cu-DOTA-C-ANF=100:1 mole ratio) and imaged with PET.
The baseline tracer uptake was measured in one healthy rabbit (4.6 kg) on a normal rabbit chow diet. The level of cholesterol in plasma was analyzed in each rabbit before imaging sessions. Samples of injured and control arteries were taken at each TP for histology and immunohistochemistry.
The presence of atherosclerotic lesions in the rabbits was confirmed by 3T MRI 1 hour after administration of a non-receptor specific plaque-targeting contrast agent (Gadofluorine M, 0.5 μmol/kg body weight, Schering AG, Germany) (Sirol, M., et al., Circulation 109:2890-2896, 2004; Zheng, J., et al., Invest. Radiol. 43:49-55, 2008; Meding, J., et al., Mol. Imaging 2:120-129, 2007). This agent has been shown to bind to extracellular matrix proteins such as collagen and proteoglycans (Meding, J., et al., Mol. Imaging 2:120-129, 2007). For scanning, the rabbit was placed supine into a plastic bed. Three micropipette tubes filled with 0.5 mL Gadofluorine M served as fiducial markers, and were taped into position on the bed. At the beginning of the PET study, these tubes were drained of the Gadofluorine by syringe, and refilled with 0.5 mL of 64Cu to serve as the PET fiducials. These helped to “co-localize” the plaque regions between the two image modalities.
Immediately after the MR scan, the rabbits were injected with 64Cu-DOTA-C-ANF (3.9±0.9 mCi) and 60 min dynamic scans were acquired on the microPET Focus-220 (Siemens Medical Solutions, Inc., Malvern, Pa.). Fiducial markers attached to the animal bed and filled with a 64Cu aqueous solution were used to correlate the MRI and MAP reconstructed PET images. In the competitive blocking experiments, 1 mg of C-ANF was co-administered with the radiotracer (100:1 mole ratio of blocking C-ANF to 64Cu-DOTA-C-ANF).
Data analysis of the microPET images was performed using the manufacturer's software (ASI Pro). The accumulation of 64Cu-DOTA-C-ANF at the injury site and on the contralateral, non-injured femoral artery (control) was calculated as standardized uptake values (SUVs) in 3D regions of interest (ROIs) by averaging the activity concentration corrected for decay over the contained voxels (multiple image slices) at selected time points post injection. (Sun, X., et al., Bioconjug Chem. 16:294-305, 2005). SUVs were not corrected for partial volume effects.
After the last PET imaging, the animals were euthanized by exsanguination and the femoral vessels were perfusion-fixed in situ with freshly prepared 4% paraformaldehyde. Tissue samples containing the injured and control arteries were harvested for histology and immunohistochemistry.
Vessel specimens were embedded in paraffin, step sectioned (10 μm) transversely at 1 mm intervals to approximate the distance between MRI slices, and the sections stained with hematoxylin and eosin (H&E) and Verhoeff's Van Gieson (VVG) stain for elastin. The sections were examined to identify the plaque components including foam cells, and vascular smooth muscle cells.
Immunohistostaining was performed on paraffin embedded sections from both the injured and non-injured arteries in each rabbit. For immunohistochemistry, we used anti-C-type natriuretic peptide receptor antibody (Abgent, San Diego, Calif.; 1:100) revealed by a secondary fluorescein isothiocyanate-conjugated anti-rabbit antibody (Invitrogen, Carlsbad, Calif.; 1:1000). Slides were viewed with a laser scanning microscope (LSM510 META, Carl Zeiss, Jena, Germany) and the image browser (Carl Zeiss, Jena, Germany). Blocking studies for NPR-C were performed by competitively blocking the primary antibody binding by pre-incubation of diluted antibody (NPR-C rabbit, Abgent, San Diego) with the cognate peptide (0.5 mg/mL) overnight at 4° C. prior to IHC staining. Also, absence of primary antibody was used as a negative control.
Results are expressed as mean and standard deviation (SD). The 2-tailed paired and unpaired Student's t test were used to test differences within animals (injured artery vs. control artery) and between animals imaged at different time points (such as TP 1 vs. TP 2), respectively. The significance level in all tests was ≦0.05. GraphPad Prism 4.0 was used for all statistical analyses.
This example illustrates copper-64 labeling and serum stability of 64Cu-DOTA-C-ANF. With C-18 Sep-Pak purification, the radiochemical purity of the 64Cu-DOTA-C-ANF was higher than 98% confirmed by radio-HPLC. The mass spectrometry of the decayed 64Cu-DOTA-C-ANF showed one DOTA conjugated to one C-ANF peptide.
64Cu-DOTA-C-ANF was highly stable in rabbit serum. The radio-HPLC analysis showed 97.7±3.9% (n=3) intact tracer after 1 h incubation at 37° C. On the contrary, only free 64Cu was detected in the control samples (64Cu-acetate incubated in rabbit serum) by radio-HPLC.
This example illustrates a tracer blood clearance study using the rabbit model for atherosclerosis.
In these experiments, the blood clearance studies were performed in normal rabbits (n=4) to evaluate the pharmacokinetics of tracer in vivo. About 20 MBq of 64Cu-DOTA-C-ANF was injected intravenously into the left ear of rabbit, and blood sample (0.2 mL) was drawn from the contralacteral ear over the period of 1 h (1 min, 3 min, 5 min, 10 min, 20 min, 40 min and 60 min). The activity of the blood samples were counted in gamma counter and presented in percent injected dose per gram (ID %/g).
This example illustrates plasma cholesterol levels in the rabbit atherosclerosis model.
At the time of imaging, compared to the reported normal baseline value of total plasma cholesterol (72±12 mg/dL), the rabbits on a high-cholesterol diet had 1111±366 mg/dL, 1451±421 mg/dL, and 1554±265 mg/dL total plasma cholesterol at TP1, TP2 and TP3, respectively.
This example illustrates histopathology in the rabbit atherosclerosis model.
Light micrographs of femoral arterial cross-sections from hypercholesterolemic rabbits were obtained at time points after injury and stained with Verhoeff's Van Gieson (VVG) for elastin (
In these experiments, as shown in
Immunohistochemistry confirmed the presence of NPR-C on the luminal surface of the neointimal (
This example illustrates MRI of Gadofluorine uptake in atherosclerotic-like lesions
In these experiments, the injured vessels in all rabbits at all time points demonstrated increased signal secondary to Gadofluorine M uptake. The site of injury visible on MRI also correlated with regions of increased radiotracer activity within the injured artery seen on PET. The control arteries showed no Gadofluorine M uptake.
This example illustrates quantitative PET imaging of 64Cu-DOTA-C-ANF in atherosclerotic-like lesions.
Specific binding of 64Cu-DOTA-C-ANF on injured arteries from rabbits from time points (TPs) is demonstrated in
In these experiments, tracer uptake in the injured femoral artery was visualized by PET.
The muscle tracer uptake around the injured artery shows low background (
Receptor blocking experiments (n=3) showed the similar uptakes on PET images for both injured artery and non-injured control artery (
This example presents results obtained using 25% CANF-Comb PET in a rabbit model.
In these experiments, rabbits were injected with 64Cu-CANF-Comb(˜2 mCi) at each time point. Animals were subjected to MicroPET scan at 1 h, 4 h, and 24 h post injection on Focus 220 scanner. At each time point, IHC and histopathology were performed on injured and control arteries to assess NPR-C presence and plaque morphology.
64Cu-DOTA-CANF
64Cu-CANF-Comb (n = 4)
The data demonstrate higher injury/control SUV values and higher injury/muscle SUV values for 64Cu-CANF-Comb compared to 64Cu-DOTA-CANF.
All references cited herein are incorporated by reference, each in its entirety.
This application claims priority from U.S. Provisional Application Ser. No. 61/351,847, filed Jun. 4, 2010, which is incorporated herein by reference in its entirety.
The Invention was made with government support under U.S.P.H.S. Grants U01 HL080729 and HHSN268201000046C awarded by the National Institutes of Health and National Cancer Institute Grant CA86307. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61351847 | Jun 2010 | US |
