WO2006082108A2

WO2006082108A2 - Imaging method and composition for imaging vascular diseases

Info

Publication number: WO2006082108A2
Application number: PCT/EP2006/001054
Authority: WO
Inventors: Philip Kaufmann; Bruno Weber; Alfred Buck
Original assignee: Schering Ag
Priority date: 2005-02-07
Filing date: 2006-02-07
Publication date: 2006-08-10
Also published as: WO2006082108A3

Abstract

The present invention relates to methods of using labeled choline or labeled choline derivatives as imaging agents for the detection and localization of vascular diseases or vascular lesions. The invention also relates to the use of labeled choline or labeled choline derivatives for the preparation of radiopharmaceutical compositions for the detection and localization of vascular diseases or vascular lesions.

Description

Schering AG

IMAGING METHOD AND COMPOSITION FOR IMAGING VASCULAR DISEASES

BACKGROUND OF THE INVENTION

Cardiovascular disease is a major health problem throughout the world, having a particularly high incidence in the United States, where during each year approximately 1,500,000 persons suffer a heart attack, approximately 400,000 to 500,000 persons suffer a stroke, and approximately 5,600,000 persons suffer angina. In the United States, heart attack (i.e., myocardial infarction) is in fact the leading cause of death, and stroke is the third leading cause of death. Unstable angina, i.e., severe constricting pain of coronary origin that occurs in response to less exercise than usually required to induce angina, may be associated with sudden cardiac death. A great need exists for early, accurate diagnosis of cardiovascular diseases.

Heart attack, angina, and stroke are caused by stenosis, or narrowing, of arteries, which is closely related to formation of atherosclerotic plaque, or atherogenesis. Until recently, cardiovascular lesions were believed to form gradually, and acute clinical episodes were believed to occur only when the stenosis exceeds 40% of the cross-sectional area of the original blood vessel's lumen. However, improved therapeutic methods such as thrombolytic therapy during acute myocardial infarction have revealed that atherosclerotic lesions most likely to precipitate a heart attack often were not associated with a high degree of stenosis. From autopsy studies, a class of atherosclerotic plaque has been identified, termed unstable or vulnerable plaque, which is associated with acute myocardial infarction and which is particularly susceptible to rupture. A large proportion of cardiovascular disease is now believed to progress through one or more subclinical episodes in which an unstable plaque is disrupted with local thrombin activation and subsequent healing. Acute myocardial infarction is now believed to result from formation of an occluding thrombus, or blood clot, at the site of a ruptured atherosclerotic plaque. Unstable angina is also believed to be associated with thrombus formation, while other forms of angina are believed to be associated with stenoses that are not associated with thrombosis. The presence of severe stenoses is now considered to be a marker for less occlusive, unstable plaques that may be prone to rupture. Presently, no means for identifying unstable plaques prior to an acute event or death is available, since current angiographic methods can only detect plaques when the extent of occlusion approaches 50% of the blood vessel luminal cross-section.

Atherogenesis is believed to begin with an initial lesion that appears as a fatty streak on the inner surface of an artery, consisting of two to five layers of lipid filled macrophages known as foam cells. Subsequently an intermediate fibrofatty lesion forms, consisting of twenty to thirty alternating layers of foam cells together with T lymphocytes and smooth muscle cells that separate the layers. Ultimately a fibrous plaque forms, in which a fibrous cap covers a central necrotic zone that may contain lipid, cells, and necrotic debris. A region containing numerous smooth muscle cells may also lay beneath the central necrotic lesion. The fibrous cap of an atherosclerotic plaque consists of layers of smooth muscle cells surrounded by a dense connective tissue matrix containing basement membrane, collagen fibers, and proteoglycan dispersed throughout the matrix. Some fibrous caps are inherently weaker than others, and these weak fibrous caps are rupture-prone, hi addition a plaque may be relatively stable or vulnerable. It is mostly the latter kind which harbors danger.

Since there are no diagnostic methods that can detect early stages of atherosclerosis and related vascular diseases, which often are clinically silent, and notably no diagnostic methods available to detect vulnerable plaques. Lifestyle changes, drug therapy, and other means, which would allow delaying or reducing vascular occlusion or the stresses on various body organs, which result from atherosclerotic lesions are not used in a timely fashion. Consequently, the early detection of vulnerable atheromatous plaques in the vascular system would be of considerable value in permitting preventive intervention at a time when it can be most effective. There is a particular need to detect vulnerable plaques early, since these are the most prone to rupture with serious, life threatening consequences. Currently available methods for diagnosing cardiovascular diseases employ a variety of imaging technologies, including conventional x-ray imaging, computerized tomography (CT), magnetic resonance imaging (MRI), ultrasound, and nuclear medicine imaging. Contrast agents and radiopharmaceuticals capable of accumulating at the site of a lesion are commercially available for use in imaging cardiovascular disease, for example in angiography and venography. However, angiograms only detect stenosis and, thus, do not detect or measure the degree of atherosclerosis accurately, since they do not detect plaques that cause no stenosis. The radiopharmaceuticals include nuclides such as ²⁰¹TI, "¹¹¹Tc, ¹³³Xe; or nuclide labeled metabolic agents like ^πC-2-deoxy-D-glucose, ¹⁸F-2-fluorodeoxy-D-glucose, [¹¹C]- and [¹²³I]-beta-methyl fatty acid analogs, ¹³N-ammonia; or infarct avid agents such as ^99mTc-tetracycline, ^99mTc- pyrophosphate, ²⁰³Hg-mercurials, ⁶⁷Ga-citrate, and the like. These imaging agents have not been suitable for diagnosis of cardiovascular lesions, especially for pre-acute event diagnosis of unstable atherosclerotic plaques.

Arteriography, the conventional approach to diagnosing vascular disease, involves catheterization and the injection of radiopaque substances into the bloodstream in order to image obstructions in the arteries. This procedure involves significant morbidity, in that infection, perforation of the artery, arrhythmia, stroke, infarction, and even death can occur. Because of the risks involved, arteriograms typically are reserved for individuals with advanced or acute atherosclerotic disease.

Other methods based on the use of ultrasonic waves and MR tomography have also been used to diagnose atherosclerosis. These methods suffer from the disadvantage that they detect atherosclerotic vascular changes based on the diminished blood flow or by detecting significant alterations of the artery wall and are, thus, only capable of detecting advanced stages of athero genesis. Moreover, these methods do not allow distinguishing between vulnerable plaques and stable plaques.

A variety of less invasive techniques for the diagnosis of vascular lesions and diseases at earlier stages have been proposed, but have not yet been put into routine clinical practice. These techniques include plethysmography, thermography, ultrasonic scanning (Lees and Myers, Adv.

Int. Med. 27:475, 1982) ), optical coherence tomography (Brezinski et al. (1997) Heart, 77: 397- 403), Raman spectroscopy (Romer et al. (1998) Circulation, 97, 878-885) and high resolution MRI tomography (Fayad et al. (1998) Circulation, 98: 1541-1547).

Non-invasive methods of diagnosing atherosclerosis using radiodiagnostic tracers have been described before. Thus, antibodies labeled with radioisotopes, or labeled low-density lipoproteins (LDL), were introduced that bond to atherosclerotic wall sections (Lees et al. (1983) J. Nucl. Med., 24: 154-156; Kaliman et al. (1985) Circulation, 72: 300; Virgolini et al. (1991) Sur. J. Nucl. Med., 18: 944-947). These methods, however, have major disadvantages, e.g. the antigenicity of the antibodies, which is a burden to the patient and prevents repeat administration of the antibody and the long period of time (several days) required to isolate, refine, and label the LDL obtained from the patient's blood. Above all, these big molecules have a long half-life in the blood, which together with high background radiation makes it difficult, if not impossible, to locate atherosclerotic lesion.

Some smaller targeting molecules have been suggested for atherosclerosis radio-imaging. Shih et al. (1990, Proc. Natl. Acad. ScL, 87, 1436-1440) have synthesized partial sequences of the LDL protein moiety (apo-B-100) that still bind to atherosclerotic plaques but have a considerably shorter half-life in the blood and an improved signal-to-noise ratio. However, due to the low affinity of the apo-B-peptides to the plaque and/or lower density of the bonding places in the plaque it was not possible to establish a successful in vivo diagnosis of atherosclerosis with the apo-B-peptides.

Endothelin peptides and derivatives have been proposed as biological carriers being able to target sites of vascular injury. Dinkelborg et al. describe in US 6,342,201 Bl radiolabeling of these endothelins and derivatives, and propose their use as imaging agents. Radiolabeled somatostatin peptides have also been proposed for radiodiagnosis of atherosclerotic lesions (Dean et al. US 5,976,496). The small (36 kDa) protein annexin V radiolabeled with ^99mTc has been shown to image apoptosis in human tumors (Mochizuke et al. (2003) J. Nucl. Med., 44: 92- 97), and has been proposed as a diagnostic agent for atherosclerosis based on promising results in a rabbit model (Kolodgie et al. (2003) Circulation, 108: 3134-3139). The glucose analog, [¹⁸F]fluoro-2-deoxy-glucose (FDG), has proven successful as a PET imaging agent for detection and localization of many forms of cancer. FDG also accumulates at sites of inflammation, and since atherogenesis has an inflammatory component, this tracer has been proposed for imaging of atherosclerosis. Preliminary clinical studies have shown accumulation of FDG in suspected atherosclerotic plaques in carotid arteries (Rudd et al. (2002) Circulation, 105: 2708-2712) and in thoracic aortas (Tatsumi, et al. (2003) Radiology, 229: 831- 837) using PET/CT. It remains to be seen whether the uptake of FDG will correlate with stability of the plaques.

Early detection of atherosclerosis would be of great importance for monitoring the therapeutic effect of diets, calcium antagonists, lipid and hypertension depressants, for monitoring restenosis after angioplastic surgery, for diagnosing coronary heart diseases, and for detecting thrombotic deposits in the vessels.

Consequently, there exists a need for better noninvasive techniques and reagents capable of detecting and mapping early, non-stenosing, non-fiow-disturbing atherosclerotic arterial lesions.

Accordingly, it is an object of the present invention to provide a method of detecting and mapping vascular lesion, including vascular lesion at its early and especially vulnerable stages.

Yet another object of the present invention is to provide means to prepare pharmaceutical compositions for detection of a lesion in a vascular system.

FDG PET has been found to have less sensitivity and/or specificity for some types of cancer, motivating efforts to develop new tracers for tumor imaging. Carbon-11

min) labeled choline (CH, [¹¹C]trimethyl-2-hydroxyethylammonium) has shown potential improved utility relative to FDG in two oncological imaging applications: brain tumors (Hara et al (1997) J. Nucl.

Med., 38: 842-847 Shinoura et al. (1997) Radiology 202: 497-503), where FDG has suboptimal specificity due to uptake by normal brain and some post-therapy responses (Marriott et al. (1998) J. Nucl. Med. 39: 1376-1390), and prostate carcinoma (Hara et al. (1998) J. Nucl. Med., 39: 990-

995), where FDG shows inadequate sensitivity due to the slow metabolism of tumor (Hoh et al.

(1998) J. Urology, 159: 347-356 and Shreve et al. (1996) Radiology 199: 751-756). The practical advantages of working with the longer lived radioisotope fluorine- 18 (T¹Z₂=I lO min) led Hara et al. ((1997) J. Nucl. Med. 38: 44P) to synthesize and preliminarily evaluate the choline analog, 2-[¹⁸ F]fluoroethyl-dimethyl-2-hydroxyethyl-ammonium.

De Grado et al. also proposed in US 6,630,125 the use of various analogs of ¹⁸F-Choline as PET imaging agent for cancer diagnosis, and notably prostate cancer.

DETAILED DESCRIPTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and KM, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in tlieir entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The present inventors have discovered that labeled choline or choline derivatives show a propensity to accumulate at sites of vascular lesions and, thus, can be employed to image vascular, preferably cardiovascular lesions. In particular, such labeled choline or choline derivatives can be used to image unstable atherosclerotic plaques and, thus, allow addressing a previously unmet need. By virtue of the present invention, the incidence of acute cardiovascular illness may be reduced through detection of unstable plaques in a patient and administration of appropriate treatment prior to the occurrence of a potentially debilitating or fatal myocardial infarction. Prior to the present invention, detection of unstable plaques has not been possible.

Thus, in one aspect the present invention provides a method for detection of a lesion in a vascular system of a subject, comprising the steps of:

(a) introducing into said subject labeled choline or choline derivative or a pharmaceutically acceptable salt of the labeled choline or choline derivative,

(b) detecting the accumulated choline or choline derivative in said vascular system.

The labeled choline or choline derivative or salt thereof, used in the methods according to the invention are particularly appropriate for detection purposes as they do not additionally and non- specifically accumulate at levels in other tissues or organs in the vicinity of the vascular lesion, which would interfere with the distinction between diseased and healthy tissue. This is decisive for their suitability as agents for diagnosis.

hi addition the labeled choline or choline derivative or salt thereof, used in the methods according to the present invention shows both higher accumulation in pathological vascular areas and better contrast characteristics due to more favorable pharmacokinetics than many of the agents for diagnosis that have previously been described for detecting vascular diseases.

The label comprised in the choline or derivative thereof can be any label known to someone of skill in the art, which allows its detection due to chemical or physical phenomenons, e.g. fluorescence; phosphorescence; α-₅ β-, γ-, or positron emission; or absorption. In a preferred embodiment the labeled choline or choline derivative comprises a label selected from the group consisting of a fluorescent label, an electron dense label, a radioactive label, and a paramagnetic label. Depending on the chemical nature of the respective label the skilled person is capable of coupling the label to choline using standard techniques. Labels capable of forming covalent bonds can be linked directly to the choline or derivative thereof or through a spacer while labels, which comprise a metal or metal ion can be complexed through a metal chelating moiety, which in turn can be covalently linked directly or indirectly to the choline or derivative thereof. A large variety of suitable metal chelating moieties are known in the art and are described in, for example, US 5,654,272, US 5,681,541, US 5,788,960, US 5,811,394, US 5,720,934, US 5,776,428, US 5,780,007, US 5,922,303, US 6,093,383, US 6,086,849, US 5,965,107, US 5,300,278, US 5,350,837, US 5,589,576, US 5,679,778 and US 5,879,659. The respectively described metal chelating residues are specifically referenced herewith and can all equally be used as metal chelating residues in the context of the choline or choline derivatives of the present invention.

The term "direct link" as used throughout the specification means a covalent bond to a residue of the choline or choline derivative, preferably to a backbone carbon, while the term "indirect link" as used herein means that one or more additional chemical residues which are connected by covalent or non-covalent bonds, preferentially by covalent bonds, are located between the choline or choline derivative and the label. These one or more additional chemical residues can also be termed "spacer" and can decrease the interaction between the choline or choline derivative and the label, which might interfere with the specific interaction between the choline or the derivative thereof and the vascular lesion.

The use of light in medical diagnosis has recently gained importance (see, e.g., Biomedical Photonics Handbook (Editor: T. Vo-Dinh), CRC Press). A wide variety of diagnostic processes are under experimental testing for application in various medical disciplines, e.g. endoscopy, mammography, surgery or gynecology. To this end dyes are fed to the tissue as exogenic contrast media for fluorescence diagnosis and imaging, and here in particular fluorescence dyes with an absorption and fluorescence maximum in the spectral range of 700-900 nm (diagnostic window of tissue), have been used for in vivo imaging. Photons of this wavelength are comparatively little absorbed by tissue and can therefore penetrate several centimeters into the tissue before the absorption process (primarily by oxyhemoglobin and deoxyhemoglobin) ends the light transport. Absorption can take place, moreover, by the fluorescence dyes that are introduced into the tissue, but that emit the absorbed energy in the form of longer-wave fluorescence radiation. This fluorescence radiation can be detected spectrally separated and makes possible the localization of dyes and the correlation with molecular structures to which the dye or a targeting residue attached thereto has bonded (see in this respect also Licha, K. (2002) In: Topics in Current Chemistry - Contrast Agents II (Editor: W. Krause), Volume 222, Springer Heidelberg, pp. 1 — 31.).

In the context of the method of the present invention a wide variety of fluorescent labels can be used. Particularly suitable fluorescent labels show absorption and/or emission in the near infrared, since light with a wavelength in the range of 700 to 900 run allows for an efficient photon migration through the tissues and has minimal autofiuorescence. Fluorescent labels are preferably used, if vascular lesions are detected in extremities, e.g. in the vascular system of arms, legs or the neck. A particular suitable vessel for performing the method of the present invention is the carotid artery, which can be penetrated by near infrared light. However, fluorescent labels, which are excited and/or emit light in a wavelength, which does not easily penetrate the skin and tissue can still be used in connection with endoscopic or arteriographic techniques. Preferred fluorescent labels, which can be used in a method of the present invention are selected from the group consisting of polymethine dyes, like dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium, holopolar cyanine, hemicyanine, oxonol and hemioxanole dyes; rhodamine dyes, phenoxazin dyes; or phenothiazin dyes. In particular, carbocyanines with indocarbocyanine, indodicarbocyanine and indotricarbocyanine skeletons have high extinction coefficients and good fluorescence quantum yields (Licha, K. (2002) supra, and the references cited therein). The synthesis of cyanine dyes useable according to the present invention can be carried out using the methods known in the state of the art and which are exemplified in, e.g. Hamer F.M. The Cyanine Dyes and Related Compounds, John Wiley and Sons, New York 1964; Ernst LA, et al. (1989) Cytometry 10:3-10; Southwick PL, et al., (1990) Cytometry 11:418-430; Lansdorp PM et al., (1991) Cytometry 12:723-730; Mujumdor RB et al., (1993) Bioconjugate Chem. 4:105-11; Mujumdor SR et al., (1996) Bioconjugate Chem. 7:356-62; Flanagan JH et al., (1997) Bioconjugate Chem. 8:751-56; KdI D et al., (1991) Dyes and Pigments 17:19-27; Terpetschnig E and Lakowicz JR (1993) Dyes and Pigments 21:227-34; Terpetschnig E et al., (1994) Anal. Biochem. 217: 197-204; Lindsey JS et al., (1989) Tetrahedron 45:4845-66; Gόrecki T et al., (1996) J. Heterocycl. Chem. 33, 1871-6; Narayanan N and Patonay G (1995) J. Org. Chem. 60:2391-5, 1995; and Terpetschnig E et al., (1993) J. Fluoresc. 3:153-155. Additional processes are described in patent publications US 4,981,977; US 5,688,966; US 5,808,044; EP 0 591 820 Al; WO 97/42976; WO 97/42978; WO 98/22146; WO 98/26077; and EP 0 800 831.

The coupling of fluorescent labels to the choline or choline derivative can be effected by art known methods involving, e.g. activated fluorescent labels. Such activated fluorescent labels carry a leaving group, which allows reaction with, for example, a hydroxyl residue on the choline or choline derivative. Suitable leaving groups include without limitation maleinimide (maleimide), bromoacetyl, chloroacetyl, iodoacetyl, chloroacetamido, bromoacetamido, iodoacetamido, chloroalkyl, bromoalkyl, iodoalkyl, pyridyl disulfide and vinyl sulfonamide.

CT and X-ray imaging is based on the principle that various substances effect different degrees of attenuation of an X-ray beam. Contrast agents useful for CT or X-ray imaging usually contain atoms which are electron dense, such as bromine or iodine, and are efficient attenuators of X-ray radiation. By far the most common CT agents are monomeric or dimeric iodinated benzene rings with various pendent groups such as ORAGRAFIN, CHOLOGRAFIN and RENOGRAFIN (Squibb Diagnostics, Princeton, NJ.). One important advance in the use of iodine-containing CT agents has been the development of non-ionic contrast agents, such as the ones described by M. T. Rneller et al., PCT Application Ser. No. WO 93/10825. Further suitable electron dense labels are known in the art. Preferably, the electron dense label is selected from the group consisting of Ag, Au, Br, Fe, and I. Particularly preferred electron dense labels comprised in the labeled choline or derivative thereof are Br and I, which can replace one or more hydrogens within the choline molecule or can be comprised in a further molecule, like iodinated benzene rings, which are directly or indirectly linked to the choline or derivative thereof. If metals or metal ions are used as electron dense labels, e.g. Ag, Au or Fe, they can be attached to choline or a derivative thereof using metal chelating moieties as outlined above. Nuclear imaging is based on the detection of radioactive isotopes. A wide variety of radioactive isotopes is known in the art, which can be used for imaging and/or therapeutic purposes. Isotopes, which can be used to label the choline or derivative thereof include without limitation ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ⁷⁶Br₅ ¹¹C, ^34mCl, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ¹⁶⁶Ho, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³N₅ ²²³Ra, ¹⁸⁶Re, ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ^94mTc, ^99mTc, and ⁹⁰Y. The isotopes ¹¹C, ^34mCl, ⁷⁶Br, ⁶⁸Ga, ¹⁸F, ¹²³I, ¹²⁴I, ¹³¹I, and ¹³N are particularly suitable for imaging and are, thus, particular preferred labels within the context of the present invention. Radioactive metals or metal ions are preferably attached to the choline or derivative thereof through a chelating residue, which is directly or indirectly linked to the choline. Radioactive isotopes capable of forming a covalent bond like, for example, ¹¹C, ^34mCl, ⁹⁶Br, ¹⁸F, ¹²³I, ¹²⁴I₅ ¹³¹I, and ¹³N can be directly or indirectly linked to the choline or derivative thereof or in case of ¹¹C and ¹³N replace one or more of the carbon or nitrogen moieties in the backbone of choline. Of course ¹¹C and ¹³N can also be comprised in a separate molecule, which is directly or indirectly linked to the choline or choline derivative. Methods of synthesizing molecules labeled with a radioactive isotope are known in the art. For example, US 6,630,125 B2 discloses methods of synthesizing ¹⁸F labeled choline and choline derivatives. Further examples for the synthesis of choline or its derivatives are given in: Hara et al. (2002) J. Nuc. Med, 43: 187-199; De Grado et al. (2000) Cancer Research, 61: 110-117; Degrado et al. (2001) J. Nuc. Med, 42: 1805-1814.

Depending on the imaging technique used, e.g. SPECT or PET, different amounts of labeled choline or derivatives thereof are required to provide an amount of radioactivity sufficient to detect the labeled choline or derivative thereof in the vascular system. Preferably the amount or radioactivity comprised within the choline or derivative thereof is within the range of about 0.1 to about 100 mCi and more preferably in the range of between about 1 and about 15 mCi, even more preferably between about 3 and about 10 mCi. These preferred and more preferred ranges of radioactivity are particular suitable in the context of ¹¹C, ^34mCl, ¹⁸F, ⁷⁶Br, ¹²³1, ¹²⁴1, ¹³¹I, and ¹³N labels, in particular ¹¹C, and ¹⁸F labels.

The term "paramagnetic label" as used herein means a metal ion which is magnetized parallel or antiparallel to a magnetic field to an extent proportional to the field. Generally, these are metal ions which have unpaired electrons; this is a term understood in the art. Examples of suitable paramagnetic metal ions, include, but are not limited to, gadolinium III (Gd³⁺ or Gd(III)), iron III (Fe³⁺ or Fe(III)), manganese II (Mn²⁺ or Mn(II)), yttrium III (Yt³⁺ or Yt(III)), dysprosium (Dy³⁺ or Dy(III)), and chromium (Cr(III) or Cr³⁺). In a preferred embodiment the paramagnetic ion is the lanthanide atom Gd(III), due to its high magnetic moment, a symmetric electronic ground state (S8), and its current approval for diagnostic use in humans.

Although the different categories of labels are only mentioned individually and are employed in different imaging and detection methods the methods of the present invention also comprise the simultaneous use of two labels of different categories, e.g. of a radioactive PET label like, e.g. ¹⁸F, and an electron dense label like, e.g. Br or I. In this case two imaging techniques, e.g. PET/CT, can be employed simultaneously or subsequently, which will allow a higher sensitivity and specificity in detecting vascular lesions in some applications. Accordingly, the present invention also comprises labeled choline or choline derivatives, which comprise two labels each detectable by a different imaging or detection technique. Preferably the two labels are selected from the group of labels comprising fluorescent labels, electron dense labels, radioactive labels, and paramagnetic labels. Alternatively, two or more different cholines or choline derivatives are administered simultaneously or subsequently (within an interval of a few minutes to a few hours), wherein each choline or derivative thereof carries a different label, preferably from categories of labels, which are detectable by different imaging or detection methods.

The term "pharmaceutically acceptable salt" refers to a salt of a choline or derivative thereof. Suitable pharmaceutically acceptable salts of choline or derivative thereof for compounds bearing a basic moiety include acid addition salts which may, for example, be formed by mixing a solution of choline or derivative thereof with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compound of the invention carries an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, foπnate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isotliionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, sub acetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like.

A "choline derivate" is an organic compound with a choline derived structure, i.e. it comprises a N or P heteroatom substituted with at least one lower alkyl (C₁ to C₅) side chain, which bears a terminal hydroxyl group and a short hydrocarbon chain (C₁ to C₅), preferably a lower alkyl chain (C₁ to C₅), having a propensity to accumulate at a site of vascular lesions, which is at least 10% of the propensity of choline at the same molar concentration. Preferably, the heteroatom of the choline derivative is further substituted with hydrogen or a short hydrocarbon chain (C₁ to C₅) optionally interspersed with a O or S residue, preferably a lower alkyl chain (C₁ to C₅). The propensity of a given derivative can be measured, for example, using an ApoE -/- mouse-model as outlined in Example 2 below. Fig. 1 shows the accumulation of a labeled choline derivative in areas of vascular lesions, while almost no label is detectable between the lesions. The ratio of the amount of label detected in healthy vascular tissue and diseased vascular tissue is a measure of the propensity of a labeled choline derivative to accumulate in vascular lesions. Preferably the choline derivative has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, 250%, 300%, 400%, 500% or more of the propensity of choline to accumulate at the site of vascular lesions.

As defined herein, a cardiovascular lesion includes atherosclerotic plaques at any stage of atherogenesis, e.g., initial fatty streaks, intermediate fibrofatty lesions, and fibrous plaque are all considered to be cardiovascular lesions which are detectable by the methods of the present invention. Any cardiovascular lesion may be detected using a labeled form of a choline or a choline derivative in accordance with the present invention. Preferably, highly stenotic plaques, i.e., plaques that occlude more than about 40% of the blood vessel luminal cross-section, are detectable using the methods of the invention. More preferably, non-critically stenotic plaques, i.e., plaques that occlude less than about 40%, preferably less than about 30%, less than about 20% of the blood vessel luminal cross-section, are detected using the methods of the present invention. Most preferably, unstable atherosclerotic plaques are detected using the methods and kits of the invention. A useful histopathologic definition of the term "unstable plaques" has been provided by the American Heart Association (AHA) on the basis of work by Herbert Stary (Stary HC, et al. (1992) Circulation, 85: 391; Stary HC, (1994) Circulation, 89: 2462-2478; Stary HC, et al. (1995) Circulation, 92: 1355—1374). This classification describes the natural history of plaque initiation and development designated by lesion types I (earliest lesion) through VI (complicated lesion). Early lesions up to type III lesions are potentially reversible. Lesion types IV and Va are called atheroma and fibro-atheroma, respectively. They can progress to vessel occlusion or to type VI lesions, that is, plaques with rupture, erosion, hematoma or haemorrhage, and thrombus formation. The difference between type IV and V plaques lies in the composition of the layer covering the lipid core. This layer is composed of preexisting intima in type IV plaques, whereas in type V plaques, the intima has been replaced by pathological fibromuscular tissue. Preferably, plaques of lesion type II or III are detected using the method of the present invention, i.e. at a stage at which a life style change or medication has the potential of reversing the disease.

Cardiovascular lesions are detected in accordance with the invention by administering an amount of labeled choline or choline derivatives which is sufficient for later detection. As used herein, this amount is defined as an amount sufficient to yield an acceptable image using equipment that is available for clinical use. An effective amount of the labeled choline or choline derivative may be administered in more than one dose, but is preferably administered in a single dose.

Effective amounts of the labeled choline or choline derivative may vary according to factors such as the degree of susceptibility of the individual; the age, sex, and weight of the individual; idiosyncratic responses of the individual; and in case of radioactively labeled cholines and derivatives thereof dosimetry. Effective amounts of the labeled choline or choline derivative may also vary with the particular instrument employed and film- and/or detector-related factors and can be determined by the physician. For example, assuming a medium body weight of 70 kg, the amount of radioactivity for diagnostic applications is preferably between 1 and 15 mCi, more preferably 3 and 10 mCi per application. Normally, a solution of the agent according to the invention is applied as an intravenous injection of 5 to 10 ml of the agent according to the invention.

Preferably the labeled choline or choline derivative or pharmaceutically acceptable salt of the choline or choline derivative is of the following formula (I):

wherein

A⁺ is N⁺ or P⁺,

B is a counteranion,

R¹ and R² independent of each other are H; a direct or indirect link to a label; or a C_1-5 straight or branched alkyl, e.g. C₁, C₂, C₃, C₄, or C₅ alkyl, in particular methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1- dimethylpropyl, or 1,2-dimethylpropyl; C_2-5 straight or branched alkenyl, e.g. C₂, C₃, C₄, or C₅ alkenyl, in particular ethenyl, propenyl, iso-propenyl, butenyl, iso-butenyl, pentenyl, 1- methylbutenyl, 2-methylbutenyl, 3-methylbutenyl, 1,1-dimethylpropyl, or 1,2-dimethylpropyl; C_2-5 straight or branched alkynyl, e.g. C₂, C₃, C₄, or C₅ alkynyl, in particular ethynyl, propynyl, butynyl, iso-butynyl, pentynyl, 1-methylbutynyl, 2-methylbutynyl, 3-methylbutynyl, or 1,1- dimethylpropynyl; C_1-5 straight or branched oxyalkyl, e.g. C₁, C₂, C₃, C₄, or C₅ oxyalkyl, wherein the O residue can be interspersed at any position between the carbon residues, in particular methoxy, ethyoxy, propoxy, butoxy, pentoxy, methylmethoxy, methylethoxy, methylpropoxy, methylbutoxy, ethylmetlαoxy, or ethylethoxy; C_2-5 straight or branched oxyalkenyl, e.g. C₂, C₃, C₄, or C₅ oxyalkenyl, wherein the O residue can be interspersed at any position between the carbon residues; C_1-5 straight or branched thioalkyl, e.g. C₁, C₂, C₃, C₄, or C₅ thioalkyl, wherein the S residue can be interspersed at any position between the carbon residues, in particular thiomethyl, thioethyl, thiopropyl, thiopentyl; or C_2-5 straight or branched tliioalkenyl e.g. C₂, C₃, C₄, or C₅ thioalkenyl wherein the S residue can be interspersed at any position between the carbon residues; the alky, alkenyl, alynyl, oxyalkyl, oxyalkenyl, thioalkyl or thioalkenyl is optionally substituted with one or more OH, aryl, heteroaryl or halogen, e.g. F, Cl, Br, I, and/or comprises at least one direct or indirect link to a label, preferably R¹ and R² independent of each other are H or substituted or unsubstituted C_1-5 straight or branched alkyl, e.g. C₁, C₂, C₃, C₄, or C₅ alkyl,

R³ is a C_1-3 straight or branched alkyl, e.g. C₁, C₂, or C₃ alkyl, in particular methyl, ethyl, propyl, iso-propyl, or alkenyl, e.g. C₂, or C₃ alkenyl, in particular ethenyl, propyl, iso-propenyl, optionally substituted with one or more OH or halogen, e.g. F, Cl, Br, I, and/or comprises at least one direct or indirect link to a label,

X¹ and X² independent of each other are H, deuterium or halogen, e.g. F, Cl, Br, I,

n= 1, 2 or 3, preferably 2 or 3;

under the proviso that at least one label is comprised within R¹, R² or R³, or that at least one carbon moiety within the choline or derivative is ¹¹C, or that at least one nitrogen moiety is ¹³N.

In a preferred embodiment R¹ and R² independent of each other are H, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₂)₂, CH^=CH₂, CH₂CH=CH₂, CH=CHCH₃, C≡≡CH, C≡≡CCH₃, CH₂C≡≡CH, CH₂CH₂CH₂CH₃, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, C(CH₃)₃, CH₂C(CH₃)==CH₂, CH=C(CH₃)CH₃, CH(C₆H₅), OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₂)₂, OCH=CH₂, OCH₂CH=CH₂, OCH=CHCH₃, OC≡≡CH, 0C≡≡CCH₃, OCH₂C≡≡CH, OCH₂CH₂CH₂CH₃, OCH₂CH(CH₃)₂, OCH(CH₃)CH₂CH₃, OC(CH₃)₃, OCH₂C(CH₃)=CH₂, OCH=C(CH₃)CH₃, OCH(C₆H₅), CH₂OCH₃, CH₂OCH₂CH₃, CH₂CH₂OCH₃ SCH₃, SCH₂CH₃, SCH₂CH₂CH₃, SCH(CH₂)₂, SCH=CH₂, SCH₂CH=CH₂, SCH==CHCH₃, SC≡≡CH, SC≡≡CCH₃, SCH₂C≡≡CH, SCH₂CH₂CH₂CH₃, SCH₂CH(CH₃)₂, SCH(CH₃)CH₂CH₃, SC(CH₃)₃, SCH₂C(CH₃)=CH₂, SCH=C(CH₃)CH₃, SCH(C₆H₅), CH₂SCH₃, CH₂SCH₂CH₃, CH₂CH₂SCH₃, optionally substituted with one or more OH, halogen, e. g. F, Cl, Br, or I, or comprises at least one direct or indirect link to a label.

In a further preferred embodiment R³ is (CX³X⁴)_mX⁵, wherein

X³ and X⁴ independent of each other are H, deuterium or halogen, e. g. F, Cl, Br, or I, preferably X³ and X⁴ are H,

X⁵ is H or a direct or indirect link to a label, and m = 1, 2 or 3, preferably 2 or 3.

hi the context of above preferred meaning of R³ it is preferred that R¹ and R² independent of each other have the meaning H; CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₂)₂, CH=CH₂, CH₂CH=CH₂, CH=CHCH₃, C=CH, C≡≡CCH₃, CH₂C≡≡CH, CH₂CH₂CH₂CH₃, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, C(CH₃)₃, CH₂C(CH₃)=CH₂, CH=C(CH₃)CH₃, CH(C₆H₅), OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₂)₂, OCH=CH₂, OCH₂CH=CH₂, OCH=CHCH₃, OC≡≡CH, OC=CCH₃, OCH₂C≡≡CH, OCH₂CH₂CH₂CH₃, OCH₂CH(CH₃)₂, OCH(CH₃)CH₂CH₃, OC(CH₃)₃, OCH₂C(CH₃)=CH₂, OCH=C(CH₃)CH₃, OCH(C₆H₅), CH₂OCH₃, CH₂OCH₂CH₃, CH₂CH₂OCH₃ SCH₃, SCH₂CH₃, SCH₂CH₂CH₃, SCH(CH₂)₂, SCH=CH₂, SCH₂CH=CH₂, SCH=CHCH₃, SC≡≡CH, SC≡≡CCH₃, SCH₂C≡≡CH, SCH₂CH₂CH₂CH₃, SCH₂CH(CH₃)₂, SCH(CH₃)CH₂CH₃, SC(CH₃)₃, SCH₂C(CH₃)=CH₂, SCH=C(CH₃)CH₃, SCH(C₆H₅), CH₂SCH₃, CH₂SCH₂CH₃, CH₂CH₂SCH₃, optionally substituted with one or more OH or halogen, e. g. F, Ce, Br, or I. If R , R and R have this meaning than at least one carbon moiety within the choline or derivative is ¹¹C and/or at least one nitrogen moiety is ¹³N, if X⁵ is H.

The counteranion is preferably selected from the group consisting of OH^", acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydro chloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate and valerate.

In a preferred embodiment of the method of the of the present invention the labeled choline or choline derivative is chosen form the group consisting of [¹⁸F] N,N-dimethyl-N- fluoroethylethanolamine (¹⁸F-fluoroethylcholine), [¹⁸F] N,N-dimethyl-N-fluoromethyl- ethanolamine (¹⁸F-fluorocholine), [¹⁸F] fluoromethyl-methylethyl-2-hydroxyethyllammonium, [¹⁸F] fluoropropyl-dimethyl-2-hydroxyethyl-ammonium, N,N-dimethyl-N-[^πC] methyl- ethanolamine (¹ ^-choline).

Administration may be accomplished by arterial or venous injection. In the context of PET imaging, analogs of the invention are preferably administered as an intravenous (IV) bolus. Typically, the patient is fasted at least 4 hours prior to administration of the analogue.

To allow at least a portion of the labeled choline or choline derivative to accumulate at the disease site it is preferred to carry out the imaging or detection method in a time interval of between 5 min and 20 hours after administration of the labeled choline of derivative thereof, preferably in a time interval of between 10 min and 10 hours, more preferably 20 min to 2 hours and even more preferably 30 min to 1 hour. The time required in each case will depend on the respective choline or choline derivative used and patient parameters and will ultimately be determined by the attending physician.

The method according to the invention can further comprise a step of imaging of a region of said vascular system at which said labeled choline or choline derivative has accumulated. In a preferred embodiment this is performed by extracorporeal monitoring, and in a more preferred embodiment, monitoring is performed with a fluorescent, CT, MRI, PET, fluorescent/PET, MRI/PET, CT/PET, ultrasound/PET, fluorescent/CT, fluorescent/MRI, fluorescent/ultrasound, CT/MRI, or CT/MRI/ultrasound scanner. The combination of two or more imaging methods is particularly preferred, if the choline or derivative thereof, which is used comprises labels of two or more categories, i.e. which is detectable with two or more different imaging methods. The PET imaging technique is particular preferred. It utilizes scanning devices that detect the 511 keV annihilation photons that are emitted after radioactive decay of the PET isotope. PET scanners are widely available for imaging of human subjects. In addition, "micro-PET" scanners that have high spatial resolution can be used for imaging of small animals or extremities of larger animals. In addition to PET scanners, positron emission radioactivity can also be monitored using one or more radiation detector probes.

In radiodiagnostic imaging, the recent development of combined PET (positron emission tomography) or SPECT (single photon emission tomography) + computed tomography modalities (PET/CT or SPECT/CT) has helped to better identify the precise location of the radiotracer uptake. In a preferred embodiment the method according to the invention includes detection of the accumulation of radiolabled choline or choline derivative by PET/CT or

SPECT/CT. PET is a radiodiagnostic technique uniquely suited to image small disease features like atherosclerotic lesions owing to its superior resolution and sensitivity relative to single photon emission tomography (SPECT). Furthermore, image reconstruction and attenuation correction techniques are better-developed for PET as compared to SPECT, allowing for better quantification. Therefore, a more preferred embodiment of the method according to the invention includes detection of the accumulation of radiolabled choline or choline derivative by PET/CT. PET can be used to detect and stage vascular diseases or vascular lesions because of its unique strength in detecting and quantifying small regions of label uptake. These advantages also make

PET particular suitable to be used to monitor a patient's response to therapy.

The labeled choline or choline derivative is preferably administered intravenously, in combination with a pharmaceutically acceptable carrier, to the subject. As used herein, a pharmaceutically acceptable carrier may include any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. The labeled choline or choline derivative is formulated as a sterile, pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.

The labeled choline or choline derivative may further be administered to the subject in an appropriate diluent or adjuvant or be co-administered with enzyme inhibitors or in a carrier such as species appropriate albumin. Pharmaceutically acceptable diluents include solutions such as saline or aqueous buffer solutions. Many such diluents are known to those of skill in the art, such as, for example, Sodium Chloride Injection and Ringer's Injection. Moreover, labeled choline and choline derivatives of the invention can also be formulated with a chemical stabilizer. In particular in the context of radiolabeled choline or derivatives thereof such stabilizers can reduce the likelihood for radiolysis-induced decomposition of the radiolabeled choline or choline derivative at high concentrations of radioactivity. Suitable stabilizers include antioxidants such as the pharmaceutically acceptable antioxidant, sodium L-ascorbate or α-tocopherol.

For administration to humans, the labeled choline or choline derivative may be administered in autologous serum or plasma. Supplementary active compounds may also be co-administered with labeled choline or choline derivative in accordance with the invention.

The invention further relates to the use of labeled choline or a labeled choline derivative or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for detection of a lesion in a vascular system of a subject, as described hereabove.

The invention further relates to the labeled choline or choline derivatives, which are suitable for carrying out the method of the present invention.

Labeled choline or choline derivatives can also be used in the noninvasive assessment of the response of vascular lesions in a patient to therapeutic interventions using PET scanning or another external radiation detection technique. The patient can be scanned at more than one time and the data from two or more scans are compared to determine potential differences in the uptake of the analogue at the site of said vascular lesion. Comparisons can involve either qualitative image comparison (e.g. contrast of vascular lesion uptake from background) or quantitative indices derived from the imaging or external radiation detection data (e.g. standardized uptake values (SUVs)).

DESCRIPTION OF THE FIGURE

Fig. 1: Plaque imaging in atherosclerotic ApoE -/- mice using ¹⁸F-Choline. The accumulation of ¹⁸F-Choline in the aorta of atherosclerotic mice is shown (ApoE ko #2 and ApoE ko #3), whereas no significant accumulation in the aorta of wild type mice (WT) has been observed (see left panel of the images for the respective animal). The right panel of the images show oil red O stained aortas for the respective animal. This suggests that ¹⁸F- Choline can be effectively used to perform PET scan of mammals in order to detect lesions in the vascular system, and particularly atherosclerotic vulnerable plaques.

EXAMPLES

The invention is further described in the following non-limiting examples.

Example 1: Synthesis of choline/choline derivative (precursors)

[¹⁸F]FCH was produced by the reaction of [¹⁸F]fluoromethyltriflate with diaminoethanol. [¹⁸F] fluoride (azeotropically dried with 2 x 0.7 ml acetonitrile) was reacted with dibromomethane in acetonitrile in the presence of Kryptofix(2.2.2) at HO⁰C to give [¹⁸F]fluorobromomethane (Bergman J, et al. (2001) Appl Radiat Isot, 54: 927-933) which was purified over a series of 4 Sep-Pak Plus silica cartridges. [¹⁸F]fluoromethyltriflate was made by passing [¹⁸F]bromofluoromethane over a silvertriflate/Graphpac GC column at 18O⁰C. [^lsF]fluoromethyltriflate was then used for the N-alkylation of 2-dimethylaminoethanol immobilized on a Sep-Pak Plus C-18 cartridge (a solution of 2-dimethylaminoethanol in ethanol (200 μl in 600 μl) was put on the cartridge), to quantitatively yield the desired product. For purification [¹⁸F]FCH was selectively trapped on a Sep-Pak Accel CM cartridge, washed with water and removed with saline and via a sterile filter added to a patient bottle with 4 ml saline, 0.5 ml 10% NaCl and 70 μl NaHC03. Quality control was by HPLC over a cation exchange column (Vassiliev D, et al. (2003) Eur. J. Nucl. Med., 30(supρl):S311). Supelcosil LC-SCX₃ 250x4.6 mm, 5 μm, eluting with 0.15 M NaH₂PO₄ in water/pyridine (1000/0.08 v/v) adjusted to pH 2.37 by 70% H₃PO₄. Radiochemical purity was > 99%. In the UV trace FCH was not visible and no quantifiable impurities were visible either.

Example 2: Plaque imaging in atherosclerotic ApoE -/- mice using ¹⁸F-Choline

Following canulation of the tail vein of ApoE -/- mice 1 MBq/g bodyweight of ¹⁸F-fluorocholine (N,N-dimethyl-N-[¹⁸F]fluoromethyl-2-hydroxyethylammom^'um) was injected as a slow bolus. Twenty minutes later the animals were sacrificed and the aorta was removed and cut open. The opened vessel was placed on a phosphor imager plate for a 4 h exposition. The autoradiographies were then correlated with the histological slices which were stained with oil red O for fat demarcation. An almost 100% correlation between re O fat stained regions and ¹⁸F-fluorocholine stained regions was observed.

Claims

Schering AGCLAIMS

1. A method for detection of a lesion in a vascular system of a subject, comprising the steps of:

(a) introducing into said subject labeled choline or choline derivative or pharmaceutically acceptable salt thereof; and

2. The method of claim 1, wherein said labeled choline or choline derivative comprises a label selected from the group of a fluorescent label, an electron dense label, a radioactive label, and a paramagnetic label.

3. The method of claim 2, wherein the fluorescent label is selected from the group consisting of a polymethine dye, like dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium, holopolar cyanine, hemicyanine, oxonol and hemioxanole dyes; a rhodamine dye, a phenoxazin dye; or a phenothiazin dye.

4. The method of claim 2, wherein the electron dense label is selected from Ag, Au, Br, Fe, and I.

5. The method of claim 2, wherein the radioactive label is selected from the group consisting Of ²²⁵Ac, ²¹¹At, ²¹²Bi₃ ²¹³Bi, ⁷⁶Br, ¹¹C, ^34mCl, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ¹⁶⁶Ho, ¹²³I, ¹²⁴1, ¹²⁵I, ¹³¹I, ¹³N, ²²³Ra, ¹⁸⁶Re, ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ^94mTc, ^99mTc, and ⁹⁰Y, in particular ⁷⁶Br, ¹¹C, ^34mCl,. ¹⁸F₅ ¹²³I, ¹²⁴1, ¹³¹I, and ¹³N.

6. The method of claim 5, wherein the radioactive label comprised in said choline or derivative thereof has a radioactivity of between 1 and 15 mCi, preferably between 3 and 10 mCi.

7. The method of claim 2, wherein the paramagnetic label is selected from the group consisting OfGd³⁺, Fe³⁺, Mn²⁺, Yt³⁺, Dy³⁺, and Cr³⁺.

8. The method of claim 1, wherein said arterial lesion in the vascular system comprises atherosclerotic plaques, initial fatty streaks, intermediate fibrofatty lesions, and fibrous plaques.

9. The method of claim 1, wherein said choline or choline derivative or pharmaceutically acceptable salt thereof is of the following formula (I):

wherein

A⁺ is N⁺ or P⁺,

B is a counteranion,

R and R independent of each other are H; a direct or indirect link to a label; or a C_1-5 straight or branched alkyl, alkenyl, alkynyl, oxyalkyl, oxyalkenyl, thioalkyl, or thioalkenyl, optionally substituted with one or more OH, aryl, heteroaryl or halogen, and/or comprises at least one direct or indirect link to a label,

R³ is a C_1-3 straight or branched alkyl or alkenyl, optionally substituted with one or more OH or halogen, and/or comprises at least one direct or indirect link to a label,

X¹ and X² independent of each other are H, deuterium or halogen, n= 1, 2 or 3; under the proviso that at least one label is comprised within R¹, R² or R³, or that at least one carbon moiety within the choline or derivative is ¹¹C, or that at least one nitrogen moiety is

10. The method of claim 9, wherein R¹ and R² independent of each other are H; CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₂)₂, CH=CH₂, CH₂CH=CH₂, CH=CHCH₃, C≡CH, C≡≡CCH₃, CH₂C≡≡CH, CH₂CH₂CH₂CH₃, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, C(CH₃)₃, CH₂C(CH₃)=CH₂, CH=C(CH₃)CH₃, CH(C₆H₅), OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₂)₂, OCH=CH₂, OCH₂CH=CH₂, OCH=CHCH₃, OC≡≡CH, OC≡≡CCH₃, OCH₂C=CH, OCH₂CH₂CH₂CH₃, OCH₂CH(CH₃)₂, OCH(CH₃)CH₂CH₃, OC(CH₃)₃, OCH₂C(CH₃)=CH₂, OCH=C(CH₃)CH₃, OCH(C₆H₅), CH₂OCH₃, CH₂OCH₂CH₃, CH₂CH₂OCH₃ SCH₃, SCH₂CH₃, SCH₂CH₂CH₃, SCH(CH₂)₂, SCH=CH₂, SCH₂CH=CH₂,

SCH=CHCH₃, SC=CH, SC≡≡CCH₃, SCH₂C≡≡CH, SCH₂CH₂CH₂CH₃, SCH₂CH(CH₃)₂, SCH(CH₃)CH₂CH₃, SC(CH₃)₃, SCH₂C(CH₃)=CH₂, SCH=C(CH₃)CH₃, SCH(C₆H₅), CH₂SCH₃, CH₂SCH₂CH₃, CH₂CH₂SCH₃, optionally substituted with one or more OH or halogen, comprises at least one direct or indirect link to a label.

11. The method of claim 9, wherein R³ is (CX³X⁴)_mX⁵, wherein X³ and X⁴ independent of each other are H, deuterium or halogen,

X⁵ is H or a direct or indirect link to a label, and m = 1, 2 or 3.

12. The method of claim 11, wherein X³ andX⁴are H.

13. The method of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate and valerate.

14. The method of claim 1, wherein said labeled choline or choline derivative is chosen form the group consisting of [¹⁸F] N,N-dimethyl-N-fluoroethylethanolamine (¹⁸F-fluoroethylcholine), [¹⁸F] N,N-dimethyl-N-fluoromethylethanolamine (¹⁸F-fluorocholine), [¹⁸F] fluoromethyl- methylethyl-2-hydroxyethyllammonium, [¹⁸F] fluoropropyl-dimethyl-2- hydroxyethylammonium, N,N-dimethyl-N- [ ^{l x} C]methylethanolamine (^{J 1} C-choline) .

15. The method of claim 1, wherein said introducing step (a) comprises the administration of said labeled choline or choline derivative or pharmaceutically acceptable salt thereof by arterial or by venous inj ection.

16. The method of claim 1, wherein said detecting step (b) further comprises imaging a region of said vascular system at which the said labeled choline or choline derivative has accumulated.

17. The method of claim 1, further comprising the step of quantifying the detected amount of said labeled choline or choline derivative.

18. The method of claim 1, wherein said detecting step (b) further includes the extracorporeal monitoring of said labeled choline or labeled choline derivative.

19. The method of claim 1, wherein said detecting step (b) comprises the extracorporeal monitoring of said label with a fluorescent, CT, MRI, PET, fluorescent/PET, MRI/PET, CT/PET, ultrasound/PET, fluorescent/CT, fluorescent/MRI, fluorescent/ultrasound, CT/MRI, or CT/MRI/ultrasound scanner.

20. Use of labeled choline or a labeled derivative or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for detection of a lesion in a vascular system of a subject.

21. Use according to claim 20 wherein said detection of vascular lesion in a vascular system of a subject comprises the steps of: (a) introducing into said subject labeled choline or choline derivative or pharmaceutically acceptable salt thereof; and

22. Use according to claim 20 or 21, wherein said labeled choline or choline derivative comprises a label selected from the group of a fluorescent label, an electron dense label, a radioactive label, and a paramagnetic label.

23. Use according to claim 22, wherein the fluorescent label is selected from the group consisting of a polymethine dye, like dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium, holopolar cyanine, hemicyanine, oxonol and hemioxanole dyes; a rhodamine dye, a phenoxazin dye; or a phenothiazin dye.

24. Use according to claim 22, wherein the electron dense label is selected from Ag, Au, Br, Fe, and I.

25. Use according to claim 22, wherein the radioactive label is selected from the group consisting Of ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ⁷⁶Br, ¹¹C, ^34mCl, ⁶⁷Cu, ¹⁸F, ⁶⁸Ga, ¹⁶⁶Ho, ¹²³I, ¹²⁴1, ¹²⁵I, ¹³¹I, ¹³N, ²²³Ra, ¹⁸⁶Re, ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ^94mTc, ^99mTc, and ⁹⁰Y, in particular⁷⁶Br, ¹¹C, ^34mCl,.

¹⁸F, ¹²³1, ¹²⁴1, ¹³¹I, and ¹³N.

26. Use according to claim 25, wherein the radioactive label comprised in said choline or derivative thereof has a radioactivity of between 1 and 15 mCi, preferably between 3 and 10 mCi.

27. Use according to claim 22, wherein the paramagnetic label is selected from the group consisting Of Gd³⁺, Fe³⁺, Mn²⁺, Yt³⁺, Dy³⁺, and Cr³⁺.

28. Use according to any of claims 22 to 27, wherein said arterial lesion in the vascular system comprises atherosclerotic plaques, initial fatty streaks, intermediate fibrofatty lesions, and fibrous plaques.

29. Use according to any of claims 22 to 28, wherein said choline or choline derivative or pharmaceutically acceptable salt thereof is of the following formula (I):

wherein

A⁺ is N⁺ or P⁺,

B is a counteranion, R and R² independent of each other are H; a direct or indirect link to a label; or a C_1-5 straight or branched alkyl, alkenyl, alkynyl, oxyalkyl, oxyalkenyl, thioalkyl, or thioalkenyl, optionally substituted with one or more OH, aryl, heteroaryl or halogen, and/or comprises at least one direct or indirect link to a label,

R³ is a Ci_-3 straight or branched alkyl or alkenyl, optionally substituted with one or more OH or halogen, and/or comprises at least one direct or indirect link to a label,

X and X independent of each other are H, deuterium or halogen, n= 1, 2 or 3; under the proviso that at least one label is comprised within R¹, R² or R³, or that at least one carbon moiety is ¹¹C, or that at least one nitrogen moiety is ¹³N.

30. Use according to claim 29, wherein R¹ and R² independent of each other are H; CH₃,

CH₂CH₃, CH₂CH₂CH₃, CH(CH₂)₂, CH=CH₂, CH₂CH=CH₂, CH=CHCH₃, C≡≡CH,

C≡≡CCH₃, CH₂C≡≡CH, CH₂CH₂CH₂CH₃, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, C(CH₃)₃,

CH₂C(CH₃)==CH₂, CH==C(CH₃)CH₃, CH(C₆H₅), OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, OCH(CH₂)₂, OCH=CH₂, OCH₂CH=CH₂, OCH=CHCH₃, OC≡≡CH, OC≡≡CCH₃,

OCH₂C≡≡CH, OCH₂CH₂CH₂CH₃, OCH₂CH(CH₃)₂, OCH(CH₃)CH₂CH₃, OC(CH₃)₃,

OCH₂C(CH₃)=CH₂, OCH=C(CH₃)CH₃, OCH(C₆H₅), CH₂OCH₃, CH₂OCH₂CH₃,

CH₂CH₂OCH₃ SCH₃, SCH₂CH₃, SCH₂CH₂CH₃, SCH(CH₂)₂, SCH=CH₂, SCH₂CH=CH₂, SCH=CHCH₃, SC≡≡CH, SC≡≡CCH₃, SCH₂C≡≡CH, SCH₂CH₂CH₂CH₃, SCH₂CH(CH₃)₂, SCH(CH₃)CH₂CH₃, SC(CH₃)₃, SCH₂C(CH₃)=CH₂, SCH=C(CH₃)CH₃, SCH(C₆H₅), CH₂SCH₃, CH₂SCH₂CH₃, CH₂CH₂SCH₃, optionally substituted with one or more OH or halogen, comprises at least one direct or indirect link to a label.

31. Use according to claim 29 or 30, wherein R³ is (CX³X⁴)_mX⁵, wherein X³ and X⁴ independent of each other are H, deuterium or halogen,

X⁵ is H or a direct or indirect link to a label, and m = 1, 2 or 3.

32. Use according to claim 31, wherein X³ and X⁴ are H.

33. Use according to any of claims 22 to 32, wherein the pharmaceutically acceptable salt is selected from the group consisting of acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2- hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N- methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate and valerate.

34. Use according to any of claims 22 to 33, wherein said labeled choline or choline derivative is chosen form the group consisting of [¹⁸F] N,N-dimethyl-N-fluoroethylethanolamine (¹⁸F- fluoroethylcholine), [¹⁸F] N,N-dimethyl-N-fluoromethylethanolamine (¹⁸F-fluorocholine), [¹⁸F] fluoromethyl-methylethyl-2-hydroxyethylammonium, [¹⁸F] fluoropropyl-dimethyl-2- hydroxyethyllammoniuni, N^-dimethyl-N-f¹ ^methylethanolamine (^πC-choline).

35. Use according to any of claims 22 to 34, wherein said introducing step (a) comprises the administration of said labeled choline or choline derivative or pharmaceutically acceptable salt thereof by arterial or by venous injection.

36. Use according to any of claims 22 to 35, wherein said detecting step (b) further comprises imaging a region of said vascular system at which the said labeled choline or choline derivative has accumulated.

37. Use according to any of claims 22 to 36, further comprising the step of quantifying the detected amount of said labeled choline or choline derivative.

38. Use according to any of claims 22 to 37, wherein said detecting step (b) further includes the extracorporeal monitoring of said labeled choline or labeled choline derivative.

39. Use according of claim 38, wherein said detecting step (b) comprises the extracorporeal monitoring of said label with a fluorescent, CT, MRI, PET, fluorescent/PET, MRI/PET, CT/PET, ultrasound/PET, fluorescent/CT, fluorescent/MRI, fluorescent/ultrasound,

CT/MRI, or CT/MRI/ultrasound scanner.