Patent Application
20130183239
The present invention describes a novel radiotracer(s) for Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) imaging of disease states related to altered choline metabolism (e.g., tumor imaging of prostate, breast, brain, esophageal, ovarian, endometrial, lung and prostate cancer—primary tumor, nodal disease or metastases). The present invention also describes intermediate(s), precursor(s), pharmaceutical composition(s), methods of making, and methods of use of the novel radiotracer(s).
The biosynthetic product of choline kinase (EC 2.7.1.32) activity, phosphocholine, is elevated in several cancers and is a precursor for membrane phosphatidylcholine (Aboagye, E. O., et al., Cancer Res 1999; 59:80-4; Exton, J. H., Biochim Biophys Acta 1994; 1212:26-42; George, T. P., et al., Biochim Biophys Acta 1989; 104:283-91; and Teegarden, D., et al., J Biol Chem 1990; 265(11):6042-7). Over-expression of choline kinase and increased enzyme activity have been reported in prostate, breast, lung, ovarian and colon cancers (Aoyama, C., et al., Prog Lipid Res 2004; 43(3):266-81; Glunde, K., et al., Cancer Res 2004; 64(12):4270-6; Glunde, K., et al., Cancer Res 2005; 65(23): 11034-43; Iorio, E., et al., Cancer Res 2005; 65(20): 9369-76; Ramirez de Molina, A., et al., Biochem Biophys Res Commun 2002; 296(3): 580-3; and Ramirez de Molina, A., et al., Lancet Oncol 2007; 8(10): 889-97) and are largely responsible for the increased phosphocholine levels with malignant transformation and progression; the increased phosphocholine levels in cancer cells are also due to increased breakdown via phospholipase C (Glunde, K., et al., Cancer Res 2004; 64(12):4270-6).
Because of this phenotype, together with reduced urinary excretion, [11C]choline has become a prominent radiotracer for positron emission tomography (PET) and PET-Computed Tomography (PET-CT) imaging of prostate cancer, and to a lesser extent imaging of brain, esophageal, and lung cancer (Hara, T., et al., J Nucl Med 2000; 41:1507-13; Hara, T., et al., J Nucl Med 1998; 39:990-5; Hara, T., et al., J Nucl Med 1997; 38:842-7; Kobori, O., et al., Cancer Cell 1999; 86:1638-48; Pieterman, R. M., et al., J Nucl Med 2002; 43(2):167-72; and Reske, S. N. Eur J Nucl Med Mol Imaging 2008; 35:1741). The specific PET signal is due to transport and phosphorylation of the radiotracer to [11C]phosphocholine by choline kinase.
Of interest, however, is that [11C]choline (as well as the fluoro-analog) is oxidized to [11C]betaine by choline oxidase (see
was developed to overcome the short physical half-life of carbon-11 (20.4 min) (DeGrado, T. R., et al., Cancer Res 2001; 61(1): 110-7) and a number of PET and PET-CT studies with this relatively new radiotracer have been published (Beheshti, M., et al., Eur J Nucl Med Mol Imaging 2008; 35(10): 1766-74; Cimitan, M., et al., Eur J Nucl Med Mol Imaging 2006; 33(12):1387-98; de Jong, I. J., et al., Eur J Nucl Med Mol Imaging 2002; 29:1283-8; and Price, D. T., et al., J Urol 2002; 168(1):273-80). The longer half-life of fluorine-18 (109.8 min) was deemed potentially advantageous in permitting late imaging of tumors when sufficient clearance of parent tracer in systemic circulation had occurred (DeGrado, T. R., et al., J Nucl Med 2002; 43(1):92-6).
WO2001/82864 describes 18F-labeled choline analogs, including [18F]Fluoromethylcholine ([18F]-FCH) and their use as imaging agents (e.g., PET) for the non-invasive detection and localization of neoplasms and pathophysiologies influencing choline processing in the body (Abstract). WO2001/82864 also describes 18F-labeled di-deuterated choline analogs such as [18F]fluoromethyl-[1-2H2]choline ([18F]FDC) (hereinafter referred to as “[18F]D2-FCH”):
The oxidation of choline under various conditions; including the relative oxidative stability of choline and [1,2-2H4]choline has been studied (Fan, F., et al., Biochemistry 2007, 46, 6402-6408; Fan, F., et al., Journal of the American Chemical Society 2005, 127, 2067-2074; Fan, F., et al., Journal of the American Chemical Society 2005, 127, 17954-17961; Gadda, G. Biochimica et Biophysica Acta 2003, 1646, 112-118; Gadda, G., Biochimica et Biophysica Acta 2003, 1650, 4-9). Theoretically the effect of the extra deuterium substitution was found to be neglible in the context of a primary isotope effect of 8-10 since the β-secondary isotope effect is ˜1.05 (Fan, F., et al., Journal of the American Chemical Society 2005, 127, 17954-17961).
[18F]Fluoromethylcholine is now used extensively in the clinic to image tumour status (Beheshti, M., et al., Radiology 2008, 249, 389-90; Beheshti, M., et al., Eur J Nucl Med Mol Imaging 2008, 35, 1766-74).
The present invention, as described below, provides a novel 11C-radiolabeled radiotracer that can be used for PET imaging of choline metabolism and exhibits increased metabolic stability and a favourable urinary excretion profile.
a is a picture of a fully assembled cassette of the present invention for the production of [18F]fluoromethyl-[1,2-2H4]choline (D4-FCH) via an unprotected precursor.
b is a picture of a fully assembled cassette of the present invention for the production of [18F]fluoromethyl-[1,2-2H4]choline (D4-FCH) via a PMB-protected precursor.
The present invention provides a compound of Formula (III):
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5;
m is an integer from 1-4;
C* is a radioisotope of carbon;
X, Y and Z are each independently hydrogen, deuterium (D), a halogen selected from F, Cl, Br, and I, alkyl, alkenyl, alkynl, aryl, heteroaryl, heterocyclyl group; and
Q is an anionic counterion; with the proviso the compound of Formula (III) is not 11C-choline.
The present invention provides a novel radiolabeled choline analog compound of formula (I):
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, Cl, Br, and I or a radioisotope; and
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not fluoromethylcholine, fluoromethyl-ethyl-choline, fluoromethyl-propyl-choline, fluoromethyl-butyl-choline, fluoromethyl-pentyl-choline, fluoromethyl-isopropyl-choline, fluoromethyl-isobutyl-choline, fluoromethyl-sec-butyl-choline, fluoromethyl-diethyl-choline, fluoromethyl-diethanol-choline, fluoromethyl-benzyl-choline, fluoromethyl-triethanol-choline, 1,1-dideuterofluoromethylcholine, 1,1-dideuterofluoromethyl-ethyl-choline, 1,1-dideuterofluoromethyl-propyl-choline, or an [18F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is provided wherein:
R1, R2, R3, and R4 are each independently hydrogen;
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, Cl, Br, and I or a radioisotope;
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not fluoromethylcholine, fluoromethyl-ethyl-choline, fluoromethyl-propyl-choline, fluoromethyl-butyl-choline, fluoromethyl-pentyl-choline, fluoromethyl-isopropyl-choline, fluoromethyl-isobutyl-choline, fluoromethyl-sec-butyl-choline, fluoromethyl-diethyl-choline, fluoromethyl-diethanol-choline, fluoromethyl-benzyl-choline, fluoromethyl-triethanol-choline, or an [18F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is provided wherein:
R1 and R2 are each hydrogen;
R3 and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, Cl, Br, and I or a radioisotope;
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not 1,1-dideuterofluoromethylcholine, 1,1-dideuterofluoromethyl-ethyl-choline, 1,1-dideuterofluoromethyl-propyl-choline, or an [18F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is provided wherein:
R1, R2, R3, and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, Cl, Br, and I or a radioisotope;
Q is an anionic counterion.
According to the present invention, when Z of a compound of Formula (I) as described herein is a halogen, it can be a halogen selected from F, Cl, Br, and I; preferably, F.
According to the present invention, when Z of a compound of Formula (I) as described herein is a radioisotope (hereinafter referred to as a “radiolabeled compound of Formula (I)”), it can be any radioisotope known in the art. Preferably, Z is a radioisotope suitable for imaging (e.g., PET, SPECT). More preferably Z is a radioisotope suitable for PET imaging. Even more preferably, Z is 18F, 76Br, 123I, 124 or 125I. Even more preferably, Z is 18F.
According to the present invention, Q of a compound of Formula (I) as described herein can be any anionic counterion known in the art suitable for cationic ammonium compounds. Suitable examples of Q include anionic: bromide (Br−), chloride (Cl−), acetate (CH3CH2C(O)O−), or tosylate (−OTos). In a preferred embodiment of the invention, Q is bromide (Br−) or tosylate (−OTos). In a preferred embodiment of the invention, Q is chloride (Cl−) or acetate (CH3CH2C(O)O−). In a preferred embodiment of the invention, Q is chloride (Cl−).
According the invention, a preferred embodiment of a compound of Formula (I) is the following compound of Formula (Ia):
wherein:
R1, R2, R3, and R4 are each independently deuterium (D);
R5, R6, and R7 are each hydrogen;
X and Y are each independently hydrogen;
Z is 18F;
Q is Cl−.
According to the invention, a preferred compound of Formula (Ia) is [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH). [18F]-D4-FCH is a more metabolically stable fluorocholine (FCH) analog. [18F]-D4-FCH offers numerous advantages over the corresponding 18F-non-deuterated and/or 18F-di-deuterated analog. For example, [18F]-D4-FCH exhibits increased chemical and enzymatic oxidative stability relative to [18F]fluoromethylcholine. [18F]-D4-FCH has an improved in vivo profile (i.e., exhibits better availability for in vivo imaging) relative to dideuterofluorocholine, [18F]fluoromethyl-[1-2H2]choline, that is over and above what could be predicted by literature precedence and is, thus, unexpected. [18F]-D4-FCH exhibits improved stability and consequently will better enable late imaging of tumors after sufficient clearance of the radiotracer from systemic circulation. [18F]-D4-FCH also enhances the sensitivity of tumor imaging through increased availability of substrate. These advantages are discussed in further detail below.
The present invention further provides a precursor compound of Formula (II):
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4.
The present invention further provides a method of making a precursor compound of Formula (II).
The present invention provides a compound of Formula (III):
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5;
m is an integer from 1-4;
C* is a radioisotope of carbon;
X, Y and Z are each independently hydrogen, deuterium (D), a halogen selected from F, Cl, Br, and I, alkyl, alkenyl, alkynl, aryl, heteroaryl, heterocyclyl group; and
Q is an anionic counterion; with the proviso the compound of Formula (III) is not 11C-choline.
According to the invention, C* of the compound of Formula (III) can be any radioisotope of carbon. Suitable examples of C* include, but are not limited to, 11C, 13C, and 14C. Q is a described for the compound of Formula (I).
In a preferred embodiment of the invention, a compound of Formula (III) is provided wherein C* is 11C; X and Y are each hydrogen; and Z is F.
In a preferred embodiment of the invention, a compound of Formula (III) is provided wherein C* is 11C; X, Y and Z are each hydrogen H; R1, R2, R3, and R4 are each deuterium (D); and R5, R6, and R7 are each hydrogen (11C-[1,2-2H4]choline or “11C-D4-choline”.
The present invention provides a pharmaceutical or radiopharmaceutical composition comprising a compound for Formula (I), including a compound of Formula (Ia), each as defined herein together with a pharmaceutically acceptable carrier, excipient, or biocompatible carrier. According to the invention when Z of a compound of Formula (I) or (Ia) is a radioisotope, the pharmaceutical composition is a radiopharmaceutical composition.
The present invention further provides a pharmaceutical or radiopharmaceutical composition comprising a compound for Formula (I), including a compound of Formula (Ia), each as defined herein together with a pharmaceutically acceptable carrier, excipient, or biocompatible carrier suitable for mammalian administration.
The present invention provides a pharmaceutical or radiopharmaceutical composition comprising a compound for Formula (III), as defined herein together with a pharmaceutically acceptable carrier, excipient, or biocompatible carrier.
The present invention further provides a pharmaceutical or radiopharmaceutical composition comprising a compound for Formula (III), as defined herein together with a pharmaceutically acceptable carrier, excipient, or biocompatible carrier suitable for mammalian administration.
As would be understood by one of skill in the art, the pharmaceutically acceptable carrier or excipient can be any pharmaceutically acceptable carrier or excipient known in the art.
The “biocompatible carrier” can be any fluid, especially a liquid, in which a compound of Formula (I), (Ia), or (III) can be suspended or dissolved, such that the pharmaceutical composition is physiologically tolerable, e.g., can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity-adjusting substances (e.g., salts of plasma cations with biocompatible counterions), sugars (e.g., glucose or sucrose), sugar alcohols (e.g., sorbitol or mannitol), glycols (e.g., glycerol), or other non-ionic polyol materials (e.g., polyethyleneglycols, propylene glycols and the like). The biocompatible carrier may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations. Preferably the biocompatible carrier is pyrogen-free water for injection, isotonic saline or an aqueous ethanol solution. The pH of the biocompatible carrier for intravenous injection is suitably in the range 4.0 to 10.5.
The pharmaceutical or radiopharmaceutical composition may be administered parenterally, i.e., by injection, and is most preferably an aqueous solution. Such a composition may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g., cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid orpara-aminobenzoic acid). Where a compound of Formula (I), (Ia), or (III) is provided as a radiopharmaceutical composition, the method for preparation of said compound may further comprise the steps required to obtain a radiopharmaceutical composition, e.g., removal of organic solvent, addition of a biocompatible buffer and any optional further ingredients. For parenteral administration, steps to ensure that the radiopharmaceutical composition is sterile and apyrogenic also need to be taken. Such steps are well-known to those of skill in the art.
The present invention provides a method to prepare a compound for Formula (I), including a compound of Formula (Ia), wherein said method comprises reaction of the precursor compound of Formula (II) with a compound of Formula (IIIa) to form a compound of Formula (I) (Scheme A):
wherein the compounds of Formulae (I) and (II) are each as described herein and the compound of Formula (IIIa) is as follows:
ZXYC-Lg (IIIa)
wherein X, Y and Z are each as defined herein for a compound of Formula (I) and “Lg” is a leaving group. Suitable examples of “Lg” include, but are not limited to, bromine (Br) and tosylate (OTos). A compound of Formula (IIIa) can be prepared by any means known in the art including those described herein.
Synthesis of a compound of Formula (IIIa) wherein Z is F; X and Y are both H and the Lg is OTos (i.e., fluoromethyltosylate) can be achieved as set forth in Scheme 3 below:
wherein: i: Silver p-toluenesulfonate, MeCN, reflux, 20 h;
Commercially available diiodomethane can be reacted with silver tosylate, using the method of Emmons and Ferris, to give methylene ditosylate (Emmons, W. D., et al., “Metathetical Reactions of Silver Salts in Solution. II. The Synthesis of Alkyl Sulfonates”, Journal of the American Chemical Society, 1953; 75:225).
Fluoromethyltosylate can be prepared by nucleophilic substitution of Methylene ditosylate from step (a) using potassium fluoride/Kryptofix K222 in acetonitrile at 80° C. under standard conditions.
When Z is a radioisotope, the radioisotope can be introduced by any means known by one of skill in the art. For example, the radioisotope [18F]-fluoride ion (18F−) is normally obtained as an aqueous solution from the nuclear reaction 18O(p,n)18F and is made reactive by the addition of a cationic counterion and the subsequent removal of water. Suitable cationic counterions should possess sufficient solubility within the anhydrous reaction solvent to maintain the solubility of 18F−. Therefore, counterions that have been used include large but soft metal ions such as rubidium or caesium, potassium complexed with a cryptand such as Kryptofix™, or tetraalkylammonium salts. A preferred counterion is potassium complexed with a cryptand such as Kryptofix™ because of its good solubility in anhydrous solvents and enhanced 18F− reactivity. 18F can also be introduced by nucleophilic displacement of a suitable leaving group such as a halogen or tosylate group. A more detailed discussion of well-known 18F labelling techniques can be found in Chapter 6 of the “Handbook of Radiopharmaceuticals” (2003; John Wiley and Sons: M. J. Welch and C. S. Redvanly, Eds.). For example, [18F]Fluoromethyltosylate can be prepared by nucleophilic substitution of Methylene ditosylate with [18F]-fluoride ion in acetonitrile containing 2-10% water (see Neal, T. R., et al., Journal of Labelled Compounds and Radiopharmaceuticals 2005; 48:557-68).
In a preferred embodiment, the method to prepare a compound for Formula (I), including a compound of Formula (Ia), is automated. For example, [18F]-radiotracers may be conveniently prepared in an automated fashion by means of an automated radiosynthesis apparatus. There are several commercially-available examples of such platform apparatus, including TRACERlab™ (e.g., TRACERlab™ MX) and FASTlab™ (both from GE Healthcare Ltd.). Such apparatus commonly comprises a “cassette”, often disposable, in which the radiochemistry is performed, which is fitted to the apparatus in order to perform a radiosynthesis. The cassette normally includes fluid pathways, a reaction vessel, and ports for receiving reagent vials as well as any solid-phase extraction cartridges used in post-radiosynthetic clean up steps. Optionally, in a further embodiment of the invention, the automated radiosynthesis apparatus can be linked to a high performance liquid chromatograph (HPLC).
The present invention therefore provides a cassette for the automated synthesis of a compound of Formula (I), including a compound of Formula (Ia), each as defined herein comprising:
In one embodiment of the invention, a method of making a compound of Formula (I), including a compound of Formula (Ia), each as described herein, that is compatible with FASTlab™ from a protected ethanolamine precursor that requires no HPLC purification step is provided.
The radiosynthesis of [18F]fluoromethyl-[1,2-2H4]choline (18F-D4-FCH) can be performed according to the methods and examples described herein. The radiosynthesis of 18F-D4-FCH can also be performed using commercially available synthesis platforms including, but not limited to, GE FASTlab™ (commercially available from GE Healthcare Inc.).
An example of a FASTlab™ radiosynthetic process for the preparation of [18F]fluoromethyl-[1,2-2H4]choline from a protected precursor is shown in Scheme 5:
wherein:
a. Preparation of [18F]KF/K222/K2CO3 complex as described in more detail below;
b. Preparation of [18F]FCH2OTs as described in more detail below;
c. SPE purification of [18F]FCH2OTs as described in more detail below;
d. Radiosynthesis of O-PMB-[18F]-D4-Choline (O-PMB-[18F]-D4-FCH) as described in more detail below; and
e. Purification & formulation of [18F]-D4-Choline (18F-D4-FCH) as the hydrochloric salt as described in more detail below.
The automation of [18F]fluoro-[1,2-2H4]choline or [18F]fluorocholine (from the protected precursor) involves an identical automated process (and are prepared from the fluoromethylation of O-PMB-N,N-dimethyl-[1,2-2H4]ethanolamine and O-PMB-N,N-dimethylethanolamine respectively).
According to one embodiment of the present invention, FASTlab™ syntheses of [18F]fluoromethyl-[1,2-2H4]choline or [18F]fluoromethylcholine comprises the following sequential steps:
(i) Trapping of [18F]fluoride onto QMA;
(ii) Elution of [18F]fluoride from a QMA;
(iii) Radiosynthesis of [18F]FCH2OTs;
(iv) SPE clean up of [18F]FCH2OTs;
(v) Reaction vessel clean up;
(vi) Drying reaction vessel and [18F]fluoromethyl tosylate retained on SPE t-C18 plus simultaneously;
(vii) Alkylation reaction;
(viii) Removal of unreacted O-PMB-precursor; and
(ix) Deprotection & formulation.
Each of steps (i)-(ix) are described in more detail below.
In one embodiment of the present invention, steps (i)-(ix) above are performed on a cassette as described herein. One embodiment of the present invention is a cassette capable of performing steps (i)-(ix) for use in an automated synthesis platform. One embodiment of the present invention is a cassette for the radiosynthesis of [18F]fluoromethyl-[1,2-2H4]choline ([18F]-D4-FCH) or [18F]fluoromethylcholine from a protected precursor. An example of a cassette of the present invention is shown in
(i) Trapping of [18F]Fluoride onto QMA
[18F]fluoride (typically in 0.5 to 5 mL H218O) is passed through a pre-conditioned Waters QMA cartridge.
(Ii) Elution of [18F]Fluoride from a QMA
The eluent, as described in Table 1 is withdrawn into a syringe from the eluent vial and passed over the Waters QMA into the reaction vessel. This procedure elutes [18F]fluoride into the reaction vessel. Water and acetonitrile are removed using a well-designed drying cycle of “nitrogen/vacuum/heating/cooling”.
Once the K[18F]Fluoride/K222/K2CO3 complex of (ii) is dry, CH2(OTs)2 methylene ditosylate in a solution containing acetonitrile and water is added to the reaction vessel containing the K[18F]fluoride/K222/K2CO3 complex. The resulting reaction mixture will be heated (typically to 110° C. for 10 min), then cooled down (typically to 70° C.).
Once radiosynthesis of [18F]FCH2OTs is completed and the reaction vessel is cooled, water is added into the reaction vessel to reduce the organic solvent content in the reaction vessel to approximately 25%. This diluted solution is transferred from the reaction vessel and through the t-C18-light and t-C18 plus cartridges—these cartridges are then rinsed with 12 to 15 mL of a 25% acetonitrile/75% water solution. At the end of this process:
The reaction vessel was cleaned (using ethanol) prior to the alkylation of [18F]fluoroethyl tosylate and O-PMB-DMEA precursor.
(Vi) Drying Reaction Vessel and [18F]Fluoromethyl Tosylate Retained on SPE t-C18 Plus Simultaneously
Once clean up (v) was completed, the reaction vessel and the [18F]fluoromethyl tosylate retained on SPE t-C18 plus was dried simultaneously.
Following step (vi), the [18F]FCH2OTs (along with tosyl-[18F]fluoride) retained on the t-C18 plus was eluted into the reaction vessel using a mixture of O-PMB-N,N-dimethyl-[1,2-2H4]ethanolamine (or O-PMB-N,N-dimethylethanolamine) in acetonitrile.
The alkylation of [18F]FCH2OTs with O-PMB-precursor was achieved by heating the reaction vessel (typically 110° C. for 15 min) to afford [18F]fluoro-[1,2-2H4]choline (or O-PMB-[18F]fluorocholine).
Water (3 to 4 mL) was added to the reaction and this solution was then passed through a pre-treated CM cartridge, followed by an ethanol wash—typically 2×5 mL (this removes unreacted O-PMB-DMEA) leaving “purified” [18F]fluoro-[1,2-2H4]choline (or O-PMB-[18F]fluorocholine) trapped onto the CM cartridge.
Hydrochloric acid was passed through the CM cartridge into a syringe: this resulted in the deprotection of O-PMB-[18F]fluorocholine (the syringe contains [18F]fluorocholine in a HCl solution). Sodium acetate was then added to this syringe to buffer to pH 5 to 8 affording [18F]-D4-choline (or [18F]choline) in an acetate buffer. This buffered solution is then transferred to a product vial containing a suitable buffer.
Table 1 provides a listing of reagents and other components required for preparation of [18F]fluoromethyl-[1,2-2H4]choline (D4-FCH) (or [18F]fluoromethylcholine) radiocassette of the present invention:
2H4]ethanolamine and O-PMB-N,N-
According to one embodiment of the present invention, FASTlab™ synthesis of [18F]fluoromethyl-[1,2-2H4]choline via an unprotected precursor comprises the following sequential steps as depicted in Scheme 6 below:
1. Recovery of [18F]fluoride from QMA;
2 Preparation of K[18F]F/K222/K2CO3 complex;
3 Radiosynthesis of 18FCH2OTs;
4 SPE cleanup of 18FCH2OTs;
5 Clean up of reaction vessel cassette and syringe;
6 Drying of reaction vessel and C18 SepPak;
7 Elution off and coupling of 18FCH2OTs with D4-DMEA;
8 Transfer of reaction mixture onto CM cartridge;
9 Clean up of cassette and syringe;
10 Washing of CM cartridge with dilute aq ammonia solution, Ethanol and water;
11 Elution of [18F]fluoromethyl-[1,2-2H4]choline from CM cartridge with 0.09% sodium chloride (5 ml), followed by water (5 ml).
In one embodiment of the present invention, steps (1)-(11) above are performed on a cassette as described herein. One embodiment of the present invention is a cassette capable of performing steps (1)-(11) for use in an automated synthesis platform. One embodiment of the present invention is a cassette for the radiosynthesis of [18F]fluoromethyl-[1,2-2H4]choline ([18F]-D4-FCH) from an unprotected precursor. An example of a cassette of the present invention is shown in
Table 2 provides a listing of reagents and other components required for preparation of [18F]fluoromethyl-[1,2-2H4]choline (D4-FCH) (or [18F]fluoromethylcholine) via an unprotected precursor radiocassette of the present invention:
The radiolabeled compound of the invention, as described herein, will be taken up into cells via cellular transporters or by diffusion. In cells where choline kinase is overexpressed or activated the radiolabeled compound of the invention, as described herein, will be phosphorylated and trapped within that cell. This will form the primary mechanism of detecting neoplastic tissue.
The present invention further provides a method of imaging comprising the step of administering a radiolabeled compound of the invention or a pharmaceutical composition comprising a radiolabeled compound of the invention, each as described herein, to a subject and detecting said radiolabeled compound of the invention in said subject. The present invention further provides a method of detecting neoplastic tissue in vivo using a radiolabeled compound of the invention or a pharmaceutical composition comprising a radiolabeled compound of the invention, each as described herein. Hence the present invention provides better tools for early detection and diagnosis, as well as improved prognostic strategies and methods to easily identify patients that will respond or not to available therapeutic treatments. As a result of the ability of a compound of the invention to detect neoplastic tissue, the present invention further provides a method of monitoring therapeutic response to treatment of a disease state associated with the neoplastic tissue.
In a preferred embodiment of the invention, the radiolabeled compound of the invention for use in a method of imaging of the invention, as described herein, is a radiolabeled compound of Formula (I).
In a preferred embodiment of the invention, the radiolabeled compound of the invention for use in a method of imaging of the invention, as described herein, is a radiolabeled compound of Formula (III).
As would be understood by one of skill in the art the type of imaging (e.g., PET, SPECT) will be determined by the nature of the radioisotope. For example, if the radiolabeled compound of Formula (I) contains 18F it will be suitable for PET imaging.
Thus the invention provides a method of detecting neoplastic tissue in vivo comprising the steps of:
The step of “administering” a radiolabeled compound of the invention is preferably carried out parenterally, and most preferably intravenously. The intravenous route represents the most efficient way to deliver the compound throughout the body of the subject. Intravenous administration neither represents a substantial physical intervention nor a substantial health risk to the subject. The radiolabeled compound of the invention is preferably administered as the radiopharmaceutical composition of the invention, as defined herein. The administration step is not required for a complete definition of the imaging method of the invention. As such, the imaging method of the invention can also be understood as comprising the above-defined steps (ii)-(v) carried out on a subject to whom a radiolabeled compound of the invention has been pre-administered.
Following the administering step and preceding the detecting step, the radiolabeled compound of the invention is allowed to bind to the neoplastic tissue. For example, when the subject is an intact mammal, the radiolabeled compound of the invention will dynamically move through the mammal's body, coming into contact with various tissues therein. Once the radiolabeled compound of the invention comes into contact with the neoplastic tissue it will bind to the neoplastic tissue.
The “detecting” step of the method of the invention involves detection of signals emitted by the radioisotope comprised in the radiolabeled compound of the invention by means of a detector sensitive to said signals, e.g., a PET camera. This detection step can also be understood as the acquisition of signal data.
The “generating” step of the method of the invention is carried out by a computer which applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset is then manipulated to generate images showing the location and/or amount of signals emitted by the radioisotope. The signals emitted directly correlate with the amount of enzyme or neoplastic tissue such that the “determining” step can be made by evaluating the generated image.
The “subject” of the invention can be any human or animal subject. Preferably the subject of the invention is a mammal. Most preferably, said subject is an intact mammalian body in vivo. In an especially preferred embodiment, the subject of the invention is a human.
The “disease state associated with the neoplastic tissue” can be any disease state that results from the presence of neoplastic tissue. Examples of such disease states include, but are not limited to, tumors, cancer (e.g., prostate, breast, lung, ovarian, pancreatic, brain and colon). In a preferred embodiment of the invention the disease state associated with the neoplastic tissue is brain, breast, lung, espophageal, prostate, or pancreatic cancer.
As would be understood by one of skill in the art, the “treatment” will be depend on the disease state associated with the neoplastic tissue. For example, when the disease state associated with the neoplastic tissue is cancer, treatment can include, but is not limited to, surgery, chemotherapy and radiotherapy. Thus a method of the invention can be used to monitor the effectiveness of the treatment against the disease state associated with the neoplastic tissue.
Other than neoplasms, a radiolabeled compound of the invention may also be useful in liver disease, brain disorders, kidney disease and various diseases associated with proliferation of normal cells. A radiolabeled compound of the invention may also be useful for imaging inflammation; imaging of inflammatory processes including rheumatoid arthritis and knee synovitis, and imaging of cardiovascular disease including artherosclerotic plaque.
The present invention provides a precursor compound of Formula (II):
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is provided wherein:
R1, R2, R3, and R4 are each independently hydrogen;
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, or —CD(R8)2;
R8 is hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is provided wherein:
R1 and R2 are each hydrogen;
R3 and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, or —CD(R8)2;
R8 is hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is provided wherein:
R1, R2, R3, and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, or —CD(R8)2;
R8 is hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4.
According to the invention, compound of Formula (II) is a compound of Formula (IIa):
In one embodiment of the invention, a compound of Formula (IIb) is provided:
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4; and
Pg is a hydroxyl protecting group.
In a preferred embodiment of the invention, a compound of Formula (IIb) is provided wherein Pg is a p-methoxybenyzl (PMB), trimethylsilyl (TMS), or a dimethoxytrityl (DMTr) group.
In a preferred embodiment of the invention, a compound of Formula (IIb) is provided wherein Pg is a p-methoxybenyzl (PMB) group.
In one embodiment of the invention, a compound of Formula (IIc) is provided:
wherein:
R1, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, —CH(R8)2, or —CD(R8)2;
R8 is independently hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4;
with the proviso that when R1, R2, R3, and R4 are each hydrogen, R5, R6, and R7 are each not hydrogen; and with the proviso that when R1, R2, R3, and R4 are each deuterium, R5, R6, and R7 are each not hydrogen.
In a preferred embodiment of the invention, a compound of Formula (IIc) is provided wherein:
R1, R2, R3, and R4 are each independently hydrogen;
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, or —CD(R8)2;
R8 is hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4; with the proviso that R5, R6, and R7 are each not hydrogen.
In a preferred embodiment of the invention, a compound of Formula (IIc) is provided wherein:
R1, R2, R3, and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, —(CH2)mR8, —(CD2)mR8, —(CF2)mR8, or —CD(R8)2;
R8 is hydrogen, —OH, —CH3, —CF3, —CH2OH, —CH2F, —CH2Cl, —CH2Br, —CH2I, —CD3, —CD2OH, —CD2F, CD2Cl, CD2Br, CD2I, or —C6H5; and
m is an integer from 1-4; with the proviso that R5, R6, and R7 are each not hydrogen.
In a preferred embodiment of the invention, a compound of Formula (IIc) is provided wherein:
R1 and R2 are each hydrogen; and
R3 and R4 are each deuterium (D).
A precursor compound of Formula (II), including a compound of Formula (IIa), (IIb) and (IIc), can be prepared by any means known in the art including those described herein. For example, the compound of Formula (IIa) can be synthesized by alkylation of dimethylamine in THF with 2-bromoethanol-1,1,2,2-d4 in the presence of potassium carbonate as shown in Scheme 1 below:
wherein i=K2CO3, THF, 50° C., 19 h. The desired tetra-deuterated product can be purified by distillation. The 1H NMR spectrum of the compound of Formula (IIa) (
A di-deuterated analog of a precursor compound of Formula (II) can be synthesized from N,N-dimethylglycine via lithium aluminium hydride reduction as shown in Scheme 2 below:
wherein i=LiAlD4, THF, 65° C., 24 h. 13C NMR analysis indicated that isotopic purity of greater than 95% in favor of the 2H isomer (relative to the 1H isotope) can be achieved.
According to the invention, the hydroxyl group of a compound of Formula (II), including a compound of Formula (IIa) can be further protected with a protecting group to give a compound of Formula (IIb):
wherein Pg is any hydroxyl protecting group known in the art. Preferably, Pg is any acid labile hydroxyl protecting group including, for example, those described in “Protective Groups in Organic Synthesis”, 3rd Edition, A Wiley Interscience Publication, John Wiley & Sons Inc., Theodora W. Greene and Peter G. M. Wuts, pp 17-200. Preferably, Pg is a p-methoxybenzyl (PMB), trimethylsilyl (TMS), or a dimethoxytrityl (DMTr) group. More preferably, Pg is a p-methoxybenyzl (PMB) group.
Stability to oxidation resulting from isotopic substitution was evaluated in in vitro chemical and enzymatic models using [18F]fluoromethylcholine as standard. [18F]Fluoromethyl-[1,2-2H4]choline was then evaluated in in vivo models and compared to [11C]choline, [18F]fluoromethylcholine and [18F]Fluoromethyl-[1-2H2]choline:
The effect of deuterium substitution on bond strength was initially tested by evaluation of the chemical oxidation pattern of [18F]fluoromethylcholine and [18F]Fluoromethyl-[1,2-2H4]choline using potassium permanganate. Scheme 6 below details the base catalyzed potassium permanganate oxidation of [18F]fluoromethylcholine and [18F]Fluoromethyl-[1,2-2H4]choline at room temperature, with aliquots removed and analyzed by radio-HPLC at pre-selected time points:
The results are summarized in
[18F]fluoromethylcholine and [18F]fluoromethyl-[1,2-2H4]choline were evaluated in a choline oxidase model (Roivainen, A., et al., European Journal of Nuclear Medicine 2000; 27:25-32). The graphical representation in
[18F]fluoromethyl-[1,2-2H4]-choline is more resistant to oxidation in vivo. The relative rates of oxidation of the two isotopically radiolabeled choline species, [18F]fluoromethylcholine and [18F]fluoromethyl-[1,2-2H4]-choline to their respective metabolites, [18F]fluoromethylcholine-betaine ([18F]-FCH-betaine) and [18F]fluoromethyl-[1,2-2H4]-choline-betaine ([18F]-D4-FCH-betaine) was evaluated by high performance liquid chromatography (HPLC) in mouse plasma after intravenous (i.v.) administration of the radiotracers. [18F]fluoromethyl-[1,2-2H4]-choline was found to be markedly more stable to oxidation than [18F]fluoromethylcholine. As shown in
Time course biodistribution was carried out for [18F]fluoromethylcholine, [18F]fluoromethyl-[1-2H2]choline and [18F]fluoromethyl-[1,2-2H4]choline in nude mice bearing HCT116 human colon xenografts. Tissues were collected at 2, 30 and 60 minutes post-injection and the data summarized in
Metabolite analysis of tissues including liver, kidney and tumor by HPLC was also accomplished. Typical HPLC chromatograms of [18F]FCH and [18F]D4-FCH and their respective metabolites in tissues are shown in
The suitable and preferred aspects of any feature present in multiple aspects of the present invention are as defined for said features in the first aspect in which they are described herein. The invention is now illustrated by a series of non-limiting examples.
The present invention provides a compound of Formula (III) as described herein. Such compounds are useful as PET imaging agents for tumor imaging, as described herein. In particular, a compound of Formula (III), as described herein, may not be excreted in the urine and hence provide more specific imaging of pelvic malignancies such as prostate cancer.
The present invention provides a method to prepare a compound for Formula (III), wherein said method comprises reaction of the precursor compound of Formula (II) with a compound of Formula (IV) to form a compound of Formula (III) (Scheme A):
wherein the compounds of Formulae (I) and (III) are each as described herein and the compound of Formula (IV) is as follows:
ZXYC*-Lg (IV)
wherein C*, X, Y and Z are each as defined herein for a compound of Formula (III) and “Lg” is a leaving group. Suitable examples of “Lg” include, but are not limited to, bromine (Br) and tosylate (OTos). A compound of Formula (IV) can be prepared by any means known in the art including those described herein (e.g., analogous to Examples 5 and 7).
Reagents and solvents were purchased from Sigma-Aldrich (Gillingham, UK) and used without further purification. Fluoromethylcholine chloride (reference standard) was purchased from ABCR Gmbh & Co. (Karlsruhe, Germany). Isotonic saline (0.9% w/v) was purchased from Hameln Pharmaceuticals (Gloucester, UK). NMR Spectra were obtained using either a Bruker Avance NMR machine operating at 400 MHz (1H NMR) and 100 MHz (13C NMR) or 600 MHz (1H NMR) and 150 MHz (13C NMR). Accurate mass spectroscopy was carried out on a Waters Micromass LCT Premier machine in positive electron ionisation (EI) or chemical ionisation (CI) mode. Distillation was carried out using a Bichi B-585 glass oven (Bichi, Switzerland).
To a suspension of K2CO3 (10.50 g, 76 mmol) in dry THF (10 mL) was added dimethylamine (2.0 M in THF) (38 mL, 76 mmol) followed by 2-bromoethanol-1,1,2,2-d4 (4.90 g, 38 mmol) and the suspension heated to 50° C. under argon. After 19 h, thin layer chromatography (TLC) (ethyl acetate/alumina/I2) indicated complete conversion of (2) and the reaction mixture was allowed to cool to ambient temperature and filtered. Bulk solvent was then removed under reduced pressure. Distillation gave the desired product (3) as a colorless liquid, b.p. 78° C./88 mbar (1.93 g, 55%). 1H NMR (CDCl3, 400 MHz) δ 3.40 (s, 1H, OH), 2.24 (s, 6H, N(CH3)2). 13C NMR (CDCl3, 75 MHz) δ 62.6 (NCD2CD2OH), 60.4 (NCD2CD2OH), 47.7 (N(CH3)2). HRMS (EI)=93.1093 (M+). C4H72H4NO requires 93.1092.
To a suspension of N,N-dimethylglycine (0.52 g, 5 mmol) in dry THF (10 mL) was added lithium aluminium deuteride (0.53 g, 12.5 mmol) and the resulting suspension refluxed under argon. After 24 h the suspension was allowed to cool to ambient temperature and poured onto sat. aq. Na2SO4 (15 mL) and adjusted to pH 8 with 1M Na2CO3, then washed with ether (3×10 mL) and dried (Na2SO4). Distillation gave the desired product (5) as a colorless liquid, b.p. 65° C./26 mbar (0.06 g, 13%). 1H NMR (CDCl3, 400 MHz) δ 2.43 (s, 2H, NCH2CD2), 2.25 (s, 6H, N(CH3)2), 1.43 (s, 1H, OH). 13C NMR (CDCl3, 150 MHz) δ 63.7 (NCH2CD2OH), 57.8 (NCH2CD2OH), 45.7 (N(CH3)2).
Methylene ditosylate (7) was prepared according to an established literature procedure and analytical data was consistent with reported values (Emmons, W. D., et al., Journal of the American Chemical Society, 1953; 75:2257; and Neal, T. R., et al., Journal of Labelled Compounds and Radiopharmaceuticals 2005; 48:557-68). To a solution of methylene ditosylate (7) (0.67 g, 1.89 mmol) in dry acetonitrile (10 mL) was added Kryptofix K222 [4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane] (1.00 g, 2.65 mmol) followed by potassium fluoride (0.16 g, 2.83 mmol). The suspension was then heated to 110° C. under nitrogen. After 1 h TLC (7:3 hexane/ethyl acetate/silica/UV254) indicated complete conversion of (7). The reaction mixture was diluted with ethyl acetate (25 mL), washed with water (2×15 mL) and dried over MgSO4. Chromatography (5→10% ethyl acetate/hexane) gave the desired product (8) as a colorless oil (40 mg, 11%). 1H NMR (CDCl3, 400 MHz) δ 7.86 (d, 2H, J=8 Hz, aryl CH), 7.39 (d, 2H, J=8 Hz, aryl CH), 5.77 (d, 1H, J=52 Hz, CH2F), 2.49 (s, 3H, tolyl CH3). 13C NMR (CDCl3) δ 145.6 (aryl), 133.8 (aryl), 129.9 (aryl), 127.9 (aryl), 98.1 (d, J=229 Hz, CH2F), 21.7 (tolyl CH3). HRMS (CI)=222.0604 (M+NH4)+. Calcd. for C8H13FNO3S 222.0600.
To a dry flask was added dimethylethanolamine (4.46 g, 50 mmol) and dry DMF (50 mL). The solution was stirred under argon and cooled in an ice bath. Sodium hydride (2.0 g, 50 mmol) was then added portionwise over 10 min and the reaction mixture then allowed to warm to room temperature. After 30 min 4-methoxybenzyl chloride (3.92 g, 25 mmol) was added dropwise over 10 min and the resulting mixture left to stir under argon. After 60 h GC-MS indicated reaction completion (disappearance of 4-methoxybenzyl chloride) and the reaction mixture was poured onto 1M sodium hydroxide (100 mL) and extracted with dichloromethane (DCM) (3×30 mL) then dried (Na2SO4). Column chromatography (0→10% methanol/DCM; neutral silica) gave the desired product (O-PMB-DMEA) as a yellow oil (1.46 g, 28%). 1H NMR (CDCl3, 400 MHz) δ 7.28 (d, 2H, J=8.6 Hz, aryl CH), 6.89 (d, 2H, J=8.6 Hz, aryl CH), 4.49 (s, 2H, —CH2—), 3.81 (s, 3H, OCH3), 3.54 (t, 2H, J=5.8, NCH2CH2O), 2.54 (t, 2H, J=5.8, NCH2CH2O), 2.28 (s, 6H, N(CH3)2). HRMS (ES)=210.1497 (M+H+). C12H20NO2 requires 210.1494.
The di- and tetra-deuterated analogs of N,N-Dimethylethanolamine(O-4-methoxybenzyl)ether can be prepared according to Example 4 from the appropriate di- or tetra-deuterated dimethylethanolamine.
To a Wheaton vial containing a mixture of K2CO3 (0.5 mg, 3.6 μmol, dissolved in 100 μL water), 18-crown-6 (10.3 mg, 39 μmol) and acetonitrile (500 μL) was added [18F]fluoride (˜20 mCi in 100 μL water). The solvent was then removed at 110° C. under a stream of nitrogen (100 mL/min). Afterwards, acetonitrile (500 μL) was added and distillation to dryness continued. This procedure was repeated twice. A solution of methylene ditosylate (7) (6.4 mg, 18 μmol) in acetonitrile (250 μL) containing 3% water was then added at ambient temperature followed by heating at 100° C. for 10-15 min., with monitoring by analytical radio-HPLC. The reaction was quenched by addition of 1:1 acetonitrile/water (1.3 mL) and purified by semi-preparative radio-HPLC. The fraction of eluent containing [18F]fluoromethyl tosylate (9) was collected and diluted to a final volume of 20 mL with water, then immobilized on a Sep Pak C18 light cartridge (Waters, Milford, Mass., USA) (pre-conditioned with DMF (5 mL) and water (10 mL)). The cartridge was washed with further water (5 mL) and then the cartridge, with [18F]fluoromethyl tosylate (9) retained, was dried in a stream of nitrogen for 20 min. A typical HPLC reaction profile for synthesis of [18F](13) is shown in FIG. 4A/4B below.
[18F]Fluorobromomethane (prepared according to Bergman et al (Appl Radiat Isot 2001; 54(6):927-33)) was added to a Wheaton vial containing the amine precursor N,N-dimethylethanolamine (150 μL) or N,N-dimethyl-[1,2-2H4]ethanolamine (3) (150 μL) in dry acetonitrile (1 mL), pre-cooled to 0° C. The vial was sealed and then heated to 100° C. for 10 min. Bulk solvent was then removed under a stream of nitrogen, then the sample remaining was redissolved in 5% ethanol in water (10 mL) and immobilized on a Sep-Pak CM light cartridge (Waters, Milford, Mass., USA) (pre-conditioned with 2 M HCl (5 mL) and water (10 mL)) to effect the chloride anion exchange. The cartridge was then washed with ethanol (10 mL) and water (10 mL) followed by elution of the radiotracer (11a) or (11c) using saline (0.5-2.0 mL) and passing through a sterile filter (0.2 μm) (Sartorius, Goettingen, Germany).
[18F]Fluoromethyl tosylate (9) (prepared according to Example 5) and eluted from the Sep-Pak cartridge using dry DMF (300 μL), was added in to a Wheaton vial containing one of the following precursors: N,N-dimethylethanolamine (150 μL); N,N-dimethyl-[1,2-2H4]ethanolamine (3) (150 μL) (prepared according to Example 1); or N,N-dimethyl-[1-2H2]ethanolamine (5) (150 μL) (prepared according to Example 2), and heated to 100° C. with stirring. After 20 min the reaction was quenched with water (10 mL) and immobilized on a Sep Pak CM light cartridge (Waters) (pre-conditioned with 2M HCl (5 mL) and water (10 mL)) in order to effect the chloride anion exchange and then washed with ethanol (5 mL) and water (10 mL) followed by elution of the radiotracer [18F]Fluoromethylcholine (12a), [18F]fluoromethyl-[1-2H2]choline (12b) or [18F]fluoromethyl-[1,2-2H4]choline [18F](12c) with isotonic saline (0.5-1.0 mL).
Commercially available diiodomethane (13) (2.67 g, 10 mmol) was reacted with silver tosylate (6.14 g, 22 mmol), using the method of Emmons and Ferris, to give methylene ditosylate (10) (0.99 g) in 28% yield (Emmons, W. D., et al., “Metathetical Reactions of Silver Salts in Solution. II. The Synthesis of Alkyl Sulfonates”, Journal of the American Chemical Society, 1953; 75:225).
Fluoromethyltosylate (11) (0.04 g) was prepared by nucleophilic substitution of Methylene ditosylate (10) (0.67 g, 1.89 mmol) of Example 3(a) using potassium fluoride (0.16 g, 2.83 mmol)/Kryptofix K222 (1.0 g, 2.65 mmol) in acetonitrile (10 mL) at 80° C. to give the desired product in 11% yield.
Adapting the method of Bergman et al (Appl Radiat Isot 2001; 54(6):927-33), commercially available dibromomethane (16) is reacted with [18F]potassium fluoride/Kryptofix K222 in acetonitrile at 110° C. to give the desired [18F]fluorobromomethane (17), which is purified by gas-chromatography and trapped by elution into a pre-cooled vial containing acetonitrile and the relevant choline precursor.
Radiochemical purity for [18F]Fluoromethylcholine, [18F]fluoromethyl-[1-2H2]choline and [18F]fluoromethyl-[1,2-2H4]choline [18F] was confirmed by co-elution with a commercially available fluorocholine chloride standard. An Agilent 1100 series HPLC system equipped with an Agilent G1362A refractive index detector (RID) and a Bioscan Flowcount FC-3400 PIN diode detector was used. Chromatographic separation was performed on a Phenomenex Luna C18 reverse phase column (150 mm×4.6 mm) and a mobile phase comprising of 5 mM heptanesulfonic acid and acetonitrile (90:10 v/v) delivered at a flow rate of 1.0 mL/min.
This method was adapted from that of Roivannen et al (Roivainen, A., et al., European Journal of Nuclear Medicine 2000; 27:25-32). An aliquot of either [18F]Fluoromethylcholine or [18F]fluoromethyl-[1,2-2H4]choline [18F](100 μL, ˜3.7 MBq) was added to a vial containing water (1.9 mL) to give a stock solution. Sodium phosphate buffer (0.1M, pH 7) (10 uL) containing choline oxidase (0.05 units/uL) was added to an aliquot of stock solution (190 uL) and the vial was then left to stand at room temperature, with occasional agitation. At selected time-points (5, 20, 40 and 60 minutes) the sample was diluted with HPLC mobile phase (buffer A, 1.1 mL), filtered (0.22 μm filter) and then ˜1 mL injected via a 1 mL sample loop onto the HPLC for analysis. Chromatographic separation was performed on a Waters C18 Bondapak (7.8×300 mm) column (Waters, Milford, Massachusetts, USA) at 3 mL/min with a mobile phase of buffer A, which contained acetonitrile, ethanol, acetic acid, 1.0 mol/L ammonium acetate, water, and 0.1 mol/L sodium phosphate (800:68:2:3:127:10 [v/v]) and buffer B, which contained the same constituents but in different proportions (400:68:44:88:400:10 [v/v]). The gradient program comprised 100% buffer A for 6 minutes, 0-100% buffer B for 10 minutes, 100-0% B in 2 minutes then 0% B for 2 minutes.
Human colon (HCT116) tumors were grown in male C3H-Hej mice (Harlan, Bicester, United Kingdom) as previously reported (Leyton, J., et al., Cancer Research 2005; 65(10):4202-10). Tumor dimensions were measured continuously using a caliper and tumor volumes were calculated by the equation: volume=(π/6)×a×b×c, where a, b, and c represent three orthogonal axes of the tumor. Mice were used when their tumors reached approximately 100 mm3. [18F]Fluoromethylcholine, [18F]fluoromethyl-[1-2H2]choline and [18F]fluoromethyl-[1,2-2H4]choline (˜3.7 MBq) were each injected via the tail vein into awake untreated tumor bearing mice. The mice were sacrificed at pre-determined time points (2, 30 and 60 min) after radiotracer injection under terminal anesthesia to obtain blood, plasma, tumor, heart, lung, liver, kidney and muscle. Tissue radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK) and decay corrected. Data were expressed as percent injected dose per gram of tissue.
[18F]FCH or [18F](D4-FCH) (80-100 μCi) was injected via the tail vein into anesthetized non-tumor bearing C3H-Hej mice; isofluorane/O2/N2O anesthesia was used. Plasma samples obtained at 2, 15, 30 and 60 minutes after injection were snap frozen in liquid nitrogen and stored at −80° C. For analysis, samples were thawed and kept at 4° C. To approximately 0.2 mL of plasma was added ice-cold acetonitrile (1.5 mL). The mixture was then centrifuged (3 minutes, 15,493×g; 4° C.). The supernatant was evaporated to dryness using a rotary evaporator (Heidoloph Instruments GMBH & CO, Schwabach, Germany) at a bath temperature of 45° C. The residue was suspended in mobile phase (1.1 mL), clarified (0.2 μm filter) and analyzed by HPLC. Liver samples were homogenized in ice-cold acetonitrile (1.5 mL) and then subsequently treated as per plasma samples. All samples were analyzed on an Agilent 1100 series HPLC system equipped with a γ-RAM Model 3 radio-detector (IN/US Systems inc., FL, USA). The analysis was based on the method of Roivannen (Roivainen, A., et al., European Journal of Nuclear Medicine 2000; 27:25-32) using a Phenomenex Luna SCX column (10μ, 250×4.6 mm) and a mobile phase comprising of 0.25 M sodium dihydrogen phosphate (pH 4.8) and acetonitrile (90:10 v/v) delivered at a flow rate of 2 ml/min.
Liver, kidney, and tumor samples were obtained at 30 min. All samples were snap-frozen in liquid nitrogen. For analysis, samples were thawed and kept at 4° C. immediately before use. To ˜0.2 mL plasma was added ice-cold methanol (1.5 mL). The mixture was then centrifuged (3 min, 15,493×g, 4jC). The supernatant was evaporated to dryness using a rotary evaporator (Heidoloph Instruments) at a bath temperature of 40° C. The residue was suspended in mobile phase (1.1 mL), clarified (0.2 Am filter), and analyzed by HPLC. Liver, kidney, and tumor samples were homogenized in ice-cold methanol (1.5 mL) using an IKA Ultra-Turrax T-25 homogenizer and subsequently treated as per plasma samples (above). All samples were analyzed by radio-HPLC on an Agilent 1100 series HPLC system (Agilent Technologies) equipped with a γ-RAM Model 3 γ-detector (IN/US Systems) and Laura 3 software (Lablogic). The stationary phase comprised a Waters μBondapak C18 reverse-phase column (300×7.8 mm) (Waters, Milford, Mass., USA). Samples were analyzed using a mobile phase comprising solvent A (acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1 mol/L sodium phosphate; 800/127/68/2/3/10) and solvent B (acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1 mol/L sodiumphosphate; 400/400/68/44/88/10) with a gradient of 0% B for 6 min, then 0→100% B in 10 min, 100% B for 0.5 min, 100→0% B in 1.5 min then 0% B for 2 min, delivered at a flow rate of 3 mL/min.
HCT116 cells were grown in T150 flasks in triplicate until they were 70% confluent and then treated with vehicle (1% DMSO in growth medium) or 1 μmol/L PD0325901 in vehicle for 24 h. Cells were pulsed for 1 h with 1.1 MBq of either [8F]D4—FCH or [18F]FCH. The cells were washed three times in ice-cold phosphate buffered saline (PBS), scraped into 5 mL PBS, and centrifuged at 500×g for 3 min and then resuspended in 2 mL ice-cold methanol for HPLC analysis as described above for tissue samples. To provide biochemical evidence that the 5′-phosphate was the peak identified on the HPLC chromatogram, cultured cells were treated with alkaline phosphatase as described previously (Barthel, H., et al., Cancer Res 2003; 63(13):3791-8). Briefly, HCT116 cells were grown in 100 mm dishes in triplicate and incubated with 5.0 MBq [18F]FCH for 60 min at 37° C. to form the putative [18F]FCH-phosphate. The cells were washed with 5 mL ice-cold PBS twice and then scraped and centrifuged at 750×g (4° C., 3 min) in 5 mL PBS. Cells were homogenized in 1 mL of 5 mmol/L Tris-HCl (pH 7.4) containing 50% (v/v) glycerol, 0.5 mmol/L MgCl2, and 0.5 mmol/L ZnCl2 and incubated with 10 units bacterial (type III) alkaline phosphatase (Sigma) at 37° C. in a shaking water bath for 30 min to dephosphorylate the [18F]FCH-phosphate. The reaction was terminated by adding ice-cold methanol. Samples were processed as per plasma above and analyzed by radio-HPLC. Control experiments were done without alkaline phosphatase.
PET Imaging Studies.
Dynamic [18F]FCH and [18F]D4-FCH imaging scans were carried out on a dedicated small animal PET scanner, quad-HIDAC (Oxford Positron Systems). The features of this instrument have been described previously (Barthel, H., et al., Cancer Res 2003; 63(13):3791-8). For scanning the tail veins, vehicle- or drug-treated mice were cannulated after induction of anesthesia (isofluorane/O2/N2O). The animals were placed within a thermostatically controlled jig (calibrated to provide a rectal temperature of ˜37° C.) and positioned prone in the scanner. [18F]FCH or [18F]D4-FCH (2.96-3.7 MBq) was injected via the tail vein cannula and scanning commenced. Dynamic scans were acquired in list mode format over a 60 min period as reported previously (Leyton, J., et al., Cancer Research 2006; 66(15):7621-9). The acquired data were sorted into 0.5 mm sinogram bins and 19 time frames (0.5×0.5×0.5 mm voxels; 4×15, 4×60, and 11×300 s) for image reconstruction, which was done by filtered back-projection using a two-dimensional Hamming filter (cutoff 0.6). The image data sets were visualized using the Analyze software (version 6.0; Biomedical Imaging Resource, Mayo Clinic). Cumulative images of 30 to 60 min dynamic data were used for visualization of radiotracer uptake and to draw regions of interest. Regions of interest were defined manually on five adjacent tumor regions (each 0.5 mm thickness). Dynamic data from these slices were averaged for each tissue (liver, kidney, muscle, urine, and tumor) and at each of the 19 time points to obtain time versus radioactivity curves. Corresponding whole body time versus radioactivity curves representing injected radioactivity were obtained by adding together radioactivity in all 200×160×160 reconstructed voxels. Tumor radioactivity was normalized to whole-body radioactivity and expressed as percent injected dose per voxel (% ID/vox). The normalized uptake of radiotracer at 60 min (% ID/vox60) was used for subsequent comparisons. The average of the normalized maximum voxel intensity across five slices of tumor % IDvox60max was also use for comparison to account for tumor heterogeneity and existence of necrotic regions in tumor. The area under the curve was calculated as the integral of % ID/vox from 0 to 60 min.
Size-matched HCT116 tumor bearing mice were randomized to receive daily treatment by oral gavage of vehicle (0.5% hydroxypropyl methylcellulose+0.2% Tween 80) or 25 mg/kg (0.005 mL/g mouse) of the mitogenic extracellular kinase inhibitor, PD0325901, prepared in vehicle. [18F]D4-FCH-PET scanning was done after 10 daily treatments with the last dose administered 1 h before scanning. After imaging, tumors were snap-frozen in liquid nitrogen and stored at ˜80° C. for analysis of choline kinase A expression. The results are illustrated in
This exemplifies use of [18F]D4-FCH-PET as an early biomarker of drug response.
Most of the current drugs in development for cancer target key kinases involved in cell proliferation or survival. This example shows that in a xenograft model for which tumor shrinkage is not significant, growth factor receptor-Ras-MAP kinase pathway inhibition by the MEK inhibitor PD0325901 leads to a significant reduction in tumor [18F]D4-FCH uptake signifying inhibition of the pathway. The figure also shows that inhibition of [18F]D4-FCH uptake was due at least in part to the inhibition of choline kinase activity.
As illustrated in
Despite the relatively high systemic stability of [18F]D4-FCH and high proportion of phosphocholine metabolites, higher tumor radiotracer uptake by PET in mice that were injected with [18F]D4-FCH compared to the [18F]FCH group was observed.
To understand further the mechanisms regulating [18F]D4-FCH uptake with drug treatment, changes in CHKA expression in PD0325901 and vehicle-treated tumors excised after PET scanning were assessed. A significant reduction in CHKA protein expression was seen in vivo at day 10 (P=0.03) following PD0325901 treatment (
Statistical analyses were done using the software GraphPad Prism version 4 (GraphPad). Between-group comparisons were made using the nonparametric Mann-Whitney test. Two-tailed P≦0.05 was considered significant.
Materials and Methods
HCT116 (LGC Standards, Teddington, Middlesex, UK) and PC3-M cells (donation from Dr Matthew Caley, Prostate Cancer Metastasis Team, Imperial College London, UK) were grown in RPMI 1640 media, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U·mL−1 penicillin and 100 μg·mL−1 streptomycin (Invitrogen, Paisley, Refrewshire, UK). A375 cells (donation from Professor Eyal Gottlieb, Beatson Institute for Cancer Research, Glasgow, UK) and were grown in high glucose (4.5 g/L) DMEM media, supplemented with 10% fetal calf serum, 2 mM
Western blotting was performed using standard techniques. Cells were harvested and lysed in RIPA buffer (Thermo Fisher Scientific Inc., Rockford, Ill., USA). Membranes were probed using a rabbit anti-human choline kinase alpha polyclonal antibody (Sigma-Aldrich Co. Ltd, Poole, Dorset, UK; 1:500). A rabbit anti-actin antibody (Sigma-Aldrich Co. Ltd, Poole, Dorset, UK; 1:5000) was used as a loading control and a peroxidase-conjugated donkey anti-rabbit IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:2500) as the secondary antibody. Proteins were visualized using the Amersham ECL kit (GE Healthcare, Chalfont St Giles, Bucks, UK). Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer; Bio-Rad, Hercules, Calif., USA) and signal quantification was performed by densitometry using scanning analysis software (Quantity One; Bio-Rad).
For analysis of tumor choline kinase expression, tumors at ˜100 mm3 were excised, placed in a Precellys 24 lysing kit 2 mL tube (Bertin Technoologies, Montigny-le-Bretonneux, France), containing 1.4 mm ceramic beads, and snap-frozen in liquid nitrogen. For homogenization, 1 mL of RIPA buffer was added to the lysing kit tubes which were homogenized in a Precellys 24 homogenizer (6500 RPM; 2×17 s with 20 s interval). Cell debris were removed by centrifugation prior to western blotting as described above.
In Vitro 18F-D4-choline Uptake
Cells (5×105) were plated into 6-well plates the night prior to analysis. On the day of the experiment, fresh growth medium, containing 40 μCi 18F-D4-choline, was added to individual wells. Cell uptake was measured following incubation at 37° C. in a humidified atmosphere of 5% CO2 for 60 min. Plates were subsequently placed on ice, washed 3 times with ice-cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific Inc., Rockford, Ill., USA; 1 mL, 10 min). Cell lysate was transferred to counting tubes and decay-corrected radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK). Aliquots were snap-frozen and used for protein determination following radioactive decay using a BCA 96-well plate assay (Thermo Fisher Scientific Inc., Rockford, Ill., USA). Data were expressed as percent of total radioactivity per mg protein. For hemicholinium-3 treatment (5 mM; Sigma-Aldrich), cells were incubated with the compound 30 min prior to addition of radioactivity and for the duration of the uptake time course.
All animal experiments were performed by licensed investigators in accordance with the United Kingdom Home Office Guidance on the Operation of the Animal (Scientific Procedures) Act 1986 and within the newly-published guidelines for the welfare and use of animals in cancer research (Workman P, Aboagye E O, Balkwill F, et al. Guidelines for the welfare and use of animals in cancer research. Br J Cancer. 2010; 102:1555-1577). Male BALB/c nude mice (aged 6-8 weeks; Charles River, Wilmington, Mass., USA) were used. Tumor cells (2×106) were injected subcutaneously on the back of mice and animals were used when the xenografts reached ˜100 mm3. Tumor dimensions were measured continuously using a caliper and tumor volumes were calculated by the equation: volume=(π/6)×a×b×c, where a, b, and c represent three orthogonal axes of the tumor.
Radiolabeled metabolites from plasma and tissues were quantified using a method adapted from Smith G, Zhao Y, Leyton J, et al. Radiosynthesis and pre-clinical evaluation of [(18)F]fluoro-[1,2-(2)H(4)]choline. Nucl Med Biol. 2011; 38:39-51. Briefly, tumor-bearing mice under terminal anaesthesia were administered a bolus i.v. injection of one of the following radiotracers: 11C-choline, 11C-D4-choline (˜18.5 MBq) or 18F-D4-choline (˜3.7 MBq), and sacrificed by exsanguination via cardiac puncture at 2, 15, 30 or 60 min post radiotracer injection. For automated radiosynthesis methodology, see Example 22. Tumor, kidney and liver samples were immediately snap-frozen in liquid nitrogen. Aliquots of heparinized blood were rapidly centrifuged (14000 g, 5 min, 4° C.) to obtain plasma. Plasma samples were subsequently snap-frozen in liquid nitrogen and kept on dry ice prior to analysis.
For analysis, samples were thawed and kept at 4° C. immediately before use. To ice cold plasma (200 μl) was added ice cold methanol (1.5 mL) and the resulting suspension centrifuged (14000 g; 4° C.; 3 min). The supernatant was then decanted and evaporated to dryness on a rotary evaporator (bath temperature, 40° C.), then resuspended in HPLC mobile phase (Solvent A: acetonitrile/water/ethanol/acetic acid/1.0 M ammonium acetate/0.1 M sodium phosphate [800/127/68/2/3/10]; 1.1 mL). Samples were filtered through a hydrophilic syringe filter (0.2 μm filter; Millex PTFE filter, Millipore, Mass., USA) and the sample (˜1 mL) then injected via a 1 mL sample loop onto the HPLC for analysis. Tissues were homogenized in ice-cold methanol (1.5 mL) using an Ultra-Turrax T-25 homogenizer (IKA Werke GmbH and Co. KG, Staufen, Germany) and subsequently treated as per plasma samples.
Samples were analyzed on an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, Calif., USA), configured as described above, using the method of Leyton J, Smith G, Zhao Y, et al. [18F]fluoromethyl-[1, 2-2H4]-choline: a novel radiotracer for imaging choline metabolism in tumors by positron emission tomography. Cancer Res. 2009; 69:7721-7728. A μBondapak C18 HPLC column (Waters, Milford, Mass., USA; 7.8×3000 mm), stationary phase and a mobile phase comprising of Solvent A (vide supra) and Solvent B (acetonitrile/water/ethanol/acetic acid/1.0 M ammonium acetate/0.1 M sodium phosphate (400/400/68/44/88/10)), delivered at a flow rate of 3 mL/min were used for analyte separation. The gradient was set as follows: 0% B for 5 min; 0% to 100% B in 10 min; 100% B for 0.5 min; 100% to 0% B in 2 min; 0% B for 2.5 min.
Dynamic 11C-choline, 11C-D4-choline and 18F-D4-choline imaging scans were carried out on a dedicated small animal PET scanner (Siemens Inveon PET module, Siemens Medical Solutions USA, Inc., Malvem, Pa., USA) following a bolus i.v. injection in tumor-bearing mice of either ˜3.7 MBq for 18F studies, or ˜18.5 MBq for 11C. Dynamic scans were acquired in list mode format over 60 min. The acquired data were then sorted into 0.5 mm sinogram bins and 19 time frames for image reconstruction (4×15 s, 4×60 s, and 11×300 s), which was done by filtered back projection. For input function analysis, data were sorted into 25 time frames for image reconstruction (8×5 s, 1×20 s, 4×40 s, 1×80 s, and 11×300 s). The Siemens Inveon Research Workplace software was used for visualization of radiotracer uptake in the tumor; 30 to 60 min cumulative images of the dynamic data were employed to define 3-dimensional (3D) regions of interest (ROIs). Arterial input function was estimated as follows: a single voxel 3D ROI was manually drawn in the center of the heart cavity using 2 to 5 min cumulative images. Care was taken to minimize ROI overlap with the myocardium. The count densities were averaged for all ROIs at each time point to obtain a time versus radioactivity curve (TAC). Tumor TACs were normalized to injected dose, measured by a VDC-304 dose calibrator (Veenstra Instruments, Joure, The Netherlands), and expressed as percentage injected dose per mL tissue. The area under the TAC, calculated as the integral of % ID/mL from 0-60 min, and the normalized uptake of radiotracer at 60 min (% ID/mL60) were also used for comparisons.
11C-choline, 11C-D4-choline (˜18.5 MBq) and 18F-D4-choline (˜3.7 MBq) were each injected via the tail vein of anaesthetized BALB/c nude mice. The mice were maintained under anesthesia and sacrificed by exsanguination via cardiac puncture at 2, 15, 30 or 60 min post radiotracer injection to obtain blood, plasma, heart, lung, liver, kidney and muscle. Tissue radioactivity was determined on a gamma counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK) and decay corrected. Data were expressed as percent injected dose per gram of tissue.
Data were expressed as mean±standard error of the mean (SEM), unless otherwise shown. The significance of comparison between two data sets was determined using Student's t test. ANOVA was used for multi-parametric analysis (Prism v5.0 software for windows, GraphPad Software, San Diego, Calif., USA). Differences between groups were considered significant if P≦0.05.
Time course biodistribution was performed in non-tumor-bearing male nude mice with 11C-choline, 11C-D4-choline and 18F-D4-choline tracers.
Tracer metabolism in tissues and plasma was performed by radio-HPLC (
In the liver, both 11C-choline and 11C-D4-choline were rapidly oxidized to betaine (
In contrast to the liver, deuteration of 11C-choline resulted in protection against oxidation in the kidney over the entirety of the 60 min time course (
11C-choline, 11C-D4-choline and 18F-D4-choline metabolism were measured in HCT116 tumors (
Choline Tracers have Similar Sensitivity for Imaging Tumors by PET
Despite the high systemic stability of 18F-D4-choline, tumor radiotracer uptake in mice by PET was no higher than with 11C-choline or 11C-D4-choline (
Despite there being no difference in overall tracer retention in the tumor, the kinetic profiles of tracer uptake varied between the three choline tracers, detected by PET (
18F-D4-choline Shows Good Sensitivity for the PET Imaging of Prostate Adenocarcinoma and Malignant Melanoma
Having confirmed that 18F-D4-choline is a more stable choline analogue for in vivo studies, with good sensitivity for the imaging of colon adenocarcinoma, it was desired to evaluate its suitability for cancer detection in other models of human cancer including malignant melanoma A375 and prostate adenocarcinoma PC3-M. In vitro uptake of 18F-D4-choline was similar in the three cell lines over 30 min (
Tumor Size Affects 18F-D4-choline Uptake and Retention but not Tumor Pharmacokinetics
For PET imaging, tumors were grown to 100 mm3 prior to imaging. One small cohort of animals with implanted PC3-M xenografts were, however, imaged when the tumor size had reached 200 mm3 (See
Kidney retention increased in the order of 11C-choline<11C-D4-choline<18F-D4-choline over the 60 min time course (
Despite systemic protection against choline oxidation with 18F-D4-choline, the reduction in the rate of choline oxidation was much more subtle in implanted HCT116 tumors (
Comparison of the three choline radiotracers by PET showed no significant differences in overall tumor radiotracer uptake and hence sensitivity (
Materials were used as purchased without further purification. 1,2-2H4-Dimethylethanolamine (DMEA) was a custom synthesis by Target Molecules Ltd (Southampton, UK). Water for irrigation was from Baxter (Deerfield, Ill., USA) and soda lime was purchased from VWR (Lutterworth, Leicestershire, UK). 0.9% sodium chloride for injection was from Hameln pharmaceuticals Ltd (Gloucester, UK) a 0.045% solution of NaCl was prepared from this stock and water for irrigation. Lithium aluminium hydride (0.1 M in THF) and hydriodic acid (57%) were from ABX (Radeburg, Germany). Methylene ditosylate was obtained from the Huayi Isotope Company (Toronto, Canada). All other chemicals were from Sigma-Aldrich Co. Ltd (Poole, Dorset, UK). For 11C-methylations on the iPhase 11C-PRO, iPhase disposable synthesis kits were obtained from iPhase Technologies Pty Ltd (Melbourne, Australia). For 18F-fluoromethylations on the GE FASTlab (GE Healthcare, Chalfont St. Giles, UK) the partly assembled GE FASTlab cassette contained a FASTlab water bag, N2 filter, pre-conditioned QMA cartridge and reaction vessel. Waters Sep-Pak Accell CM light, tC18 light and tC18 Plus cartridges were obtained from Waters Corporation (Milford, Ma., USA).
Synthesis of 11C-Choline and 11C-[1,2-2H4]-choline
11C-Methyl iodide was prepared using a standard wet chemistry method. Briefly, 11C-carbon dioxide was transferred to the iPhase platform via a custom attached cryogenic trap and reduced to 11C-methane using lithium aluminium hydride (0.1 M in THF) (200 uL) over 1 min at RT. Concentrated hydroiodic acid (200 μL) was then added to the reactor vessel and the mixture heated to 140° C. for 1 min. 11C-methyl iodide was then distilled through a short column containing soda lime and phosphorus pentoxide desiccant into a 2 mL stainless steel loop containing the precursor dimethylethanolamine or 1,2-2H4-dimethylethanolamine (201). The methylation reaction was allowed to proceed at room temperature for 2.5 min. The crude product was then flushed on to a CM cartridge using ethanol (20 mL) at a flow rate of 5 mL/min. The CM cartridge had previously been pre-conditioned with 0.045% sodium chloride (5 mL) then water (5 mL). The CM cartridge was then washed with aqueous ammonia (0.08%, 15 mL) then water (10 mL). The choline product was then eluted from the cartridge using sodium chloride solution (0.045%, 10 mL).
Synthesis of 18F-fluoromethyl-[1,2-2H4]-choline
The system was configured with an eluent vial comprising of 1:4 K2CO3 solution in water:Kryptofix K222 solution in acetonitrile (1.0 mL), 180 mg K2CO3 in water (10.0 mL) and 120 mg Kryptofix K222 in acetonitrile (10.0 mL), methylene ditosylate (4.2-4.4 mg) in acetonitrile (2% water; 1.25 mL), precursor 1,2-2H4-dimethylethanolamine (150 μl) in anhydrous acetonitrile (1.4 mL).
Fluorine-18 drawn onto system and immobilised on Waters QMA light cartridge then eluted with 1 mL of a mixture of carbonate and kryptofix into the reaction vessel. After the K[18F]F/K222/K2CO3 drying cycle was complete, methylene ditosylate in acetonitrile (2% water; 1.25 mL) was added and reaction vessel heated to 110° C. for minutes. The reaction was quenched with water (3 mL) and the resulting mixture was passed through both t-C18 light and t-C18 plus cartridges (pre-conditioned with acetonitrile and water; 2 mL each); 15% acetonitrile in water was then passed through the cartridges. After completion of the clean-up cycle, methylene ditosylate was trapped on the t-C18 light cartridge and 18F-fluoromethyl tosylate (together with 18F-tosyl fluoride) was retained on the t-C18 plus, with other reactants going to waste. The washing cycles ethanol→vacuum→nitrogen were employed to clean the reaction vessel after this first stage of radiosynthesis. The reaction vessel and the t-C18 plus cartridge with immobilized 18F-fluoromethyl tosylate were then simultaneously dried under a stream of nitrogen. 18F-fluoromethyl tosylate was then eluted from the t-C18 plus cartridge with 150 μl of 1,2-2H4-dimethylethanolamine in 1.4 mL of acetonitrileinto the reaction vessel. The reactor vessel was then heated to 110° C. for 15 minutes then cooled and the reaction vessel contents washed with water on to a CM cartridge (conditioned with 2 mL water). The cartridge was washed by withdrawing ethanol from the bulk ethanol vial and passing it through CM cartridge; the washing cycle was repeated once followed by 0.08% ammonia solution (4.5 mL). The CM cartridge then was subjected to final washes with ethanol followed by water. The product, 18F-fluoro-[1,2-2H2]choline, was washed off the CM cartridge with 0.09% sodium chloride solution (4.5 mL) to afford 18F-fluoro-[1,2-2H2]choline in sodium chloride buffer as the final formulated product.
11C-Choline, 11C-[1,2-2H4]-choline and 18F-fluoro-[1,2-2H2]choline were analyzed for chemical/radiochemical purity on a Metrohm ion chromatography system (Runcorn, UK) with a Metrosep C4 cation column (250×4.0 mm) attached. The mobile phase was 3 mM Nitric acid:Acetonitrile (75:25 v/v) running in isocratic mode at 1.5 mL/min. All radiotracers were >95% radiochemical purity after formulation.
A 2-tissue irreversible compartmental model was employed to fit the TACs, as has been previously established for 11C-choline (Kenny L M, Contractor K B, Hinz R, et al. Reproducibility of [11C]choline-positron emission tomography and effect of trastuzumab. Clin Cancer Res. Aug. 15 2010; 16(16):4236-4245; and Sutinen E, Nurmi M, Roivainen A, et al. Kinetics of [(11)C]choline uptake in prostate cancer: a PET study. Eur J Nucl Med Mol. Imaging. March 2004; 31(3):317-324). An estimate of the whole blood TAC (wbTAC(t)) was derived from the PET image itself, as described above. As the wbTAC was obtained from one voxel only it was relatively noisy. Therefore it was fitted with a sum of 3 exponentials from the peak on and the fitted function was used as input function in the kinetic modeling (after metabolite correction, see below). The parent fraction values, pf, were calculated from plasma metabolite analysis: at 2, 15, 30 and 60 minutes they were [0.96, 0.55, 0.47, 0.26] for 18F-D4-choline, [0.92, 0.25, 0.20, 0.12] for 11C-choline and [0.91, 0.18, 0.08, 0.03] for 11C-D4-choline, respectively. The pf values were fitted to a sum of two exponentials with the constraint pf(t=0)=1 to obtain the function pf(t). The parent whole blood TAC wbTACPAR(t) was then computed by multiplying wbTAC(t) and pf(t) and used as input function to estimate the parameters K1 (mL/cm3/min), k2 (1/min), k3 (1/min) and Vb (unitless). The steady state net irreversible uptake rate constant Ki (mL/cm3/min) was calculated from the estimated microparameters as K1k3/(k2+k3). Because the quality of fits obtained using the wbTACPAR(t) as only input function to the model was poor, and because 18F-D4-choline, 11C-choline and 11C-D4-choline are quickly metabolized in vivo in the mouse, a double input (DI) model accounting for the contribution of metabolites to the tissue TAC was also considered (Huang S C, Yu D C, Barrio J R, et al. Kinetics and modeling of L-6-[18F]fluoro-dopa in human positron emission tomographic studies. J Cereb Blood Flow Metab. November 1991; 11(6):898-913). In the DI model the metabolite whole blood TAC wbTACMET(t) computed as wbTAC(t)x[1-pf(t)] together with wbTACPAR(t) was employed as input function; the parent tracer was modeled with a 2-tissue irreversible model whereas a simple 1-tissue reversible model was used to describe the metabolite kinetics, thus computing the metabolite influx and efflux K1′ and k2′ in addition to the parameters estimated for the parent. The standard Weighted Non-Linear Least Squares (WNLLS) was used as estimation procedure. WNLLS minimizes the Weighted Residual Sum of Squares (WRSS) function
with C(ti) and ti indicating respectively the decay-corrected concentration computed from the PET image and the mid-time of the i-th frame and n denoting number of frames. In Eq.1 weights wi were set to
with Δi and 2 representing the duration of the i-th frame and the half-life of 18F (for 18F-D4-choline) or 11C (for 11C-choline and 11C-D4-choline) (Tomasi G, Bertoldo A, Bishu S, Unterman A, Smith C B, Schmidt K C. Voxel-based estimation of kinetic model parameters of the L-[1-(11)C]leucine PET method for determination of regional rates of cerebral protein synthesis: validation and comparison with region-of-interest-based methods. J Cereb Blood Flow Metab. July 2009; 29(7):1317-1331). WNLLS estimation was performed with the Matlab function lsqnonlin; parameters were constrained to be positive but no upper bound was applied.
Supplemental Table 1.
Kinetic parameters from dynamic 18F-D4-choline PET in tumors. Decay-corrected uptake values at 60 min (NUV60) and the area under the curve (AUC) were taken from tumor TACs. Flux constant measurements, K1′, Ki and k3 were obtained by fitting tumor TAC and derived input function, corrected for radioactive plasma metabolites of 18F-D4-choline, to a 2-tissue irreversible model of tracer delivery and retention. Mean values (n=3)±SEM are shown.
All patents, journal articles, publications and other documents discussed and/or cited above are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/052275 | 9/20/2011 | WO | 00 | 3/18/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61384895 | Sep 2010 | US | |
| 61531119 | Sep 2011 | US |
