PET/SPECT agents for applications in biomedical imaging

Abstract

Tracers that can be used for PET or SPECT imaging of the distribution of Pgp are disclosed. The tracers are metalloprobes that can comprise a radioactive metal ion such as .sup.67Ga or .sup.68Ga. Methods of synthesizing the tracers, and methods of imaging heart and other tissues are also disclosed. The tracers can be used to obtain high signal-to-background ratios for imaging tissues in vivo such as heart or tumor tissue. In various embodiments, disclosed tracers can exhibit, a) enhanced first pass extraction into heart tissue compared to presently available probes, b) linearity with true blood flow, c) enhanced detection of myocardial viability compared to presently available probes, d) reduced liver retention compared to presently available probes, and e) more efficient clearance from non-cardiac and adjoining tissues compared to presently available probes.

Claims

1. A compound comprising a structure ##STR00016## Wherein each of R.sub.1 and R.sub.2 is H; each of R.sub.7 and R.sub.8 is methyl; each R.sub.3 is isopropoxy; each of R.sub.4, R.sub.5 and R.sub.6 is H; and each R.sub.9 is H.

2. A salt of structure ##STR00017## wherein A is an anion.

3. The salt in accordance with claim 2, wherein the anion is an I.sup..

4. The salt in accordance with claim 2, wherein the Ga.sup.+ is a .sup.67Ga.sup.+.

5. The salt in accordance with claim 2, wherein the Ga.sup.+ is a .sup.68Ga.sup.+.

6. The salt in accordance with claim 2, wherein the anion is an I.sup. and the Ga.sup.+ is a .sup.68Ga.sup.+.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 presents a projection view of cationic gallium (III) complex [ENBDMP-3-isopropoxy-PI-Ga].sup.+ (I).

(2) FIG. 2 presents HPLC data for [ENBDMP-3-isopropoxy-PI-.sup.67Ga].sup.+ (1A) co-injected with the unlabeled complex 1.

(3) FIG. 3 presents a characterization of [ENBDMP-3-isopropoxy-PI-.sup.67Ga].sup.+ (1A).

(4) FIG. 4 illustrates NanoSPECT/CT imaging using [ENBDMP-3-isopropoxy-PI-.sup.67Ga].sup.+ (1A) 30 min post injection into a rat.

(5) FIG. 5 illustrates NanoSPECT/CT imaging using [ENBDMP-3-ispropoxy-PI-.sup.67Ga].sup.+ (1A) 250 min post injection into a rat.

(6) FIG. 6 illustrates MicroPET imaging of myocardial perfusion using [ENBDMP-3-isopropoxy-PI-.sup.68Ga].sup.+ (1B) 60 min post injection into a rat.

DETAILED DESCRIPTION

(7) As used herein, a compound is an organic covalent structure.

(8) As used herein, a chelate is a covalent structure than can bond non-covalently with a cation.

(9) As used herein, a complex is a covalent structure or chelate bonded with a cation.

(10) As used herein, a salt is a complex combined with an anion.

(11) As used herein, a metal salt comprises a metal cation and an anion. The anion can be organic or inorganic.

(12) As used herein, with regard to chemistry procedures, contacting can include mixing, combining, stirring in, or the like, and can include, e.g., mixing chemicals under conditions that promote or result in a chemical reaction.

(13) In some configurations, a gallium(III) agent such as 1, 1A or 1B incorporating an organic scaffold comprising six donor atoms, e.g. 2 or 2A, can result in an octahedral geometry.

(14) In various aspects, compounds, chelates, complexes and salts of the present teaching can be used as tracers for imaging cardiac tissue in mammals such as humans. In various aspects, compounds, chelates, complexes and salts of the present teaching can be used as fertilizer. In some configurations, a complex or salt of the present teachings can comprise an iron ion, and can be used to provide iron to plants. In some configurations, a complex or salt of the present teachings can comprise an iron ion, and can be useful in the treatment of anemia.

(15) The present teachings, including descriptions provided in the Examples, are not intended to limit the scope of any claim. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The examples are not intended to limit the scope of the aspects. The methods described herein utilize laboratory techniques well known to skilled artisans, and guidance can be found in laboratory manuals and textbooks such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; and Harlow, E. Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999; Hedrickson et al. Organic Chemistry 3rd edition. McGraw Hill, New York, 1970; Carruthers. W., and Coldham, I., Modern Methods of Organic Synthesis (4th Edition), Cambridge University Press, Cambridge, U.K., 2004; Curati, W. L., Imaging in Oncology, Cambridge University Press, Cambridge, U.K., 1998; Welch, M. J., and Redvanly, C. S., eds. Handbook of Radiopharmaceuticals: Radiochemistry and Applications, J. Wiley, New York, 2003.

EXAMPLES

Example 1

(16) This Example illustrates the structure of a complex of the present teachings. The crystal structure of [ENBDMP-3-isopropoxy-PI-Ga]+ displayed in FIG. 1 shows a symmetrical engagement of the four nitrogen atoms in the equatorial plane and two axial phenolate atoms. FIG. 1 presents a projection view of cationic gallium (II) complex [ENBDMP-3-isopropoxy-PI-Ga].sup.+ (1), but without iodide (I.sup.) as the counter anion. FIG. 1 includes the crystallographic numbering scheme. Atoms are represented by thermal ellipsoids corresponding to 50% probability. .sup.1H NMR, proton-decoupled .sup.13C NMR, and HRMS analysis can also be used to validate the structure.

Example 2

(17) This Example illustrates HPLC data confirming synthesis and radiolabeling of [ENBDMP-3-isopropoxy-PI-.sup.67Ga].sup.+. In these experiments, the .sup.67Ga-labeled complex (1A) was synthesized and characterized via HPLC. FIG. 2 presents HPLC data for [ENBDMP-3-isopropoxy-PI-.sup.67Ga].sup.+ 1A co-injected with unlabeled 1. In FIG. 2, peaks have been offset for visualization.

Example 3

(18) This Example illustrates characterization of [ENBDMP-3-isopropoxy-PI-.sup.67Ga].sup.+ for 1A, FIG. 3 shows cellular accumulation of 1A in KB-3-1 cells (Pgp). MCF-7 cells (Pgp), MDR KB-8-5 (+Pgp), KB-8-5-11 (Pgp++) cells and stably transfected MCF-7/MDR1 cells as indicated. Shown is net uptake at 90 minutes (fmol (mg protein).sup.1 (nM.sub.0).sup.1) using control buffer in the absence or presence of MDR1Pgp inhibitor LY335979 (1 M). Each bar represents the mean of 4 determinations; line above the bar denotes +SEM.

Example 4

(19) This Example presents in cellulo and in vivo bioassays to illustrate some functions of some disclosed complexes. In these experiments, the .sup.67Ga-labeled salt 1A

(20) ##STR00015##
was evaluated using cell transport studies and quantitative biodistribution studies in mdr1a/1b.sup.(/) gene-deleted mice and their wild-type (WT) counterparts. In these experiments, radiolabeled .sup.67Ga-analogue showed high accumulation in human epidermal carcinoma drug-sensitive KB-3-1 cells (Pgp.sup.) and human breast carcinoma MCF-7 (Pgp.sup.) cells, and low accumulation in MDR KB-8-5 (+Pgp), KB-8-5-11 (++Pgp) cells and stably transfected MCF-7/MDR1 (+Pgp) cells. Pgp inhibitor LY335979 (Zosuquidar trihydrochloride, Selleck Chemicals, Houston, Tex.) (1 M), enhanced accumulation in multidrug resistant (MDR, Pgp.sup.+) KB-8-5, KB-8-5-11 cells, and stably transfected MCF-7/MDR1 cells, thus demonstrating its responsiveness to Pgp-mediated functional transport activity in cellulo (FIG. 3). In mdr1a/1b.sup.(/) gene-deleted mice, the .sup.67Ga-labelled complex showed 16-fold greater brain penetration and retention (% ID/g=0.96) compared with WT counterparts (% ID/g=0.06), 2 h post injection of 1A (Tables 1 & 2). Additionally, 1A also showed 2.6 fold higher retention in blood of mdr1a/1b.sup.(/) gene-deleted mice compared with WT counterparts (Table 1 & 2), consistent with Pgp expression in white cells of WT mice. These data indicated the ability of 1A to be transported out of cells expressing Pgp and to serve as a probe of the Pgp-mediated component of the blood-brain barrier (BBB) function.

Example 5

(21) This Example discloses synthesis of [ENBDMP-3-isopropoxy-PI-Ga].sup.+ I.sup. 1. The ligand (100 mg, 0.18 mmol) was dissolved in methanol (5 mL) and was treated with dropwise addition of gallium(II) acetylacetonate (66.2 mg, 0.18 mmol) dissolved in methanol. The contents were refluxed for 3 h. Then, potassium iodide (30 mg, 0.18 mmol) dissolved in hot water (0.5 mL) was added and the reaction mixture was refluxed further for 15 min, brought to room temperature slowly. Slow evaporation over a few days yielded crystalline material, 30% yield. .sup.1H NMR (300 MHz, DMSO-d6) : 0.79 (s, 6H), 0.96 (s, 6H), 1.30-1.33 (dd, 12H), 2.63 (d, 2H), 2.79 (d, 4H) 2.94 (br, s, 21), 3.61-3.75 (m 4H), 4.63 (quintet, 2H), 4.79 (br, s, 2H), 6.62 (t, 2H), 6.87 (d, 2H), 7.04 (d, 2H), 8.18 (s, 2H); .sup.13C NMR (300 MHz, DMSO-d6) : 22.0, 22.1, 22.2, 26.2, 35.6, 47.7, 59.2, 68.9, 69.5, 115.7, 119.2, 119.5, 125.8, 148.6, 158.1, 170.3. MS (HRESI) Calcd for [C32H48N4O4Ga]+; 621.2926. found: m/z=621.2930 and Calcd for [.sup.13C.sub.32H.sub.48N.sub.4O.sub.4Ga].sup.+; 622.2959. found: m/z=622.2967.

Example 6

(22) This Example discloses preparation of preparation of .sup.67Ga-metalloprobe 1A.

(23) Radiolabeled .sup.67Ga-metalloprobe was synthesized by following a procedure described earlier and slight modifications. .sup.67Ga was obtained as a commercial citrate salt in water (Mallinckrodt, Inc., Saint Louis, Mo.), converted into chloride, and finally into .sup.67Ga(acetylacetonate).sub.3 by reacting with acetylacetone using standard procedures. Radiolabeled .sup.67Ga-metalloprobes were obtained through a ligand exchange reaction involving either .sup.67Ga(acetylacetonate).sub.3 or .sup.67GaCl.sub.3 and hexadentate(2) or heptadentate (2A) Schiff-base ligands dissolved in ethanol at 100 C. for 40 min. Reaction was followed using thin-layer chromatography plates (C-18) employing a radiometric scanner (Bioscan), using an eluent mixture of ethanol/saline (90/10; R.sub.f: 0.23). Finally, .sup.67Ga-metalloprobe 1A was purified by radio-HPLC using a Vydac TP C-18 reversed-phase column (10 m, 300 ) (Grace Discovery Sciences, Deerfield, Ill.) using an eluent mixture of ethanol and saline as a gradient system. The fraction eluting at a retention time of 16.8 min (1A) was collected, concentrated, and employed for bioassays.

Example 7

(24) This Example discloses preparation of .sup.68Ga-metalloprobe 1B.

(25) Radiolabeled .sup.68Ga-metalloprobe was synthesized by following a procedure described earlier and slight modifications. .sup.68Ga was obtained from the generator as its chloride salt, converted into .sup.68Ga(acetylacetonate).sub.3 by reacting with acetylacetone (0.01% solution in ethanol) using standard procedures. Radiolabeled .sup.67Ga-metalloprobe were obtained through a ligand exchange reaction involving either .sup.68Ga(acetylacetonate).sub.3 or .sup.68GaCl.sub.3 and hexadentate or heptadentate Schiff-base ligands (2 or 2A) dissolved in ethanol at 100 C. for 40 min. Reaction was followed using thin-layer chromatography plates (C-18) employing a radiometric scanner (Bioscan), using an eluent mixture of ethanol/saline (90/10; Rf: 0.23). Finally, .sup.68Ga-metalloprobe 1B was purified by radio-HPLC using Vydac TP C-18 reversed-phase column (10 m, 300 ) using an eluent mixture of ethanol and saline as a gradient system. The fraction eluting at a retention time of 16.8 min (1B) was collected, concentrated, and employed for bioassays.

Example 8

(26) This Example discloses preparation of 1,2-ethylenediamino-bis[1-{(3-isopropoxyphenylene-2-ol)methylenimino-2,2-dimethyl}propane](2).

(27) To obtain 2, the starting precursor amine, 1,2-ethylenediamino-bis(2,2-dimethylaminopropane) was synthesized as described (Sivapackiam, J., et al., Dalton Transactions 39, 5842-5850, 2010). Additionally, the second starting precursor, 2-hydroxy-3-isopropoxy-1-benzaldehyde was also obtained using a procedure described below:

(28) 3-isopropoxyphenol (1.34 mmol), anhydrous magnesium chloride (6.73 mmol), and anhydrous triethylamine (13.4 mmol) were suspended in anhydrous acetonitrile (50 mL), and suspension was stirred for 1 h at room temperature. Then, p-formaldehyde (6.72 mmol) was added to the mixture and the contents were heated at reflux for 4 h. The reaction mixture was cooled to room temperature, hydrolyzed, acidified with 10% HCl (50 mL), and extracted with ether (3200 mL). The combined organic extract was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified on silica gel GF254 (Analtech, USA) using hexane/ethyl acetate (70/30) as eluent mixture, 57% yield. .sup.1H NMR (300 MHz, CDCl.sub.3) : 1.28 (d, 611), 4.48 (quintet, 1H), 6.84 (t, 1H), 7.03-7.11 (dd, 2H), 9.81 (s, 1H), 10.87 (s, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) : 22.1, 72.2, 119.6, 121.4, 122.8, 125.3, 146.5, 153.0, 196.6; MS (HRESI) Calcd for [C.sub.10H.sub.12O.sub.3].sup.+: 163.0754. found: 163.0759.

(29) Finally, for obtaining 2, starting precursors, 2-Hydroxy-3-isopropoxy-1-benzaldehyde (1.80 mmol) and 1,2-ethylenediamino-bis(2,2-dimethylaminopropane) (0.90 mmol) were dissolved in ethanol (10 mL) refluxed for 45 min, and purified by methods described previously. .sup.1H NMR (300 MHz, CDCl.sub.3) : 0.90 (s, 12H), 1.39 (d, 12H), 2.05-2.80 (m, 12H), 2.75 (bs, 2H) 4.60 (q, 2H), 6.80 (t, 2H), 6.850-6.95 (dd, 4H), 7.45 (d, 2H), 8.28 (s, 2H); MS (HRESI) Calcd for [C32H50N4O4]; 554.3832. found: m/z=555.3918.

Example 9

(30) This Example discloses a kit formulation of ligands 2 and 2A.

(31) 2 or 2A (10 mg) was dissolved in ethanol (500 l) and treated with potassium acetate (1 mM, 15 ml, pH 5.5) and contents were stirred in an argon flushed amber colored vial. The mixture was filtered through a nylon syringe filter (0.2 M), and aliquoted into amber colored sterile vials (5 ml), and lyphilized at 50 C. These kits were stored in a refrigerator for 3 months without any appreciable decomposition and used for preparation of 1 and its .sup.67Ga-labeled counterpart (1A) or .sup.68Ga-counterpart (1B) as described above.

Example 10

(32) This example discloses formation and analysis of a crystal.

(33) In these experiments, crystals suitable for X-ray crystallography were grown by dissolving 1 in refluxing methanol, slowly bringing solution to room temperature and extremely slow concentration of the methanol solution overnight. A single crystal with approximate dimensions 0.280.180.17 mm; was mounted on a glass fiber in a random orientation. Preliminary examination and data collection were performed using a Bruker Kappa Apex II (Charge Coupled Device (CCD) Detector system, Bruker AXS, Inc., Madison, Wis.) single crystal X-Ray diffractometer, equipped with an Oxford Cryostream LT device.

Example 11

(34) This example discloses bioassays.

(35) All bioassays were performed as described in earlier publications. (Sivapackiam, J., et al., Dalton Transactions 39, 5842-5850, 2010; Harpstrite, S. E., et al., J. Inorg. Biochem. 101, 1347-1353, 2007; Sharma, V., et al., J. Nucl. Med. 46, 354-364 2005).

(36) Gallium(III) agent (1) incorporates a compound possessing six donor atoms and results in an octahedral geometry (FIG. 1). Suitable crystals for analysis were obtained via slow evaporation of a methanol solution of the gallium(III) complex 1. Crystal structure showed a symmetrical engagement of the four equatorial nitrogen atoms and two phenolic oxygen atoms. Upon chemical characterization using routine analytical tools such as .sup.1H NMR, proton-decoupled .sup.13C NMR, and HRMS analysis, the agent was validated via multiple bioassays in cellulo and in vivo. The radiolabeled .sup.67Ga-agent (1A) was obtained via ligand-exchange reaction using .sup.67Ga(acac).sub.3 and ligand 2 or 2A. The product was purified via HPLC using a -radiodetector (FIG. 2) and characterized via multiple bioassays. .sup.67Ga-labeled counterpart (1A) was evaluated via cell transport studies using human epidermal carcinoma (Pgp.sup.; Pgp.sup.+) cells and quantitative biodistribution studies in mdr1a/1b.sup.(/) gene-deleted mice and their wild-type (WT) counterparts. Radiolabeled .sup.67Ga-analogue (1A) showed high accumulation in human epidermal carcinoma drug-sensitive KB-3-1 cells (Pgp.sup.), human breast carcinoma MCF-7 (Pgp.sup.) cells; an inhibitor (LY335979, 1 M) induced accumulation in multidrug resistant (MDR, Pgp.sup.) KB-8-5, KB-8-5-11 cells, and stably transfected MCF-7/MDR1 cells, thus demonstrating its ability to interrogate Pgp-mediated functional transport activity in cellulo (FIG. 3). In mdr1a/1b.sup.(/) gene-deleted mice, the .sup.67Ga-metalloprobe showed 16-fold greater brain uptake and retention compared with WT counterparts (Table 1 and Table 2). Additionally, the agent permeated the heart tissue accompanied by a facile clearance from the livers of mice (Table 1 and Table 2) and rats (Table 3), thus leading to extremely high target to background ratios (Table 4 and Table 5), showing the potential of the agent for heart perfusion imaging. Thus, molecular imaging of the functional transport activity of MDR1 Pgp (ABCB1) using the disclosed 67/68Ga-metalloprobe enables noninvasive monitoring of the blood-brain barrier in neurodegenerative diseases, assessment of tumors to stratify patient populations for chemotherapeutic treatments, as well as probe the presence or absence of Pgp tissues in vivo, probing depolarization of the membrane potential, and can also provide a myocardial perfusion PET/SPECT imaging agent. Additionally, our synthesis, purification, and formulation of the agent could be accomplished in less than 60 minutes.

Example 12

(37) This Example illustrates NanoSPECT/CT imaging using the .sup.67Ga-radiopharmaceutical (1A). In these experiments. .sup.67Ga-radiopharmaceutical 1A was injected intravenously into a rat tail-vein: NanoSPECT/CT images were obtained 30 min. (FIG. 4) and 250 min. (FIG. 5) post-injection. The arrows indicate heart uptake.

Example 13

(38) This Example illustrates MicroPET imaging of myocardial perfusion in a rat. In these experiments, .sup.68Ga-radiopharmaceutical 1B was injected intravenously into a rat tail-vein; MicroPET images were obtained 60 min. (FIG. 6) post-injection. Note low level of signal from liver compared to the heart.

(39) All references cited herein are incorporated by reference each in its entirety. Tables

(40) TABLE-US-00001 TABLE 1 Biodistribution data (% ID/g) for .sup.67Ga-Agent 1a in WT mice (n = 3). time(min) P.I. 5 15 60 120 Aver- Aver- Aver- Aver- % ID/g age SEM age SEM age SEM age SEM blood 1.26 0.33 0.29 0.04 0.10 0.01 0.07 0.01 liver 44.95 1.24 33.80 1.80 7.44 0.44 2.90 0.24 kidneys 81.04 17.46 83.46 10.00 93.35 14.49 67.91 5.59 heart 9.21 1.64 8.37 0.98 11.98 0.74 9.81 1.90 brain 0.14 0.01 0.12 0.02 0.09 0.01 0.06 0.01

(41) TABLE-US-00002 TABLE 2 Biodistribution data (% ID/g) for .sup.67Ga-Agent 1a in mdr 1a/1b (/) (dKO) mice (n = 3). time(min) P.I. 5 15 60 120 Aver- Aver- Aver- Aver- % ID/g age SEM age SEM age SEM age SEM blood 1.54 0.22 0.64 0.10 0.34 0.03 0.19 0.05 liver 46.34 3.71 46.45 3.42 42.54 5.61 29.82 2.66 kidneys 86.12 4.33 84.19 7.62 95.60 10.38 115.23 10.09 heart 17.02 2.42 10.59 0.58 14.61 0.64 20.29 4.45 brain 1.05 0.03 0.65 0.08 0.99 0.10 0.96 0.13

(42) TABLE-US-00003 TABLE 3 Biodistribution data (% ID/g) for .sup.67Ga-Agent 1a in rats (n = 3). time(min) P.I. 5 15 60 120 Aver- Aver- Aver- Aver- % ID/g age SEM age SEM age SEM age SEM blood 0.175 0.016 0.041 0.003 0.028 0.003 0.012 0.002 lung 0.814 0.069 0.626 0.018 0.559 0.023 0.591 0.006 liver 2.615 0.148 0.657 0.029 0.328 0.017 0.203 0.028 kidneys 8.142 0.620 5.168 0.144 4.201 0.101 3.543 0.108 heart 1.443 0.046 1.306 0.038 1.386 0.026 1.516 0.013 brain 0.024 0.003 0.015 0.001 0.018 0.001 0.016 0.001

(43) TABLE-US-00004 TABLE 4 Heart to Tissue Ratio of .sup.67Ga-Agent 1a in rats (n = 3). time(min) P.I. 5 60 120 Aver- Aver- Aver- % ID/g age SEM age SEM age SEM Heart/Blood 8.386 0.017 49.677 4.275 138.825 32.596 Heart/Liver 0.554 0.069 4.246 0.166 7.782 1.127

(44) TABLE-US-00005 TABLE 5 Heart to Tissue Ratio of .sup.67Ga-Agent 1a in WT mice (n = 3). time(min) P.I. 5 60 120 Aver- Aver- Aver- % ID/g age SEM age SEM age SEM Heart/Blood 7.772 1.13 126.918 18.45 146.618 24.53 Heart/Liver 0.206 0.04 1.617 0.12 3.526 0.88