Alpha-emitting complexes
09724436 · 2017-08-08
Assignee
Inventors
Cpc classification
A61K51/1045
HUMAN NECESSITIES
A61K51/1051
HUMAN NECESSITIES
A61K51/0478
HUMAN NECESSITIES
A61K51/1072
HUMAN NECESSITIES
International classification
A61K51/00
HUMAN NECESSITIES
Abstract
The present invention provides a tissue-targeting complex comprising a tissue targeting moiety, an octadentate hydroxypyridinone-containing ligand and the ion of an alpha-emitting thorium radionuclide. The invention additionally provides therapeutic methods employing such complexes, methods of their production and use, and kits and pharmaceutical compositions comprising such complexes.
Claims
1. A tissue-targeting complex consisting of a tissue targeting moiety selected from antibodies, antibody constructs, fragments of antibodies, constructs of fragments or a mixture thereof, peptide, amino acid, steroidal or non-steroidal hormone, folate, estrogen, testosterone or biotin, an octadentate hydroxypyridinone-containing ligand, and an ion of an alpha-emitting 227-thorium radionuclide, wherein said octadentate ligand is selected from the group consisting of formula VI and VII ##STR00022## a coupling moiety which links said formula VI or said formula VII to said tissue targeting moiety.
2. The tissue targeting complex of claim 1, wherein said tissue targeting moiety is an antibody.
3. A method of treatment of a human or non-human animal body having a hyperplastic or a neoplastic disease, comprising administration of at least one tissue-targeting complex to said human or non-human animal body, wherein said tissue-targeting complex is a complex as claimed in claim 1.
4. A pharmaceutical composition comprising a tissue-targeting complex together with at least one pharmaceutical carrier or excipient, wherein said tissue-targeting complex is a complex as claimed in claim 1.
5. A method of treatment of a human or non-human animal body having a hyperplastic or a neoplastic disease, comprising administration of at least one tissue-targeting complex to said human or non-human animal body, wherein said tissue-targeting complex is a complex as claimed in claim 2.
6. A pharmaceutical composition comprising a tissue-targeting complex together with at least one pharmaceutical carrier or excipient, wherein said tissue-targeting complex is a complex as claimed in claim 2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(22) The invention will now be illustrated by the following non-limiting Examples. All compounds exemplified in the examples form preferred embodiments of the invention (including preferred intermediates and precursors) and may be used individually or in any combination in any aspect where context allows. Thus, for example, each and all of compounds 2 to 4 of Example 2, compound 10 of Example 3 and compound 7 of Example 4 form preferred embodiments of their various types.
Example 1—Isolation of Pure Thorium-227
(23) Thorium-227 is isolated from an actinium-227 cow. Actinium-227 was produced through thermal neutron irradiation of Radium-226 followed by the decay of Radium-227 (t1/2=42.2 m) to Actinium-227. Thorium-227 was selectively retained from an Actinium-227 decay mixture in 8 M HNO.sub.3 solution by anion exchange chromatography. A column of 2 mm internal diameter,
(24) length 30 mm, containing 70 mg of AG®1-X8 resin (200-400 mesh, nitrate form) was used. After Actinium-227, Radium-223 and daughters had eluted from the column, Thorium-227 was extracted from the column with 12 M HCl. The eluate containing
(25) Thorium-227 was evaporated to dryness and the residue resuspended in 0.01 M HCl.
Example 2—Synthesis of ALG-DD-NCS
(26) ##STR00012## ##STR00013##
(27) 3-benzyloxy-1-methyl-4-(2-thioxothiazolidin-1-yl)carbonyl-2(1H)-pyridinone (synthesized according to Raymond, K.; Xu, J., U.S. Pat. No. 5,624,901) (2.22 mmol, 0.8 g), triethylamine (0.31 mL, 2.22 mmol) and DMAP (5 mg) was added to a solution of [5-Amino-6-((2-amino-ethyl)-{2-[bis-(2-amino-ethyl)-amino]-ethyl}-amino)-hexyl]-carbamic acid tert-butyl ester (BocLys-H(2,2)amine) (synthesized according to Raymond, K.; Corneillie, T. M.; Xu. J. WO 2008/063721 A2) (0.204 g, 0.505 mmol) in dichloromethane (80 mL). This mixture was stirred at room temperature overnight and then evaporated to dryness. The residue was dissolved in dichloromethane and loaded onto a flash silica gel column and eluted with a gradient of 2-8% methanol in dichloromethane. The appropriate fractions were collected and evaporated to dryness to give ALG-001 (1) (˜0.4 g) as pale beige thick oil.
(28) MS (ESI, pos): m/z 1369 [M+H].sup.+, m/z 1391 [M+Na].sup.+
(29) ALG-001 (1) (280 mg, 0.205 mmol) was dissolved in glacial acetic acid (20 mL), 20% Pd(OH).sub.2 and charcoal catalyst (60 mg) was added, and the mixture was hydrogenated under 40-45 psi at room temperature overnight. Filtration followed by rotary evaporation gave ALG-DEBN (2) (˜260 mg, with traces of acetic acid) as wine red colored thick oil.
(30) MS (ESI, pos): m/z 1007 [M+H].sup.+, m/z 1029 [M+Na].sup.+
(31) ALG-DEBN (2) (70 mg) was dissolved in 2:1 MeOH/dichloromethane (15 mL) at ambient temperature. Then 0.5 g of cleaned Amberlyst-15 resin was added, and the mixture was gently stirred overnight. The resin was then separated by filtration and washed with hexane (10 mL), tetrahydrofuran (10 mL), and methanol (10 mL), successively. This amine-bound resin was transferred to 4 M ammonia methanolic solution (20 mL) and was gently stirred for 50 min. Tetrahydrofuran (10 mL) was added, to dissolve all of the deprotected product. The resin was then removed by filtration, and the solution was evaporated, yielding ALG-DEBN-DEBOC (3) (37 mg).
(32) MS (ESI, pos): m/z 909 [M+H].sup.+, m/z 931 [M+Na].sup.+
(33) A suspension of 40 mg ALG-DEBN-DEBOC (3) in 6:1 isopropanol-water (v/v, 7 mL) was reacted with a solution of 1,4-phenylendiisothiocyanate (4.1 equiv.) in chloroform (2.5 mL). The reactants were stirred at room temperature for 30 minutes, when a sample was withdrawn for MS analysis. Peaks corresponding to the expected mass (1100) were observed. About 95 mg of a light brown solid was isolated after removal of the volatiles under vacuum. The light brown solid was suspended in acetonitrile, and the mixture heated under reflux for 15 min. The mixture was cooled down to room temperature, followed by isolation of 26 mg of ALG-DD-NCS (4) as brown solid by filtration.
Example 3—Synthesis of a Symmetric 3,2-HOPO Containing Chelator
(34) ##STR00014## ##STR00015## ##STR00016##
(35) Sodium hydride (60.1 g as 60% dispersion in mineral oil, 1.5 mol, 5 eq.) was charged in a flask and tetrahydrofuran (1 L) was added. Dimethyl malonate (172 mL, 1.5 mol, 5 eq.) was added drop-wise over 1.5 h; the temperature of the reaction mixture was kept below +10° C. The reaction mixture was then diluted with tetrahydrofuran (400 mL). A solution of 4-nitrobenzyl bromide (65.0 g, 0.3 mol, 1 eq.) in tetrahydrofuran (170 mL) was slowly added over 30 min to the above-prepared mixture under vigorous shaking. After 30 min of stirring at 0° C., the reaction mixture was poured into brine (1 L saturated NaCl solution) and left stirring overnight at ambient temperature, yielding a white precipitate. The mixture was then diluted with methyl tert-butyl ether, and the precipitate was filtered off and dissolved in hot ethanol. The ethanolic mixture was filtrated, and the filtrate was concentrated to the small volume, from which 24.8 g (31%) of dimethyl (4-nitrobenzyl)propanedioate (1) precipitated as a white, crystalline solid.
(36) LC Purity: >90% (254 nm)
(37) MS (APCI pos) m/z 285.1 [M+NH.sub.4].sup.+
(38) Compound 1 (24.8 g, 92.7 mmol) was dissolved in tetrahydrofuran (230 mL) and added borane-dimethyl sulfide complex (28.5 mL, 301 mmol, 3.3 eq.). The reaction mixture was refluxed for 24 h, and then allowed to stand at ambient temperature overnight. Methanol (250 mL) was added at 0° C., poured into brine (600 mL) and extracted with ethyl acetate. The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was co-evaporated with methanol. The crude product was purified by flash chromatography, giving 14.5 g (74%) 2-(4-nitrobenzyl)propane-1,3-diol (2) as a yellow oil.
(39) LC Purity: >92% (254 nm)
(40) Compound 2 (10.0 g, 47.3 mmol) was dissolved in dichloromethane (100 mL) and added triethylamine (14.5 mL, 104 mmol, 2 eq.). The reaction mixture was cooled in an ice-bath and methanesulfonyl chloride (8.0 mL, 104 mmol, 2 eq.) was added in portions. The final mixture was allowed to reach ambient temperature overnight. Additional triethylamine (1.5 mL) and methanesulfonyl chloride (0.8 mL) were then added, and stirring was continued for 30 min. The reaction mixture was then diluted with dichloromethane, and the formed precipitate was filtered off. The dichloromethane filtrate was washed with saturated aqueous NaHCO.sub.3 solution, 0.5 M aq. HCl and brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo, giving 14.5 g (83%) 2-(4-nitrobenzyl)propane-1,3-diyl dimethanesulfonate (3) as an orange oil, which was used in the next step without additional purification.
(41) LC Purity: >87% (254 nm)
(42) MS (APCI pos) m/z 385.3 [M+NH.sub.4].sup.+
(43) Compound 3 (14.5 g, 39.5 mmol) was dissolved in methyl ethyl ketone (100 mL), sodium iodide (16.0 g, 107 mmol, 2.7 eq.) was added, and the mixture was heated at 95° C. for 1 h. The resulting white precipitate was filtered off and washed with methyl ethyl ketone, and the filtrate was concentrated in vacuo. The crude product was triturated from methyl tert-butyl ether and purified by flash chromatography, giving 10.6 g (63%) 1-[3-iodo-2-(iodomethyl)propyl]-4-nitrobenzene (4) as a yellow solid.
(44) LC Purity: >90% (254 nm)
(45) MS (APCI pos) m/z 431.1 [M+H].sup.+
(46) Diethylene triamine (10.8 mL, 100 mmol) and triethylamine (42 mL, 300 mmol, 3 eq.) were dissolved in tetrahydrofuran (500 mL) and cooled in ice-bath. A solution of Boc-ON (49.5 g, 200 mmol, 2 eq.) in tetrahydrofuran (190 mL) was then added drop-wise over 2.5 h. The reaction mixture was stirred at 0° C. for an additional 1 h, before it was allowed to reach ambient temperature over night. The reaction mixture was then concentrated in vacuo. The residue was dissolved in dichloromethane and washed with 1 M aq. NaOH. The organic phase was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by flash chromatography, giving 20.0 g (66%) di-tert-butyl (iminodiethane-2,1-diyl)biscarbamate (5) as a yellow viscous oil.
(47) MS (APCI pos) m/z 304.3 [M+H].sup.+
(48) Compound 5 (26.0 g, 86 mmol, 4 eq.) was dissolved in dry toluene (40 mL) and added N-ethyl morpholine (5.4 mL, 42 mmol, 2 eq.). Compound 4 (9.2 g, 21 mmol) was dissolved in dry toluene (35 mL) and added to the reaction mixture. The mixture was incubated in a pressure reactor at 105° C. for 5 days. The reaction mixture was then cooled down to ambient temperature, diluted with ethyl acetate and washed with saturated aqueous NaHCO.sub.3 solution. The combined organic extracts were washed with water and brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product was purified by flash chromatography, yielding 11.4 g (70%) of compound 6 as a yellow solid foam.
(49) LC Purity: >96% (210 nm)
(50) MS (APCI pos) m/z 782.8, [M+H].sup.+; 682.7 [M+H-Boc].sup.+; 582.6 [M+H-2Boc].sup.+; 382.1 [M+H-4Boc].sup.+
(51) Compound 6 (2.0 g, 2.6 mmol) was dissolved in dioxane (16 mL), and a 9 M solution of HCl in dioxane (22 mL) was added. The reaction mixture was stirred at ambient temperature overnight, and then concentrated to dryness in vacuo, giving 1.9 g of the HCl salt of compound 7 as a beige solid.
(52) MS (APCI pos) m/z 382.5 [M+H].sup.+
(53) Compound 7 (1.9 g, 2.6 mmol) was added DMPU (1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone) (9.5 mL) and 3-(benzyloxy)-1-methyl-4[(2-thioxo-1,3-thiazolidin-3-yl)carbonyl]pyridine-2-(1H)-one (3.7 g, 10.3 mmol, 4 eq.). A solution of DBU (2.3 mL, 15.5 mmol, 6 eq.) in DMPU (4.7 mL) was then added slowly over 40 min. Stirring was continued overnight at ambient temperature. The reaction mixture was then diluted with dichloromethane and washed with saturated aqueous NaHCO.sub.3 solution. The combined organic extracts were washed with water and semi-saturated brine, then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product was purified by flash chromatography to obtain 4.0 g of compound 8 as yellow oil that was used in the next step without additional purification.
(54) MS (APCI pos) m/z 1347.7 [M+H].sup.+
(55) Compound 8 (4.0 g, 2.6 mmol) was dissolved in acetic acid (200 mL) and Pd(OH).sub.2 (20% w/w on C, 50% wetted) (800 mg, 10% w/w) was added. The reaction mixture was stirred in a Parr pressure reactor at 30 bar hydrogen pressure at ambient temperature overnight. The reaction mixture was then filtered through a pad of Celite®, and the filtrate was concentrated in vacuo. The crude product was purified by flash chromatography, giving compound 9 as a beige foamy solid.
(56) Compound 9 (996 mg) was dissolved in acetic acid (5.5 mL), followed by addition of 6 M aqueous HCl (2.5 mL) and 5% Pd/C (99 mg, 10% w/w). The reaction mixture was stirred in a pressure reactor at 8 bar hydrogen pressure overnight, and then filtered through a pad of Celite®. The filtrate was concentrated in vacuo and co-evaporated with toluene and methanol. The residue was dissolved in methanol and precipitated by addition of diethyl ether. The formed beige precipitate was filtered off and dried in vacuo overnight, yielding 700 mg of the HCl salt of compound 10, ALG1005-38.
(57) MS (APCI pos) m/z 956.7 [M+H].sup.+
Example 4—Synthesis of an Alternative Symmetric 3,2-HOPO Containing Chelator
(58) ##STR00017## ##STR00018##
(59) Boc anhydride (24.4 g, 119 mmol) was added in portions to a solution of imidazole (8.0 g, 117 mmol) in 50 mL dichloromethane at room temperature. The reaction mixture was stirred for one hour. The reaction mixture was washed twice with 50 mL water, dried over Na.sub.2SO.sub.4, filtered and reduced in vacuo. The residue was dissolved in 25 mL toluene and tris(2-aminoethyl)amine (8.19 g, 56 mmol) was added. The reaction mixture was stirred for 2 hours at 60° C. and reduced in vacuo. The residue was dissolved in 125 mL dichloromethane and washed with water (4 times with 50 mL), dried over Na.sub.2SO.sub.4, filtered and reduced in vacuo. Dry flash chromatography (0-15% methanol in dichloromethane with 1% triethylamine) yielded di-tert-butyl (((2-aminoethyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate (1) (13.51 g, 39.0 mmol, 70%) as a thick colorless oil.
(60) Spectroscopic data was in accordance with data reported by Frullano et al. (Chem. Eur. J. 2004, 10, 5205-17).
(61) N-Cbz-β-alanine (8.04 g, 36 mmol), 4-(dimethylamino)pyridine (4.40 g, 36 mmol) and EDC.HCl (6.90 g, 36 mmol) was dissolved in 25 mL tetrahydrofuran and stirred for 10 minutes before Compound 1 (10.4 g, 30 mmol) dissolved in 75 mL tetrahydrofuran was added. The reaction mixture was stirred for 4 hours and reduced in vacuo. Purification by dry flash chromatography (0-40% tetrahydrofuran in dichloromethane) yielded di-tert-butyl (((2-(3-(Cbz-amino)propanamido)ethyl)azanediyl)bis(ethane-2,1-diyl)dicarbamate (2) (15.11 g, 27.4 mmol, 91%) as a very viscous slightly yellow oil.
(62) .sup.1H-NMR (300 MHz, CDCl.sub.3): 1.43 (s, 18H), 2.42-2.56 (m, 8H), 3.04-3.17 (m, 4H), 3.19-3.32 (m, 2H), 3.41-3.57 (m, 2H), 5.08 (s, 2H), 5.22 (bs, 2H), 5.83 (bs, 1H), 7.23 (bs, 1H), 7.26-7.43 (m, 5H)
(63) MS (ESI, pos): m/z 574.3[M+Na].sup.+
(64) di-tert-butyl (((2-(3-(Cbz-amino)propanamido)ethyl)azanediyl)bis(ethane-2,1-diyl)dicarbamate (10.30 g, 18.7 mmol) was dissolved in 100 mL methanol and acetyl chloride (25 mL, 0.35 mol) was added drop wise. The reaction mixture was stirred for one hour at ambient temperature. The reaction mixture was reduced to approximately ⅓ of the volume in vacuo before 100 mL ether was added, leading to precipitation of a colorless solid. The solids were filtrated, washed with ether and dried in vacuo, giving benzyl (3-((2-(bis(2-aminoethyl)amino)ethyl)amino)-3-oxpropyl)carbamate tri-hydrochloride (3) (8.70 g, 18.7 mmol, ˜100%) as a colorless solid.
(65) .sup.1H-NMR (300 MHz, MeOD): 3.19-3.28 (m, 2H), 3.38-3.62 (m, 14H), 5.10 (s, 2H), 7.26-7.39 (m, 5H)
(66) MS (ESI, pos): m/z 352.2[M+H].sup.+
(67) Triethylamine (6.75 mL, 48.4 mmol) was added to a suspension of Compound 3 (10.56 g, 11.4 mmol) in 320 mL tetrahydrofuran and 320 mL N,N-dimethylformamide at room temperature. N-boc-2-aminoacetaldehyde (16.0 g, 100.5 mmol) and sodium triacetoxyborohydride (32.00 g, 151 mmol) was added. The reaction mixture was stirred for 20 hours. 200 mL brine and 500 mL chloroform was added, the phases were separated, and the aqueous phase was extracted with 3×100 mL chloroform. The combined organic phases were washed with 100 mL brine, dried over Na.sub.2SO.sub.4, filtered and reduced in vacuo. Flash chromatography (0-10% methanol in dichloromethane) yielded tetra-tert-butyl (((((2-3-Cbz-aminopropanamido)ethyl)azanediyl)bis(ethane-2,1-diyl))bis(azanetriyl)tetrakis(ethane-2,1-diyl)tetracarbamate (4) (3.40 g, 3.7 mmol, 32% as a yellow solid.
(68) MS (ESI, pos): m/z 946.7[M+Na].sup.+
(69) Compound 4 (3.40 g, 3.7 mmol) was dissolved in 60 mL methanol and acetyl chloride (12 mL, 0.17 mol) was added drop wise. The reaction was stirred at ambient temperature for one hour. The reaction mixture was reduced to approximately 10 mL in vacuo and 50 mL acetonitrile was added, leading to precipitation. The solid was filtered, washed with acetonitrile and dried in vacuo, giving benzyl (3-((2-(bis(2-aminoethyl)amino)ethyl)amino)ethyl)amino)-3-oxopropyl)carbamate hepta-hydrochloride (5) (2.88 g, 3.7 mmol, ˜100%) as a colorless solid.
(70) .sup.1H-NMR (300 MHz, MeOD): 2.44-2.61 (m, 2H), 2.68-3.00 (m, 4H), 3.08-3.40 (m, 16H), 3.44-3.52 (m, 4H), 3.54-3.77 (m, 6H), 5.12 (s, 2H), 7.30-7.47 (m, 5H)
(71) MS (ESI, pos): m/z 524.5[M+H].sup.+
(72) Triethylamine (2.52 mL, 18.1 mmol) and 4-(dimethylamino)pyridine (12 mg, cat.) was added to a suspension of Compound 5 (0.81 g, 1.04 mmol) and 3-(benzyloxy)-1-methyl-4-(2-thioxothiazolidine-3-carbonyl)pyridine-2(1H)-one (1.50 g, 4.16 mmol) in 180 mL Dichloromethane. The reaction mixture was stirred over night and reduced in vacuo. Flash chromatography (0-10% methanol in dichloromethane) yielded benzyl (1-(3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-5-(2-(3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)-8-(2-(bis(2-(3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)ethyl)-1,12-dioxo-2,5,8,11-tetraazatetradecan-14-yl)carbamate (6) (673 mg, 0.45 mmol, 43% as a light brown solid.
(73) .sup.1H-NMR (300 MHz, CDCl.sub.3): 2.10-2.40 (m, 20H), 3.02-3.21 (m, 10H), 3.32-3.46 (m, 2H), 3.50 (s, 12H), 4.99 (s, 2H), 5.25 (s, 8H), 5.95 (bs, 1H), 6.62 (d, 7.17 Hz, 4H), 7.01 (d, 7.17 Hz, 4H), 7.15-7.40 (m, 25H), 7.89 (bs, 4H)
(74) .sup.13C-NMR (75 MHz, CDCl.sub.3): 11.48, 35.71, 37.18, 37.35, 37.50, 37.66, 46.18, 51.55, 52.15, 52.82, 53.28, 66.34, 74.64, 74.75, 77.43, 104.63, 127.90, 127.94, 128.40, 128.65, 128.75, 128.94, 130.81, 132.32, 136.27, 136.76, 146.19, 156.45, 159.51, 163.30, 171.31
(75) MS (ESI, pos): m/z 766.9[M+2Na].sup.2+
(76) Compound 6 was dissolved and debenzylated essentially as described in Example 2, to yield Bb-1-HOPO-1-DEBN (7).
Example 5—Chelation Experiments with ALG-DD-NCS
(77) A reaction solution was prepared by dissolving solid ALG-DD-NCS (4 in Example 2) in DMSO (Biotech grade solvent 99.8%) to 1 mg/mL.
(78) Chelation reactions with different metals were conducted by mixing the 1 mg/mL reaction solution with selected metal solutions, using a ligand-to-metal ratio of 3:2. Metal ions for testing came from the following: .sup.232Th-solution 2% HNO.sub.3 (Perkin Elmer Pure Plus), Fe.sup.3+-solution in 2% HNO.sub.3 (Perkin Elmer Pure Plus), and InCl.sub.3 (anhydrous powder 99.999+%, Aldrich) was dissolved in 0.01 M HCl. The reagents were allowed to react for 15 minutes at room temperature, before injecting 5 μL of the reaction mixture on a LC-MS for analysis.
(79) The results showed that metal-ALG-DD-NCS complexes were formed quantitatively for the metals .sup.232Th.sup.4+, Fe.sup.3+ and In.sup.3+. LC-MS chromatograms and spectra of the .sup.232Th-ALG-DD-NCS, Fe-ALG-DD-NCS and In-ALG-DD-NCS complexes are shown in
(80) The LC-MS conditions were as follows: separation was done on a 1.7 μm, 2.1×50 mm Acquity UPLC BEH C18 column, at 50° C., using 0.05% formic acid in H.sub.2O as mobile phase A and 0.05% formic acid in acetonitrile as mobile phase B. The gradient composition and flow are shown in Table 3.
(81) TABLE-US-00003 TABLE 3 Time Flow (min) (mL/min) % A % B 0.0 0.6 95 5 2.0 0.6 95 5 9.0 0.8 40 60 9.1 0.8 10 90 10.0 0.8 10 90 10.1 0.8 95 5 11.4 0.8 95 5 11.5 0.6 95 5 12.0 0.6 95 5
(82) Mass spectroscopic analysis was performed on a Xevo-ToF-1instrument, using an acquisition mass range of ˜450-1950 Da, a molarity ES+, a 3.0 kV capillary, scanning over 1.000 sec with a cone voltage of 20 V.
Example 6—Chelation Experiments with ALG1005-38
(83) A stock solution was prepared by dissolving ALG1005-38 (10 in Example 3) in MeOH (LC-MS grade) to 10 mg/mL. The 10 mg/mL stock solution was diluted with MeOH and 0.5 M NaOAc-buffer (pH 5.5) in the proportions 1:8:1, to obtain a 1 mg/mL ALG1005-38 reaction solution. Chelation reactions with different metals were conducted by mixing the 1 mg/mL reaction solution with the selected metals, as specified in Example 5 and in addition GaCl.sub.3 (anhydrous beads 99.99%, Aldrich) dissolved in 0.01 M HCl, giving ligand-to-metal ratios from 3:1 to 1:2. The reagents were allowed to react for 10 minutes at room temperature. Finally, the reaction mixtures were diluted 10-fold with formic acid (0.1%) before injecting 5 μL on a LC-MS for analysis. The LC-MS separation and analysis was performed as described in Example 5, except that the cone voltage was 35 V.
(84) The results showed that metal-ALG1005-38 complexes were formed in significant amounts for metals like .sup.232Th.sup.4+, Fe.sup.3+, In.sup.3+ and Ga.sup.3+. LC-MS chromatograms and spectra of the .sup.232Th-ALG1005-38, Fe-ALG1005-38, In-ALG1005-38 and Ga-ALG1005-38 complexes are shown in
Example 7—Chelation Experiment with Bb-1-HOPO-1-DEBN
(85) A stock solution was prepared by dissolving Bb-1-HOPO-1-DEBN (made in Example 4) in DMSO (Biotech grade solvent 99.8%) to 1 mg/mL. A chelation experiment was conducted by mixing the 1 mg/mL solution with .sup.232Th 0.01 M HCl (Perkin Elmer) as specified in Example 5. The LC-MS separation and analysis was performed as described in Example 6. The results showed that the expected .sup.232Th-Bb-1-HOPO-1-DEBN complex was rapidly formed,
Example 8—Conjugation of ALG-DD-NCS to Trastuzumab, Labeling of the Conjugate with Thorium-227, and Confirming Retained Target Binding
(86) Conjugation
(87) Pharmaceutical grade of the monoclonal antibody trastuzumab (Herceptin®, Roche) was used. The antibody was buffer exchanged into 0.9% NaCl to a concentration of 6.3 mg/mL. A stock solution of ALG-DD-NCS was prepared in DMSO, containing 1 mg/mL (disregarding impurities). DMSO stock solution was added to the antibody solution corresponding to a chelator to antibody molar ratio of 6.5:1, or less. A 0.07 M borax solution was added to the reaction solution to give pH 9 following incubation at 37° C. over night. The resulting conjugate was purified and buffer exchanged on an Amicon Ultra-4 (30 k MWCO) centrifugal filter unit. Aliquots of 1 mg in 100 μL 0.9% NaCl were frozen and stored at −18° C. until use.
(88) Labeling
(89) 100 μL 0.5 M NaOAc-buffer (pH 5.5) was added to one vial of 1 mg ALG-DD-NCS-trastuzumab conjugate to obtain a pH suitable for labeling. This solution was mixed with 4.2-16.4 MBq .sup.227Th in 0.01 M HCl, and the pH was checked with pH paper. The Eppendorf tube cap was wrapped in aluminum foil and the tube was placed in a thermomixer at room temperature for 15 minutes with gentle mixing.
(90) The tube was further incubated at room temperature for 5 minutes after adding 10 μL saturated DTPA in MF-H.sub.2O, intended to capture remaining free thorium-227.
(91) The result was purified on a NAP-5 column using PBS to elute the labeled ALG-DD-NCS-trastuzumab conjugate, leaving the majority of free radionuclides (thorium-227 and daughter nuclides) and DTPA-thorium chelate on the column.
(92) The column and eluted fractions were measured on a HPGe-detector GEM(15) to determine reaction yields and specific activity of the product. The product fraction was sterile filtered and stored at 4° C. over night.
(93) A second NAP-5 column purification of the product fraction was performed before use.
(94) Determining the Immunoreactive Fraction
(95) SK-OV-3 cells (ATCC) were cultured under standard conditions. Fixed SK-OV-3 cells were prepared and examined by flow cytometry before use to confirm the presence of HER2 on the surface (data not shown). Regular medium was prepared with McCoy's 5A medium (GIBCO) added 10% FBS (PAA) and 1% Pen/Strep (BioChrom). Incubation of cells were performed in a CO.sub.2-incubator at 37° C. and 5% CO.sub.2, using T25 (25 cm.sup.2) and T75 (75 cm.sup.2) cell flasks.
(96) Each experiment was performed in duplicate. Fixed SK-OV-3 cells were suspended in PBS and transferred to Eppendorf tubes (10 million cells/tube). Non-conjugated trastuzumab (Herceptin®, 10 μL from the stock solution) was added to the two reference tubes, and the blocking reaction was conducted at 37° C. for 30 minutes. Equal amounts of .sup.227Th-ALG-DD-NCS-trastuzumab (ca 500 cpm) were added to each tube, followed by incubation at 37° C. for 2.5 hours. The samples were diluted with PBS, followed by centrifugation and transfer of half of the supernatant to new Eppendorf tubes. All tubes were measured for 5 minutes on a Wizard gamma counter and the amount of thorium-227 determined by applying a .sup.227Th-protocol.
(97) The binding in % was calculated as: 100*[(P+½S)−(½S)]/[(P+½S)+(½S)], where P and S are the activities measured in the cell pellet and the supernatant, respectively. The calculation is performed on the un-blocked samples to obtain total binding (%) and the blocked samples to obtain unspecific binding (%), and the IRF is calculated as: IRF (%)=total binding (%)−unspecific binding (%).
(98) The data from this quality control, summarized in Table 4, show that .sup.227Th-ALG-DD-NCS-trastuzumab binds the target cells.
(99) TABLE-US-00004 TABLE 4 Results for the quality control performed on .sup.227Th-ALG-DD-NCS- trastuzumab batches A and B, subsequently used in Examples 9 and 10 respectively. Average Specific Average total Average Yield Recovery activity IRF binding unspecific (%).sup.i (%).sup.ii (Bq/μg).sup.iii (%).sup.iv (%).sup.iv binding (%).sup.iv Batch A 60 95 10 166 65 84 20 Batch B 64 92 2 700 60 74 15 .sup.iYield (%) is calculated after NAP-5 purification of the reaction mixture. .sup.iiRecovery (%) is calculated after a new NAP-5 purification of the product fraction. .sup.iiiSpecific activity (Bq/μg) is calculated using the amount of .sup.227Th (Bq) found in the product fraction divided on the amount of ALG-DD-NCS-trastuzumab (μg) used in the reaction mixture. .sup.ivIRF is estimated according to standard procedures.
Example 9—In Vitro Stability and Efficacy Studies of Thorium-227 Labeled ALG-DD-NCS-Trastuzumab
(100) Assessment of Binding Over Time
(101) The stability of binding to SK-OV-3 cells was evaluated for the .sup.227Th-ALG-DD-NCS-trastuzumab construct by measuring the amount of .sup.227Th-activity associated with SK-OV-3 cell pellets over 7 days. The results were compared to data obtained after incubation with free .sup.227Th.
(102) SK-OV-3 cells were cultured as described in Example 8 were initially treated with 700 kBq .sup.227Th-ALG-DD-NCS-trastuzumab or 700 kBq free .sup.227Th, resulting in respectively 77 kBq (11%) and 7 kBq (1%) .sup.227Th-activity on the cell pellet after 1 hour incubation (HPGe-detector GEM(50)). The .sup.227Th-activity on SK-OV-3 cells was measured daily using the Wizard gamma counter (.sup.227Th-protocol). The measured radioactivity was corrected for decay. The results presented in
(103) Assessment of Cytotoxicity
(104) The effect on tumor cell growth was investigated in two complementary assays. In the first experiment, the metabolic activity in cells was measured using a luminescent reagent binding to ATP molecules. Thus, a decreased luminescence signal indicates loss of metabolic activity. In the second experiment, the number of colonies formed was counted.
(105) Luminescence was measured daily for 9 days, and the results from exposure to .sup.227Th-ALG-DD-NCS-trastuzumab were compared to the luminescence of SK-OV-3 cells exposed to trastuzumab and free .sup.227Th, respectively. SK-OV-3 cells growing in regular medium were used as control.
(106) On day 0, 35 mL of 500 000 SK-OV-3 cells/mL in regular medium were transferred to 4 T75 cell flasks. These cell flasks were used to prepare the following sample solutions: (1) Regular medium, (2) 20 μg/mL trastuzumab, (3) 20 kBq/mL .sup.227Th-ALG-DD-NCS-trastuzumab (10166 Bq/μg), (4) 20 kBq/mL free .sup.227Th. The flasks were incubated in a CO.sub.2-incubator for 1 hour. The supernatants were discarded, and the cells were trypsinized with 0.25% Trypsin-EDTA solution, washed with regular medium and centrifuged. Cell pellets were suspended in fresh regular medium and measured on a Wizard gamma counter (.sup.227Th-protocol). Each incubation solution was distributed into 7 new T25 cell flasks, and incubated in a CO.sub.2-incubator.
(107) On days 1 and 2 the supernatants from one flask of each of the four sample solutions were discarded. The cell pellets were prepared as on day 0 and radioactivity measured on a Wizard gamma counter (.sup.227Th-protocol) and on a HPGe-detector GEM(50). All samples were diluted 4-fold in regular medium, and 5×100 μL of each cell suspension were transferred into 5 wells of a View plate-96. Five wells were added 100 μL regular medium (without cells) for measurements of background luminescence. Finally, 100 μL of Cell Titer-Glo Reagent were added to each well, and the solutions were mixed on an orbital shaker to induce lysis. The luminescence signal was allowed to stabilize at room temperature for 10 minutes, then luminescence was measured three times on an Envision multiple plate reader, using the LUM-single program.
(108) On day 3 cells were prepared and measurements on a Wizard gamma counter and Envision multiple plate reader, as described for day 1-2. Then, each cell suspension was diluted due to dense cell growth; ¾ cell suspension was transferred into a new T75 cell flask, added regular medium and incubated in a CO.sub.2-incubator over night.
(109) On each of days 4-9 the four T75 cell flasks were trypsinized with 0.25% Trypsin-EDTA solution, washed with regular medium and centrifuged. Cell pellets were diluted 4-fold in regular medium, and 100 μL portions taken out and measurements performed as above. Then, ¾ of the cell suspensions were diluted in regular medium, transferred into new T75 cell flasks and incubated in a CO.sub.2-incubator over night.
(110)
(111) A clonogenic assay was performed to evaluate cell colony growth after exposing single cells to potentially cytotoxic conditions. SK-OV-3 cells were incubated in different concentrations of .sup.227Th-ALG-DD-NCS-trastuzumab, trastuzumab or free .sup.227Th for 1 hour, followed by incubation for 12 days to induce colony growth.
(112) Preparation of cells was done as above, and in each sample 12000 cells in 5 mL regular medium was transferred into a T25 flask. The effect of 3 amounts of each reagent was investigated, for .sup.227Th-ALG-DD-NCS-trastuzumab and .sup.227Th, 5, 10 and 20 kBq/mL, and for trastuzumab 5, 10 and 20 μg/mL, (final concentrations) respectively. One control flask was prepared. All 10 T25 cell flasks were incubated in a CO.sub.2-incubator for 1 hour. The supernatants were discarded, and the cells were washed with regular medium, trypsinized with 0.25% Trypsin-EDTA solution, washed with regular medium and centrifuged. Cell pellets were suspended in regular medium, and each of the ten cell suspensions were divided on 6 new T25 cell flasks; 3 cell flasks à 1000 cells and 3 cell flasks à 3000 cells. The resulting 60 T25 cell flasks were incubated in a CO.sub.2-incubator until colonies reached a visible size (ca 50 cells/colony). After incubation for 12 days, the medium was removed. Cells were washed with PBS, fixed with ethanol, stained with 0.25% Trypan Blue in PBS, washed with tap water and finally dried at 45° C. over night. Colonies were counted manually, and the average number of colonies in each incubation solution was plotted graphically.
(113) The results shown in
Example 10—In Vivo Tumor Targeting of Thorium-227 Labeled ALG-DD-NCS-Trastuzumab
(114) 100 μL (15 kBq) of sterile filtered .sup.227Th-ALG-DD-NCS-trastuzumab (2700 Bq/μg) was injected into the lateral tail vein of ten Balb/c nude mice bearing SK-OV-3 xenografts. Five mice were sacrificed after 24 hours and the other five after 4 days, and organs of interest were excised and weighed. All samples and 3 standard solutions containing 10% ID/g were measured for 5 minutes on a Wizard gamma counter (.sup.227Th-protocol). The results were expressed as % injected dose per gram tissue (% ID/g).
(115) The results shown in
(116) The uptake in skull and femur also increased somewhat between 24 hours and 4 days, but these values were generally low (2.8-3.2%, 24 h), indicating very little free .sup.227Th circulating in the system, and thus suggesting a high degree of stability of the .sup.227Th-ALG-DD-NCS-trastuzumab complex. Further support for this conclusion is lended by the high likelihood that a fraction of the activity measured in skull and femur is caused by uptake of the daughter nuclide .sup.223Ra. Some of this .sup.223Ra may be detected within the gated window specified for .sup.227Th-measurements on a Wizard gamma counter, hence the calculated % ID/g-values for skull and femur may be overestimates of the amount of .sup.227Th-ALG-DD-NCS-trastuzumab accumulated in these tissues.
Example 11—Preparation of a Chelating Moiety
Preparation of 3-benzyloxy-1-methyl-4-(2-thioxothiazolidin-1-yl)carbonyl-2(1H)-pyridinone (Structure A)
Step 1)—1-Methyl-3-hydroxy-2(1H)-pyridinone
(117) 3-hydroxy-2(1H)-pyridinone (34.44 g, 0.31 mol) and iodomethane (75 g, 0.53 mol) are placed in an 80 mL capped Teflon container, and heated to 150° C. for about 48-60 hours in a Parr bomb. The cooled bomb is opened and the excess iodomethane decanted. The resultant thick dark oil is mixed with sodium sulfite (64 g, 0.5 mol) and dissolved in 300 mL water to form a pale brown solution. The solution is neutralized to pH 7-8 and filtered to remove insoluble impurities. The filtrate is then extracted with methylene chloride (4×100 mL). The combined extracts are then dried, applied to a flash silica gel plug (6 cm.×8 cm) and eluted with 4% methanol in methylene chloride. The solvent is removed to give the title compound (24.3 g, 62.6%) as colourless crystals.
Step 2_—4-Carboxy-1-methyl-3-hydroxy-2(1H)-pyridinone
(118) 1-Methyl-3-hydroxy-2(1H)-pyridinone (1) (6.25 g, 50 mmol) is mixed with anhydrous potassium carbonate (36 g, 0.26 mol) and vacuum dried. The mixture is then heated to 175-185° C. for 3 days in a Parr bomb under dry carbon dioxide gas (850 psi). The cooled bomb is opened and the resultant pale yellow solid dissolved in iced water and acidified with 6N HCl to produce a beige crystalline product.
Step 3—3-Benzyloxy-4-carboxy-1-methyl-2(1H)-pyridinone
(119) 4-Carboxy-1-methyl-3-hydroxy-2(1H)-pyridinone (6.8 g, 0.04 mol) is mixed with benzyl chloride (12.1 g, 0.088 mol) and anhydrous potassium carbonate (13.8 g, 0.1 mol) in anhydrous dimethyl-formamide (DMF) (120 mL). The mixture is heated in darkness under nitrogen to 75-80° C. for 16 hours. The resulting mixture is then filtered and the solvent evaporated to yield a dark oil. The oil is purified by application to a silica gel plug (6 cm.×8 cm) and elution with 4% methanol in methylene chloride resulting in 3-benzyloxy-4-benzyloxycarbonyl-1-methyl-2(1H)-pyridinone as a pale yellow, thick oil. This is taken up in methanol (50 mL) and a 6M NaOH solution (10 mL), and the mixture stirred at room temperature for 4 hours, followed by evaporation to dryness. The residue is dissolved in water (100 mL), and acidified with 6M HCl solution to pH 2 to give the title compound (9.3 g 88.7%), as a white crystalline product.
Step 4—3-benzyloxy-1-methyl-4-(2-thioxothiazolidin-1-yl)carbonyl-2(1H)-pyridinone
(120) To a solution of 3-benzyloxy-4-carboxy-1-methyl-2(1H)-pyridinone (1.05 g, 4 mmol), 2-mercaptothiazoline (0.50 g, 4.2 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP) in dry dichloromethane (50 mL), is added N,N′-dicyclohexylcarbodiimide (DCC) (0.86 g, 4.2 mmol). Alter stirring for 4 hours, the dicyclohexylurea (DCU) solids are removed by filtration. The yellow filtrate is then rotary evaporated to provide a yellow solid. Crystallization from isopropanol-methylene chloride gives the title compound (Structure A, 1.16 g, 80.4%) as bright yellow crystalline plates.
(121) ##STR00019##
Example 12—Preparation of 3,2-HOPO Precursor for Formula VIII
(122) Preparation of the Cyclic Salt (Structure 5) is Done According to the Following Scheme:
(123) ##STR00020##
(124) The cesium fluoride (10 mol %) assisted Michael reaction of 2,3-dihydroxypyridine (2,3-DHP) 1 with ethyl acrylate in refluxing acetonitrile gave the corresponding ester 2 in good yields. Subsequent O-benzylation of 2 using standard conditions K2COz3/acetonitrile/reflux) followed by reduction of the ester moiety (BH3.THF,rt) gave alcohol 3 in 90% yield after chromatography. Treatment of the alcohol 3 with methanesulfonic anhydride in dichloromethane in the presence of triethylamine led directly to the formation of the desired cyclic salt 5 (˜85-90%) along with some of the intermediate mesylate 4 (˜10-15%), as determined by .sup.1H NMR spectral analysis. Complete conversion to the cyclic salt 5 could be achieved by stirring the crude product mixture from the mesylation in chloroform at room temperature. After trituration with hot ethyl acetate, the salt 5 was isolated as a pale white solid in 92% yield in high purity.
Example 13—Preparation of Compound of Formula VIII
(125) Salt 5 (0.2 g) is added to 1.2 equivalents of cyclene (1,4,7,10-tetraazacyclododecane) in the presence of triethylamine in acetonitrile (3 ml) and heated at 60° C. for 2 days under nitrogen. The reaction is then diluted with dichloromethane (50 ml) and washed with saturated NaHCO.sub.3 (50 ml). The aqueous layer is extracted once more with dichloromethane (25 ml). The combined organic extracts is dried with sodium sulphate, the solvent is removed in vacuo and the excess N-methylbenzylamine is removed via vacuum destillation to give VIII.
Example 14—Preparation of Compound of Formula IX
(126) To a solution of 3-benzyloxy-1-methyl-4-(2-thioxothiazolidin-1-yl)carbonyl-2(1H)-pyridinone in methylene chloride the corresponding functionalized N,N,N′,N′-tetrakis(2-aminoethyl)ethylenediamine (Structure B, Z being a protecting group) will be added. After stirring for four hours, the mixture is filtered and taken to dryness.
(127) ##STR00021##
(128) The appropriate benzyl-protected precursor product will be isolated from the reaction mixture on a flash silica column with propanol in methylene chloride. The final product with structure IX will be available after acidic deprotection of the hydroxyl groups.
Example 15
(129) Structure VIII will be conjugated to a targeting molecule using standard methods to those skilled in the art. For example will the N-hydroxysuccinimide (NHS) ester be prepared and conjugated to proteins and peptides using standard conditions and the resulting conjugated protein will be isolated using gel filtration.
Example 16
Thorium-227 Labeling of the Conjugated Protein
(130) A 10 mg/mL solution of the conjugated protein (having an octadentate ligand attached by means of a coupling moiety) will be made in a suitable buffer solution, eg. 0.5 M NaOAc-buffer (pH 5.5), and labeled with purified thorium-227 at 25° C. for 1 h followed by a purification step on a gel filtration column.