Compositions, methods of synthesis and use of carbohydrate targeted agents

Abstract

The invention provides D02S derivatives conjugated to monosaccharide ligands directly or through a linker and optionally chelated to a metal, wherein the D02S derivatives having the following structure: wherein R.sub.1, R.sub.2 are each independently OH or O-alkyl; R.sub.1 is a hydrogen, a linker, or a ligand; R.sub.3 is a linker and/or a ligand; and n is an integer from 1 to 10; the linker is an amino acid, a peptide, an amino alcohol, a polyethylylene glycol, an alkyl, an alkenyl, an alkynyl, an azide, an aromatic compound, a carboxylic acid, or an ester, the alkyl, alkenyl, or alkynyl is optionally substituted with an alkyl, a halogen, a nitro group, a hydroxyl group, an amino group, or a carboxyl group; the ligand is a GLUT1 targeting moiety.

Claims

1. A composition comprising a DO2S derivative conjugated to a monosaccharide ligand directly or through a linker and optionally chelated to a metal, wherein the DO2S derivative has the following structure: ##STR00006## wherein R.sub.1, and R.sub.2 are each independently OH; R.sub.1, and R.sub.3 comprise a ligand, optionally attached to the DO2S derivative via a linker; n is 1; the linker is an amino acid, a peptide, an amino alcohol, a polyethylene glycol, an alkyl, an alkenyl, an alkynyl, an azide, an aromatic compound, a carboxylic acid, or an ester, the alkyl, alkenyl, or alkynyl is optionally substituted with an alkyl, a halogen, a nitro group, a hydroxyl group, an amino group, or a carboxyl group; and the ligand is a GLUT1 targeting moiety, wherein the GLUT-1 targeting moiety is 2-deoxyglucose, a 12-diaminosugar, genistein, and/or scutellarin.

2. The composition of claim 1, wherein the linker comprises (CH2).sub.mX, wherein X is a hydroxyl, an amino, or a carboxyl group, and m is an integer from 1 to 10, and wherein the linker may be optionally substituted with an alkyl, a halogen, a nitro group, a hydroxyl group, an amino group, or a carboxyl group.

3. The composition of claim 1, wherein the DO2S derivative further comprises a radiometal.

4. The composition of claim 3, wherein the radiometal is .sup.68Ga or .sup.177Lu.

5. A method for synthesizing a composition comprising a DO2S derivative conjugated to a monosaccharide ligand directly or through a linker and optionally chelated to a metal, wherein the DO2S derivative has the following structure: ##STR00007## wherein R.sub.1, and R.sub.2 are each independently OH; R.sub.1, and R.sub.3 comprise a ligand, optionally attached to the DO2S derivative via a linker; n is 1; the linker is an amino acid, a peptide, an amino alcohol, a polyethylene glycol, an alkyl, an alkenyl, an alkynyl, an azide, an aromatic compound, a carboxylic acid, or an ester, the alkyl, alkenyl, or alkynyl is optionally substituted with an alkyl, a halogen, a nitro group, a hydroxyl group, an amino group, or a carboxyl group; and the ligand is a GLUT1 targeting moiety comprising a 2-deoxyglucose, a 12-diaminosugar, genistein, and/or scutellarin, the method comprising the following steps: providing the ligand bound to a branch linker at a proximal end of the branch linker, a distal end of the branch linker having two different reactive group ends; attaching a first free reactive group end of the branch linker to a solid support; deprotecting a second reactive group end of the branch linker; conjugating the deprotected second reactive group end of the branch linker to a chelator to form a chelator-linker ligand conjugate DO2S derivative, immobilized on the solid support; deprotecting functional carboxyl groups on the chelator; radiolabeling the DO2S derivative immobilized on the solid support with an isotope; and releasing the radiolabeled DO2S derivative from the solid support by cleavage of the branch linker from the solid support.

6. The composition of claim 1, wherein the DO2S derivative further comprises a paramagnetic substance, the paramagnetic substance selected from the group consisting of Gd, Mn, Cu, and Fe.

7. The composition of claim 3, wherein the radiometal is selected from the group consisting of a -emitter, a -emitter, a -emitter, and a /-emitter.

8. The composition of claim 3, wherein the radiometal is selected from the group consisting of .sup.45Ti, .sup.59Fe, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.89Sr, .sup.90Y, .sup.86Y, .sup.94mTc, .sup.99mTc, .sup.111In, .sup.149Pm, .sup.153Gd, .sup.153Sm, .sup.166Ho, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Bi, and .sup.225Ac.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 illustrates the structure of compound 1a

(2) FIG. 2 illustrates the ESI spectra of the t-Bu ester protected compound 1a before the final deprotection (a) and after final deprotection (b). Compound was analyzed using electrospray ionization mass spectroscopy (ES) in the presence of MeOH: 0.2% of formic acid. The ESI spectra showed mass peak corresponding to compound m/z=566.2 (calculated MW+H=565.37). The m/z=734.45 corresponds to compound 1a before the deprotection.

(3) FIG. 3 illustrates radio-TLC of compound 1a. Retention factor for radiolabeled Glucomedix and .sup.68GaCl.sub.3 were 0.9 and 0.1, respectively. Radio-TLC (Bioscan) analysis showed the radiochemical purity of tracer was >97%.

(4) FIG. 4 illustrates results of cellular uptake study of .sup.68Ga-labeled compound 1a.

(5) FIG. 5 illustrates results of cellular uptake study of .sup.177Lu-labeled compound 1a.

(6) FIG. 6 illustrates the structure of compound 1b.

(7) FIG. 7 illustrates a method of synthesis for compound 1b.

(8) FIG. 8 illustrates the ESI-MS spectra of compound 1b. m/z=593.62 for [M+H] and calculated for C.sub.24H.sub.43N.sub.5O.sub.12 [M+1]=595.5

(9) FIG. 9 illustrates the radio-TLC of compound 1b.

(10) FIG. 10 illustrates the structure of compound 2a

(11) FIG. 11 illustrates the ESI-MS spectra of compound 2a before (a) and after purification (b) shown below m/z=727.33 for [M+H] and calculated for C.sub.28H.sub.50N.sub.6O.sub.16 [M]=726.73;

(12) FIG. 12 illustrates the radio-TLC of compound 2a

(13) FIG. 13 illustrates the cellular accumulation of .sup.68Ga-2a.

(14) FIG. 14 illustrates the cellular accumulation and blocking of .sup.68Ga-2a

(15) FIG. 15 shows blocking of accumulation of .sup.68Ga-2a by glucose transporter inhibitors

(16) FIG. 16 illustrates the results of cellular uptake and blocking study of .sup.177Lu-2a.

(17) FIG. 17 illustrates the structure of compound 3

(18) FIG. 18 illustrates methods of synthesis of compound 3

(19) FIG. 19 illustrates loading efficiency

(20) FIG. 20 illustrates the radio-TLC of compound 3

(21) FIG. 21 illustrates the results of cellular accumulation of .sup.68Ga-3.

(22) FIG. 22 illustrates the structure of compound 4

(23) FIG. 23 illustrates of a method of synthesis of compound 4

(24) FIG. 24 illustrates the ESI-MS spectra of compound 4, m/z=720.73 for [M+H] and calculated for compound 4 m/z=719.2

(25) FIG. 25 illustrates the radio-TLC of compound 4

(26) FIG. 26 illustrates cellular accumulation of .sup.68Ga-4

(27) FIG. 27 illustrates cellular accumulation of .sup.177Lu-4

(28) FIG. 28. Illustrates results of biodistribution study of .sup.177Lu-4

(29) FIG. 29 illustrates general approach to the synthesis of DO2S-linker-carbohydrates.

(30) FIG. 30 illustrates an example of a method used for the synthesis of carbohydrate-DO2S-linker-carbohydrate by SPPS.

(31) FIG. 31. illustrates an example of method used for the synthesis of di-substituted DO2S-[carbohydrate]2 by SPPS.

(32) FIG. 32. illustrates structure of compound 5 and predicted physicochemical properties

(33) FIG. 33. illustrates the ESI-MS spectra of the t-Bu ester protected compound 5 purified by HPLC (C18 column)

(34) FIG. 34. illustrates the ESI-MS spectra of the t-Bu ester protected compound 5 after purification (precipitation with Et2O) and collecting of supernatant.

(35) FIG. 35. illustrates the ESI-MS spectra of the t-Bu ester protected compound 5 after purification (precipitation with Et2O) and collecting of pellet.

(36) FIG. 36. illustrates radio-HPLC chromatogram (UV/Vis detection: 220 nm 275 nm) of the deprotected compound 5.

(37) FIG. 37. illustrates cellular accumulation of 68Ga-5 in SKBr3 breast cancer cell line and results of blocking studies using cold GLUT-1 competitors (genistein, scutellarin, cytochalasin B).

(38) FIG. 38. illustrates concentration dependent accumulation studies of 68Ga-5 in SKBr3 breast cancer cell line.

(39) FIG. 39. illustrates an example of method used for the synthesis of DO2S-linker-[carbohydrate]-genistein compound 6

(40) FIG. 40. illustrates an example of method used for the synthesis of DO2S-linker-[carbohydrate]-compound 7a,b.

(41) FIG. 41. illustrates structure of compound 7a

(42) FIG. 42. illustrates the ESI-MS spectra of the compound 7a.

(43) FIG. 43. illustrates structure of compound 7b

(44) FIG. 44. illustrates the ESI-MS spectra of the compound 7b.

(45) FIG. 45. illustrates radio-HPLC chromatogram of compound 7b.

(46) FIG. 46. illustrates cellular accumulation of .sup.68Ga-7b in SKBr3 breast cancer cell line. Specificity of binding was confirmed in blocking studies using cold GLUT-1 competitors (glucose, cytochalasin B).

(47) FIG. 47. illustrates cellular accumulation of .sup.68Ga-7b, .sup.68Ga-1 and .sup.68Ga-copper catalyzed glucosamine conjugate in SKBr3 breast cancer cell line.

DETAILED DESCRIPTION

(48) Embodiments of the invention relate to chelator-based carbohydrate derivatives and method of their synthesis. Chelator-carbohydrate (DO2S-carbohydrate) maybe synthesized from the amino-sugar is directly conjugated to DO2S compound, or it can be coupled to DO2S through any type of the linker (DO2S-linker-carbohydrate).

(49) In accordance with some embodiments of the invention, synthesis of DO2S-carbohydrate can be performed manually on solid phase and using automated instrument, such as a peptide synthesizer.

(50) Resins used for the solid phase synthesis maybe selected from the group consisting of chloro- and bromofunctionalized (Merrifield, 4-bromomethylphenoxy)methyl polystyrene, 2-(4-bromomethylphenoxy)ethyl polystyrene, trityl, 2-chlortrityl chloride, NovaSyn TGT alcohol, NovaSyn TGT bromo), amino- and hydrazine functionalized (AM polystyrene and N-methyl aminomethylpolystyrene, NovaSyn TG amino, MBHA polystyrene, Rink Amide, Siber, amino trityl, sulfamyl-based, WeinrebAM, Fmoc-4-hydrazinobenzoyl, NOVAGel, alkylaminomethyl-indole, hydroxylamine Wang), hydroxyl functionalized (NovSyn hydroxyl, hydroxymethyl-phenyl, oxime), carboxy, aldehyde (benzyloxybenzaldehyde, FMPB AM, FMPB NovaGel, NOVAPEG FMBP, FMPE, DFPE, 3-formylindoyl)acetamidomethyl polystyreneFIA AM resin), enol functionalized (DHP HM resins), thiol functionalized (mercaptomethyl, 3-[4-tritylmercapto)phenylpropionyl AM resins), carbonate functionalized, alkenylcarbonyl functionalized.

(51) Linkers used for the solid phase synthesis of maybe selected from the group of amino acids, peptides, amino alcohols, polyethylylene glycols, alkanes, alkenes, alkynes, azide aromatic compounds, carbohydrates, carboxylic acids, esters, fosfororganic compounds, sulfonates.

(52) A targeting ligand may be selected from the group consisting of a carbohydrate, peptide, protein, antibody, nucleoside, nucleotide, heterocyclic compound, or alcohol.

(53) Preferred targeting ligands include carbohydrates, such as 2-deoxyglucose, 1amino-sugar, 2amino-sugar, 12-aminosugar, 2-amino-methylglycoside etc.

(54) The radiometal may be a transition metal ion or lanthanide series element. For example, it may be .sup.45Ti, .sup.59Fe, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.89Sr, .sup.90Y, .sup.99mTc, .sup.111In, .sup.153Gd, .sup.153Sm, .sup.166Ho, .sup.186Re, .sup.177Lu, .sup.188Re, .sup.211At, .sup.212Bi, .sup.225Ac.

(55) Generally, it is believed that virtually any -emitter, -emitter, -emitter, or /-emitter can be used in conjunction with embodiments of the invention. Preferred -emitters include .sup.211At, .sup.212Bi and .sup.223Ra. Preferred -emitters include .sup.90Y and .sup.225Ac. Preferred /-emitters include .sup.67Cu, .sup.89Sr, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.186Re and .sup.188Re. Preferred -emitters include .sup.62Cu, .sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.94mTc, .sup.99mTc and .sup.111In. It is also envisioned that paramagnetic substances, such as Gd, Mn, Cu or Fe can be chelated with DO2S derivatives for use in conjunction with embodiments of the present invention.

(56) Embodiments of the present invention provide compositions for tissue specific disease imaging and radiotherapy. The disease may be cardiovascular disease, infection, diabetes, or cancer. In a preferred embodiment, the disease is cancer. The cancer may be a solid tumor. In other specific embodiments, the tumor derives, either primarily or as a metastatic form, from cancers such as of the liver, prostate, pancreas, head and neck, breast, brain, colon, adenoid, oral, skin, lung, testes, ovaries, cervix, endometrium, bladder, stomach, and epithelium.

(57) Embodiments of the invention also provide kits for preparing a radiopharmaceutical preparation. The kit generally includes a sealed vial or bag, or any other kind of appropriate container, containing a predetermined quantity of DO2S-carbohydrate conjugate. The components of the kit may be in any appropriate form, such as in liquid, frozen or dry form. In preferred embodiments, the kit components may be provided in lyophilized form. The kit may also include an antioxidant and/or a scavenger. The antioxidant may be any known antioxidant but is preferably vitamin C. Scavengers may be DTPA, EDTA or DOTA.

EXAMPLES

(58) The following examples are provided to illustrate various aspects of the invention, and are not to be construed as limiting the scope of the invention in any manner.

Example 1

A. Synthesis of 1-N-[1,4,7,10-Tetraazacyclododecane-4,7,10-tetra(acetyl)]-2-amino-2-deoxyglucopyranoside (Compound 1a)

A.1. Direct Coupling of glucosamine hydrochloride to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS)

(59) Sodium methanolate (3eq.) in methanol was added to glucosamine hydrochloride (1eq. Sigma Aldrich) and stirred for 30 min at room temperature. Solvent was evaporated and free glucosamine was added to a solution of DOTA-NHS (1 eq., Macrocyclics) in ddH.sub.2O in phosphate buffer pH=7. Reaction was stirred at room temperature for 48 h, followed by evaporation of solvent under high vacuum. Product was purified by dialysis using Sep-Pak dialysis filters with MW cut off 500 and precipitated by addition of diethyl ether.

A2. Coupling of 1,3,4,6-tetra-O-acetyl--D-glucosamine hydrochloride to DOTA tris-t-butyl ester

Step a. Preactivation of DOTA Chelating Agent

(60) To activate the carboxyl groups, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(t-butyl acetate)-10-acetic acid (1eq.) was dissolved in 2 mL dimethylformamide (DMF), and then N-hydroxybenzotriazole (HOBT) and O-Benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU) coupling agents (4eq. each) were added in the presence of 4eq. of N,N-diisopropylamine (DIPEA). Reaction was left at room temperature for 30 min.

Step b. Coupling of Glucosamine Hydrochloride to Activated Chelating Agent

(61) Glucosaminehydrochloride (4eq) was dissolved in DMF in the presence of DIPEA (4eq) and added to the solution of pre-activated DOTA. Reaction was left stirring for 48 h at room temperature and was monitored by TLC (chloroform:methanol 1:10) and visualized using anisidine solution or dichlorofluoresceine. Conjugate was purified by extraction using CH.sub.2Cl.sub.2:H.sub.2O and organic fraction was collected. The tert-butyl ester protecting groups were removed in the presence of 30% TFA:CH.sub.2Cl.sub.2:H.sub.2O:TIS (950:250:250), and the product was dialyzed using Sep Pak in millipore water for 48 h at room temperature.

A.3. 68Ga-Radiolabeling of Compound 1a

(62) Compound 1a (1-100 ug) dissolved in 100 ul of ultra pure ddH.sub.2O and 500 ul of 0.5M NaOAc buffer (pH=4.4). 0.5-10 mCi of .sup.68GaCl.sub.3 was added (eluted from the ITG .sup.68Ge/.sup.68Ga generator using 0.05M HCl). The final pH of the reaction was 4.1-4.4. The reaction mixture was heated at 70-75 C. for 20 min. After cooling to room temperature, the reaction was analyzed by radio-TLC.

A.4. In Vitro Evaluation of 68Ga-Compound 1a

(63) a) Cellular uptake study of .sup.68Ga-compound 1a. A549 cells were plated in 12 well plates at a density of 1.510.sup.5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO.sub.2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5 microCi .sup.68Ga-1a or .sup.68Ga DOTA or DMEM media containing 10 mmD-glucose (or other GLUT1 inhibitors such as genistein, scutellarin, cytochalasin B) and 0.5 microCi.sup.68GA-1a. Cells were incubated at 37 C., 5% CO.sub.2 for the indicated time. The radioactive media were then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivities were counted. Data is expressed as % ID (cpm cells/cpm media*100). Error bars represent Standard deviation.

(64) b). Cellular uptake study of .sup.177Lu-compound 1a. LS174T cells were plated in 12 well plates at a density of 1.510.sup.5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO.sub.2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media were removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5 microCi .sup.177Lu-1a or .sup.177Lu-DOTA or DMEM media containing 10 mmD-glucose and 0.5 microCi .sup.177Lu-1a. Cells were incubated at 37 C., 5% CO.sub.2 for the indicated time. The radioactive media were then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivities were counted. Data is expressed as % ID (cpm cells/cpm media*100). Error bars represent Standard deviation.

Example 2

B.1. Synthesis of N-[1,4,7,10-Tetraazacyclododecane-1,7-bis-(propionic acid)-4,10-bis(acetic acid)]-2-amino-2-deoxyglucopyranoside (Compound 1b)

Step. 1. Modification of 1,4,7,10-tetraazacyclododecane-1,7-bis(t-butyl acetate)

(65) 1,4,7,10-tetraazacyclododecane-1,7-bis(t-butyl acetate), DO2A tBu ester (1eq., Macrcyclics) was dissolved in CH3CN (5 ml) and benzyl 3-bromopropanoate was added in the presence of Et3N (2.1 ml, 12 mmol) at temp. 0 C. Reaction was slowly warmed up to room temp. and left stirring for 24 h. After evaporation of the solvent under vacuum, product was purified by flash chromatography using 60N silica gel. Deprotection of benzyl ester groups was completed by catalytic hydrogenation under high pressure in the presence of Pd/C during 24 h.

Step 2. Preactivation and Functionalization of 1,4,7,10-tetraazacyclododecane-1,7-bis(t-butyl acetate)-4,10-bis(propionic acid)

(66) Modified chelating agent was dissolved in DMF (3 mL) in the presence DIPEA (3eq) and coupling agent, HBTU (3eq.) was added to the solution. The mixture was left for 20 min at r.t. GlcNAc-b-NH.sub.2 (1.3 eq.) was dissolved in DMSO (1 ml). The solution was heated slightly to dissolve the monosaccharide and added to the pre-actived DO2A chelating agent. The mixture was allowed to react at room temp. with constant stirring 12 h. After solvent evaporation, product was purified by extraction using CHCl.sub.3:MeOH and flash silica gel 60N column.

Step 3. Removal of tBu Ester Protecting Groups

(67) The tert-butyl ester protecting groups were removed in the presence of 30% TFA:CH.sub.2Cl.sub.2:H.sub.2O:TIS (950:250:250).

B.2. 68Ga-Labeling of Compound 1b

(68) Radiolabeling was performed in 0.5M NaOAc pH=4.4 at 80 C. for 20 min. iTLC was developed in standard running buffer (0.5M NH4OAC:methanol 1:1 v/v). Radio-TLC (Bioscan) analysis showed the radiochemical purity of tracer was >98%.

Example 3

C.1. Synthesis of N,N-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-(acetyl)]-1,7-di-(2-amino-2-deoxy--D-glucopyranoside (Compound 2a)

(69) This is a two step synthesis performed by coupling of glucosamine hydrochloride to 4,7,10-tetraazacyclodo decane-1,4,7,10-tetraacetic acid.

Step a. Preactivation of DOTA Chelating Agent

(70) To activate the carboxyl groups, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacid acetic acid (1eq.) was dissolved in 2 mL N-methylpyrolidine (NMP) and HATU coupling agent (4eq.) was added in the presence of 4eq. of N,N-diisopropylamine (DIPEA). Reaction was left at room temperature for 20 min.

Step b. Coupling of Glucosamine Hydrochloride to Activated Chelating Agent

(71) Glucosamine hydrochloride (4eq) was dissolved in NMP in the presence of DIPEA (4eq) and added to the solution of pre-activated DOTA. Reaction was left stirring for one day at room temperature and was traced by TLC (chloroform:methanol 1:10) visualized using anisidine solution or dichlorofluoresceine. After concentration of the reaction under high vacuum, product was extracted using CH2Cl2:H2O and aqueous fraction was collected. Final product was precipitated by addition of ether diethyl on ice.

C.2. 68Ga-Labeling of Compound 2a

(72) Radiolabeling was performed in 0.5M NaOAc pH=4.4 at 65 C. for 20 min. iTLC was developed in standard running buffer (0.5M NH4OAC:methanol 1:1 v/v) and is shown below on FIG. 8. Radio-TLC (Bioscan) analysis showed the radiochemical purity of tracer was >99%

(73) C.3. Cellular Accumulation of .sup.68Ga-2a

(74) SKBr3 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5-1 Ci 68Ga-2 or 68Ga DOTA. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation.

C.4. Cellular Accumulation and Blocking of 68Ga-2a

(75) SKBr3 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma Aldrich) and 0.5-1 Ci 68Ga-2. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

C.5. Blocking of Accumulation of 68Ga-2a by Glucose Transporter Inhibitors

(76) SKBr3 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma Aldrich) or other GLUT1 inhibitors (genistein, cytochalasin B, scutellarin) and 0.5-1 Ci 68Ga-2. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

C.6. Cellular Uptake and Blocking Study of 177Lu-2a

(77) A549 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5 microCi .sup.177Lu-1a or .sup.177Lu-DOTA or DMEM media containing 10 mg/ml D-glucose and 0.5 microCi .sup.177Lu-1a. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is expressed as % ID (cpm cells/cpm media*100). Error bars represent Standard deviation.

Example 4

D.1. Synthesis of N-[1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra-(acetic acid)]-2-amino-2-deoxyglucopyranoside

Step 1. Coupling

(78) Fmoc-Asn-Glc (0.015 eq.) was immobilized on the 2-chlorotrityl chloride resin (0.01 eq) in the presence of DIPEA (4 eq) in DMF. Reaction is carried out in presence of excess of DIPEA in order to prevent possible hydrolysis of chloro-resin bond and to neutralize HCl, that form during esterification reaction. Reaction is completed with 1-2 h at r.t.

Step 2. Capping

(79) After attachment of Fmoc-Asn-Glc, the unmodified chloride resin was deactivated by addition of ethanol or TFE.

Step 3. Loading Efficiency (Fmoc Titration)

(80) The Fmoc concentration on the resin was determined by addition of 20% piperidine in DMF to the tested resin. The resultant fulvene-piperidine adduct had UV absorption max at 301 nm. Free Fmoc amino acid of know concentration was used as a standard. Loading efficiency of the compound 3 on the resin was close to 15% after 1 h. Loading increased to 79% when reaction was left for more than 24 h. The lower loading of Fmoc-Asn-Glc conjugate relatively to unprotected Asn is probably caused by steric hindrance and close proximity of carboxyl group and Fmoc group. Loading efficiency was also determined using Kaiser test, that measure the content of free amines after Fmoc deprotection.

Step 4. Deprotection-Removal of Fmoc-Protecting

(81) To Fmoc-Asn-Glc attached to the resin 20% piperidine/DMF was added and lefty for 30 min followed by wash with 1 ml of DMF.

Step 4. 68Ga-Labeling on the Resin

(82) Radiolabeling was performed on the compound still attached to the resin. 200 uCi of 68Ga was added to the suspension of resin in buffer pH=4.4. reaction was mildly heated at 55 C. for 10-12 min.

Step 5. Cleavage from Resin

(83) Conjugate was released from in acid-mediated in mild conditions by addition of AcOH in DCM:H2O:TIS at r.t. in 20-25 min. This process can additionally accelerated in higher temp. The after addition of AcOH to the resin and incubation at room, flow through is collected and pH of the eluate is adjusted to 4.4 by addition of NaOAc buffer.

D.2. Cellular Accumulation of 68Ga-3

(84) A549 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5 microCi 68Ga-3 or 68Ga DOTA. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is presented as % cpm cells/cpm media.

D.3. Cellular Accumulation of 68Ga-3

(85) A549 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5 microCi 68Ga-3 or 68Ga DOTA. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is presented as % cpm cells/cpm media.

Example 5

E.1. Synthesis. N-1-(-aminocaproyl)--N-acetyl-2-deoxyglucopyranosyl N-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-(acetyl acid)]

(86) Step 1. Synthesis of -N-Acetylglucosaminylamine (FIG. 23, compound 3). 2-Acetamido-2-deoxy-beta-D-glucopyranosyl azide 3,4,6-triacetate was obtained from Aldrich and was converted to amine (2) by using PPh3 and DCM following the general literature procedure (Carbohyd. Research, 2001, 331, 439). Deacetylation: To a solution of an acetyl protected amine (2) in dry MeOH, 1-2 drops of 1M methanolicNaOMe solution were added, and the reaction mixture was kept at rt until completion of the transformation (monitored by tlc). Amberlyst 15 (H+ form) resin was then added to remove sodium ions, the resin was filtered off, and the solvent removed in vacuo.

(87) Step 2. Synthesis of N-(-aminocaproyl)--N-acetylglucosaminylamine (FIG. 23, compound 5). 6-Trifluoroacetamido-hexanoic acid (1 eq.) was dissolved in DMSO (0.6 ml). DIEA (6 eq.) and HBTU (1.3 eq.) were added to the solution. The mixture was allowed to pre activate for approximately 10 min. In a separate vial GlcNAc-b-NH2 (1.3 eq) was dissolved in DMSO (1.3 ml). The solution was heated slightly to dissolve the monosaccharide. After the solution was cooled to rt, the monosaccharide solution was added to the activated solution. The mixture was allowed to react to rt with constant stirring for 3 h. Final product was purified by flash using silica gel 60N.

(88) Step 3. Synthesis of N-1-(-aminocaproyl)--N-acetyl-2-deoxyglucopyranosyl N-[1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetra-(acetyl acid)] (FIG. 23). DOTA conjugate (6) was dissolved in DMSO (1 ml), Et3N (0.3 ml) and coupling agent HATU were added to the solution. The mixture was allowed to preactivate for approx 30 min. In a separate vial N(-aminocaproyl)-N-acetylglucosaminylamine (5) was dissolved in DMSO and added to the preactivated DOTA solution. The mixture was allowed to react at rt with constant stirring for 24 h. The reaction mixture was then concentrated under reduced pressure and purified by flash chromatography. Deprotection of tbu ester groups will be performed by addition of TFA (0.5 ml)/DCM (0.5 ml) to the DOTA ester at 0 C. Then the reaction was stirred for 3 h. The solvent was then removed under reduced pressure, precipitated with anhydrous diethyl ether and dried under high vacuum.

(89) E.2. 68Ga-labeling of compound 4. Radiolabeling was performed in 0.5M NaOAc pH=4.4 at 700 C for 20 min iTLC was developed in standard running buffer (0.5M NH4OAC:methanol 1:1 v/v). Radio-TLC (Bioscan) analysis showed the radiochemical purity of tracer was >99%.

(90) E.3. Cellular accumulation of 68Ga-4. A549 and SKBr3 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5-1Ci 68Ga-4 or 68Ga DOTA. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Black lines represent linear regression analysis. Studies were preformed in triplicate.

(91) E.4. Cellular accumulation of 177Lu-4. A549 cells were plated in 12 well plates at a density of 2*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 0.5 microCi 177Lu-4 or 177Lu-DOTA. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is presented as % cpm cells/cpm media

(92) E.5. Biodistribution study of 177Lu-4. Tumor xenografts were generated in 6 week old Swiss nu/nu mice using the human lung adenocarcinoma cell line A549. Xenografts were generated by subcutaneous inoculation of 2*10^6 cells per mouse into the right shoulder. Xenografts were allowed to grow for 7 days. Mice were fasted for 8-12 hours prior to the start of the study. On the day of the study, mice were anesthetized with Isofluorane and a tail vein cannula was inserted. The tracer was delivered via tail vein injection. Mice were placed on a warming pad or under a warming lamp to maintain body temperature. Mice were sacrificed at the indicated timepoints and organs were removed, weighed and counted. Data is expressed as % injected dose/gram tissue.

Example 6

F.1. Synthesis of Compound 5

(93) To activate the carboxyl groups, carbohydrate-genistein (1 eq., 20 ug) was dissolved in 0.5 mL dimethylformamide DMF and dimethylsulfoxide DMSO and N-hydroxybenzotriazole (HOBT) and O-Benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU) coupling agents (1.5eq. each) were added in the presence of 4eq. of N,N-diisopropylamine (DIPEA). Reaction was left at room temperature for 30 min.

Step b. Coupling of DOTA-Linker-NH2 to Activated Carbohydrate-Genistein Derivative

(94) Pre-activated carbohydrate-genistein (1.5eq) was dissolved in DMF in the presence of DIPEA (4eq) and added to the solution DOTA-linker-NH2. Reaction was left stirring for 48 h at room temperature and was traced by TLC (chloroform:methanol 1:10) visualized using anisidine solution or dichlorofluoresceine. After completion of reaction, solution was evaporated and purified on HPLC (C18 column). The tert-butyl ester protecting groups were removed in the presence of 30% TFA:CH.sub.2Cl.sub.2:H.sub.2O:TIS (950:250:250) and product was purified again by HPLC.

(95) Alternatively, protected compound 5 was purified by precipitation using Et.sub.2O and fraction of supernatant was collected. The tert-butyl ester protecting groups were removed in the presence of 30% TFA:CH.sub.2Cl.sub.2:H.sub.2O:TIS (950:250:250) and product was purified again by precipitation with Et.sub.2O.

F.2. 68Ga-Radiolabeling of Compound 5

(96) Compound 5 (1-10 ug) dissolved in 100 ul of 0.5M NaOAc buffer (pH=4.4). 0.5-4 mCi of .sup.68GaCl.sub.3 was added (eluted from the ITG .sup.68Ge/.sup.68Ga generator using 0.05M HCl). The final pH of the reaction was 4.1-4.4. The reaction mixture was heated at 95 C for 20 min After cooling to room temperature reaction was analyzed by radio-TLC.

F.3. In Vitro Evaluation of 68Ga-Compound 5

(97) a) Cellular uptake study of .sup.68Ga-compound 7. A549 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 12 uCi of .sup.68Ga-5/ml or .sup.68Ga DOTA or DMEM media containing 10 mmD-glucose and 12 uCi/ml of .sup.68GA-5. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is expressed as % ID (cpm cells/cpm media*100). Error bars represent Standard deviation.

F.4. Cellular Accumulation and Blocking of 68Ga-5

(98) SKBr3 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma alrich) and 12 uCi/ml of radiolabeled compound. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

(99) F.4. Blocking of accumulation of .sup.68Ga-5 by glucose transporter inhibitors. SKBr3 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing glucose transporters inhibitors (scutellarin, glucose, genistein, cytochalasin B) and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma alrich) and 12 uCi/ml of radiolabeled compound. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes.

(100) Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

Example 7

G.1. Synthesis of Compound 6

(101) To activate the carboxyl groups, tris-tBu ester DOTA (1 eq., 20 ug) was dissolved in 0.5 mL dimethylformamide DMF and dimethylsulfoxide DMSO and N-hydroxybenzotriazole (HOBT) and O-Benzotriazole-N,N,N,N-tetramethyl-uronium-hexafluoro-phosphate (HBTU) coupling agents (1.5eq. each) were added in the presence of 4eq. of N,N-diisopropylamine (DIPEA). Reaction was left at room temperature for 30 min.

Step b. Coupling of Linker-Genistein to Activated DOTA-tris-tBu Ester

(102) Pre-activated DOTAtris-tBuester (1.5eq) was dissolved in DMF in the presence of DIPEA (4eq) and combined with solution of genistein-linker-NH2. Reaction was left stirring for 48 h at room temperature and was traced by TLC (chloroform:methanol 1:10) visualized using anisidine solution or dichlorofluoresceine. After completion of reaction, solution was evaporated and purified on rHPLC (C18). The tert-butyl ester protecting groups were removed in the presence of 30% TFA:CH.sub.2Cl.sub.2:H.sub.2O:TIS (950:250:250) and product was purified again by HPLC.

G.2. 68Ga-Radiolabeling of Compound 6

(103) Compound 6 (1-10 ug) dissolved in 100 ul of 0.5M NaOAc buffer (pH=4.4). 0.5-4 mCi of .sup.68GaCl.sub.3 was added (eluted from the ITG .sup.68Ge/.sup.68Ga generator using 0.05M HCl). The final pH of the reaction was 4.1-4.4. The reaction mixture was heated at 95 C for 20 min. After cooling to room temperature reaction was analyzed by radio-TLC.

G.3. In Vitro Evaluation of 68Ga-Compound 6

(104) a) Cellular uptake study of .sup.68Ga-compound 6.A549 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 12 uCi of .sup.68Ga-6/ml or .sup.68Ga DOTA or DMEM media containing 10 mmD-glucose and 12 uCi/ml of .sup.68GA-6. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is expressed as % ID (cpm cells/cpm media*100). Error bars represent Standard deviation.

G.4 Cellular Accumulation and Blocking of 68Ga-6

(105) SKBr3 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma alrich) and 12 uCi/ml of radiolabeled compound. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

(106) G.5. Blocking of accumulation of .sup.68Ga-6 by glucose transporter inhibitors. SKBr3 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml DMEM (Cellgro) containing GLUT-1 inhibitors (scutellarin, glucose, genistein, cytochalasin B) and 12 uCi/ml of radiolabeled compound. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

Example 8

H.1. Synthesis of Compound 7

Step.a. Synthesis of Compound 7a

(107) 2-acetamido-N-(-minocaproyl)-2-deoxy--D-glucosylamine (1eq., 30 ug) was dissolved in 0.5 mL dimethylformamide DMF and dimethylsulfoxide DMF/H2O and were added to MFCO-NHS dissolved in DMF/H2O. Reaction was left at room temperature for 24 h.

Step b. Synthesis of Compound 7b

(108) Compound 7a (1.5eq) and DOTA-linker-N3(10eq) were dissolved in DMF/H2O and reaction was left stirring for 48 h at room temperature. Progress of reaction was monitored by TLC (chloroform:methanol 1:10) visualized using anisidine solution or dichlorofluoresceine. Final product was purified by rHPLC.

H.2. 68Ga-Radiolabeling of Compound 7b

(109) Compound 7b (1-10 ug) dissolved in 100 ul of 0.5M NaOAc buffer (pH=4.4). 0.5-4 mCi of .sup.68GaCl.sub.3 was added (eluted from the ITG .sup.68Ge/.sup.68Ga generator using 0.05M HCl). The final pH of the reaction was 4.1-4.4. The reaction mixture was heated at 95 C for 20 min. After cooling to room temperature reaction was analyzed by radio-TLC.

H.3. In Vitro Evaluation of 68Ga-Compound 7b

(110) a) Cellular uptake study of .sup.68Ga-compound 7b. A549 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 30 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) containing 12 uCi of .sup.68Ga-7b/ml or .sup.68Ga DOTA or DMEM media containing 10 mmD-glucose and 12 uCi/ml of .sup.68GA-7b. Cells were incubated at 37 C., 5% CO2 for the indicated time. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and radioactivity was counted. Data is expressed as % ID (cpm cells/cpm media*100). Error bars represent Standard deviation.

H.4 Cellular Accumulation and Blocking of 68Ga-7b

(111) SKBr3 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing 5.4 mg/ml glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma Alrich) and 12 uCi/ml of radiolabeled compound. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.

H.5. Blocking of Accumulation of 68Ga-7b by Glucose Transporter Inhibitors

(112) SKBr3 cells were plated in 12 well plates at a density of 1.5*10^5 cells per well and grown overnight in DMEM containing glucose and 10% FBS at 37 C., 5% CO2. Cells were fasted for 20 min prior to the study with glucose free DMEM. At the start of the study, media was removed from each well and replaced with 0.5 ml glucose free DMEM (Cellgro) or media that contained 100 mg/ml D or L glucose (Sigma alrich) or other GLUT1 inhibitors (genistein, scutellarin, cytochalasin B) and 12 uCi/ml of radiolabeled compound. Cells were incubated at 37 C., 5% CO2 for 1 hour. The radioactive media was then removed and cells were washed twice with 1 ml PBS. Cells were then trypsinized and transferred to counting tubes. Radioactivity in cells and media were counted at 511 keV using a Perkin Elmer Wizard gamma counter. Cells were then collected and lysed with RIPA buffer (Invitrogen) and protein concentration was determined by Pierce BCA protein assay kit (ThermoFisher). Data is expressed as % ID (cpm cells/cpm media*100)/mg protein. Error bars represent Standard deviation. Data represents the average of four separate studies performed in triplicate.