Reagents
All chemicals were purchased from Sigma-Aldrich Chemical Co. (ST. Louis, MO, USA) or Thermo Fisher Scientific (Pittsburgh, PA, USA), unless otherwise specified. DOTA-tris(tert-butyl ester) was purchased from Macrocyclics, Inc. (Dallas, TX, USA). Indium-111 ([111In]InCl3) was purchased from BWZXT ITG Canada, Inc. (Ottawa, ON, Canada). Actinium-225 nitrate was purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA).
Chemical Syntheses. DOTA-tris(t-Bu ester)-TDA was synthesized as previously described with modifications [20]. Briefly, the DOTA-tris(t-Bu) ester (50 mg, 0.09 mmol) and HBTU (0.168 mg, 0.44 mmol) were dissolved in 2 mL of DMF. The resulting solution was stirred for 30 minutes at room temperature (RT) then 1-tetradecylamine (TDA) (22 mg, 0.11 mmol) and DIPEA (76 µl, 0.44 mmol) were added to the solution, which was stirred overnight at RT. The solution was extracted with ethyl acetate washed with water (2x) then brine (1x). The ethyl acetate solution was dried over MgSO4, filtered and the ethyl acetate was evaporated under vacuum to yield a yellow powder. The DOTA-tris(t-Bu ester)-TDA was purified by silica gel chromatography (eluent: hexane/ethyl acetate, 3/1 v/v), purity confirmed by TLC and the product was used without further confirmation.
DOTA-TDA. DOTA-tris(t-Bu ester)-TDA (30 mg, 0.04 mmol) was dissolved in trifluoroacetic acid (125 µl) and dichloromethane (400 µl) and stirred overnight at RT. The solvent was evaporated under vacuum yield a pure white solid, 51.3% was recovered. DOTA-TDA was successfully synthesized (overall yield 53.1%) and confirmed by mass spectrometry. DOTA-TDA was confirmed by using a Thermo Scientific Q Exative Plus Orbitrap Mass Spectrometer coupled with a Vanquish UPLC system at the Metabolomics Facility at Johns Hopkins School of Medicine (Director – Dr. Anne Le) (M+H): calculated 600.4336, found 600.4316; (M-H) calculated 598.4185 found 598.4203.
Radiolabeling of DOTA-TDA. For radiolabeling the following were added to acid wash Eppendorf tube 1 µl of Indium-111, 40 µl of 0.4M NaOAc buffer pH=4.5, and 1 µl of DOTA-TDA (1 µg/µl). The solution was heated at 95ºC for 15 minutes and purified using a Waters Sep-Pak C18 (Milford, MA, USA). [111In]In-DOTA-TDA was eluted with ethanol, dried, and resuspended in sterile PBS with greater that 95% radiopurity as determined by thin layer chromatography (TLC).
For Actinium-225, 5 µl of Actinium-225 solution, 5 µl of 2M Tris buffer pH=7, and 2.5 µL of DOTA-TDA were added to an acid washed tube, heated at 95ºC for 15 minutes. Radiolabeling yields were ≥95%; [225Ac]Ac-DOTA-TDA was used without further purification.
Partition Coefficient (Log P). Log P were determined for the radiolabeled conjugates, [111In]In- or [225Ac]Ac-DOTA-TDA, were dissolved in 1 mL of octanol in a centrifuge tube. The centrifuge tube was vigorously vortexed for a minute with 1 mL of 0.9% saline. The tube was centrifuged at 1000 rpm for 5 minutes and allowed to sit for 30 minutes until the phases were clearly separated. The organic phase (Octanol) and the aqueous phase (Saline) were collected separately and counted at equilibrium of Actinium-225 in an automatic γ-well counter (Perkin-Elmer 2470 WIZARD2® Automatic Gamma Counter, Waltham, MA, USA). The Log P values were calculated using the following formula: Log P = Log ([Counts]octanol/[Counts]saline).
Animal Studies
Animal studies were performed using 7-8-week-old healthy male NCG mice obtained from Charles River Laboratories (Wilmington, MA, USA). Adult New Zealand rabbits were used weighing approximately 4 kg (Myrtle’s Rabbitry, Thompson Station, TN, USA). All animal studies were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine. For tumor retention and survival studies the mice were injected subcutaneously in the right flank with approximately 1 x 106 HEP2G cells in 100 μL of sterile PBS and Matrigel (1:1).
Mouse Model
Tumor Retention. Biodistribution studies were carried out as previously described in healthy NCG male mice (n=3/group) bearing HEP2G subcutaneous tumors [21, 22]. To assess tumor retention the mice were divided into two groups that were injected intratumorally with 111In-DOTA-TDA (37kBq, 60µL) alone or 111In-DOTA-TDA (37kBq, 60µL) emulsified in Lipiodol. At 48-hours post-injection (p.i.), the mice were euthanized. The blood, heart, lungs, liver, spleen, kidneys, stomach (with content), intestines (with content), bone, muscle, and tumors were harvested, weighed, and measured in an automatic γ-well counter. The percentage of injected dose per gram (%ID/g) was calculated by comparison with a weighed, diluted standard.
Therapeutic Efficacy: Survival Studies. Subcutaneous liver cancer models were used to evaluate the therapeutic efficacy of [225Ac]Ac-DOTA-TDA/Lipiodol emulsions. The following treatments were administered in subcutaneously HEP2G tumors (266.5 ± 23.2 mm3): 1. Saline (60 μL) 2. Lipiodol alone (60 μL) or 3. [225Ac]Ac-DOTA-TDA/Lipiodol (37 kBq, 60 μL). Mice were observed for signs of pain and distress (lethargy, hunched back, paralysis, etc.), weight loss, and tumor size [volume=0.5(length x width2)]; mice were euthanized if one of the following conditions were met: 1. weight loss (>20%); 2. primary tumor reaching 1,500 mm3; 3. signs of pain or distress; or 4. 180 days post-treatment. Survival fractions were plotted as a Kaplan-Meier survival curve using Prism 8 (GraphPad; La Jolla, CA, USA).
VX2 Rabbit Model
Anesthesia. Rabbits were administered a mixture of ketamine (30-50 mg/kg) and xylazine (3-5 mg/kg) intramuscularly prior to tumor harvesting, implantation, and imaging. Anesthesia was maintained with 3% isoflurane in oxygen as well as additional injections of the ketamine/xylazine solution.
Ultrasound Guided Tumor Implantation. The implantation of the VX2 tumor was done as previously described with modifications[23]. Briefly, the solid VX2 tumor for implantation was obtained from a carrier rabbit that had been injected intramuscularly in the thigh approximately 2 weeks prior. The VX2 tumor was harvested, placed in 0.9% sodium chloride, and minced into small pieces. The minced tumor was prepared in a 16-gauge Angiocath (2 inches long), using ultrasound guidance, the needle punctured the liver and the tumor pieces were pushed into the liver with a guide wire. Manual compression was administered after the needle was removed.
Intrahepatic Arterial Injection including Agents and Dosing. Hepatic angiography was performed as previously described[23]. Briefly, rabbits with VX2 hepatic tumors, confirmed by ultrasound were anesthetized and surgical cutdowns were performed to expose the femoral artery. A 4-F sheath (Cook) was inserted to allow a 2-F catheter tip (JB1 catheter; Cook) to access the common hepatic artery. Fluoroscopy was utilized to select the arterial branch and radioembolization was performed.
SPECT Imaging in VX2-bearing Rabbit. SPECT/CT imaging was performed on a Siemens Symbia T16 SPECT/CT system. The anesthetized rabbit (n=1) was placed supine on the imaging bed with the whole body in the field-of-view. The images were acquired at 24 hours, 72 hours and 6 days p.i.. Projection data were acquired in 120 views over 360° with a 128 x 128 matrix and 4.795 mm pixel size. The imaging time were 75 seconds per view for the 24-hour imaging, and 90 seconds per view for the 72-hour and 6-day imaging. The high-energy collimators were used. Data were binned into two energy windows - 20% centered at 440.45 keV peak of Bismuth-213 and 20% centered at 218 keV peak of Francium-221, respectively. Both isotopes are daughters of Actinium-225 decay and emit gamma photons that can be imaged by SPECT. Francium-221 has a short half-life of 4.9 minutes, therefore its distribution represents those of Actiniun-225’s. Bismuth-213 has a half-life of approximately 45.6 minutes and could have redistributed from the parent isotope. After SPECT acquisition, a CT scan was performed to provide anatomical images and an attenuation map. The SPECT images were reconstructed with Siemens Flash3D OSEM algorithm with compensation for attenuation and resolution[24]. A total of 6 iterations with 15 subsets per iteration were used.
Ex Vivo Biodistribution VX2-bearing and non-tumor bearing Rabbits. Biodistribution studies were carried out following the SPECT imaging approximately 6-days post-injection of [225Ac]Ac-DOTA-TDA (0.49 MBq) emulsified in 1 mL of Lipiodol in the VX2-bearing rabbit (n=1) and at approximately 24 hours for non-tumor bearing rabbits (n=2). At the end timepoint the rabbits were euthanized (Ethansol®, 1 mL minimum then 1mL/4.5 kg). The blood, heart, lungs, liver, spleen, kidneys, stomach, intestines, marrow, femur+marrow, muscle, gall bladder, bile, and if applicable the tumor were harvested, weighted, and measure in an automatic γ-well counter and/or a Capintec CRC-7 dose calibrator. The percentage of injected dose per gram (%ID/g) was calculated based on the dose injected and converted to Bq/g using an efficiency coefficient.
Stastical Analysis was performed using the software Graphpad Prism 8. All data are presented as mean ± SD. Tumor rententions were compared using 2-way ANOVA. Survival studies used Kaplan-Meier curves that were analyzed using the Log Rank (Mantel-Cox) test. Values were considered significant when P<0.05.