Technetium-99m (99mTc) radiopharmaceuticals are widely used for diagnostic imaging of heart, kidney, and liver disease, and cancer. Evolution from perfusion type tracers to targeted agents however has proven difficult. 99mTc labeled antibodies for imaging specific disease biomarkers would be of great interest, however the disparity between the isotopes half-life (6 hours) and the long circulation time of most antibodies (multiple days) has been a significant barrier. Furthermore, the conjugation of bifunctional 99mTc-chelate complexes to small molecules often has a detrimental impact on targeting. The use of bioorthogonal chemistry derived from tetrazines and trans-cyclooctene derivatives, along with pretargeting has the potential to overcome these issues and create a new generation of targeted 99mTc radiopharmaceuticals.
Initially, the synthesis of three generations of imidazole based tridentate chelates linked to a tetrazine was completed. These new ligands were labeled with 99mTc under mild conditions (60 °C, 20 min, pH 3.5) with modest to good radiochemical yields ranging from 31 to 83%. Biodistribution studies revealed that compound 14, which contains a polyethylene glycol 5 (PEG5) linker had the best clearance from non-target tissues. Compound 14 was also used successfully in a pretargeting strategy along with a transcyclooctene (TCO) derivative of the bone targeting bisphosphonate, alendronate (ALN). One hour following the administration of TCO-ALN to BALB/c mice, compound 14 was injected intravenously where uptake at sites of high calcium turn over (i.e. the joints) was observed. At 6 hours post injection, for example, uptake reached as high as 20.1 ± 4.91 and 16.1 ± 4.84 %ID/g in the knee and shoulder, respectively.
Pretargeted imaging studies were performed subsequently with a TCO-functionalized huA33 antibody in mice bearing SW122 xenografts. The TCO-huA33 antibody was injected 24 hours before the administration of two radiolabeled tetrazines at high and low specific activities. At 6 hours post injection tumour uptake was minimal, with tumour: blood ratios <1 in all cases. Blood clearance studies determined that the tetrazines were being cleared rapidly, with a blood residence half-life of 1.3-2.1 minutes. The hypothesis is that the low concentration of the antibody (owing to its high molecular weight), combined with the rapid clearance of the tetrazine and significant off-target uptake resulted in unfavorable kinetics and low tumor binding.
Studies of the clearance pathway of 14 were investigated with clinically approved hepatobiliary transport inhibitors to help understand the mechanism of clearance, which could in turn be used to optimize the pharmacokinetics of the tetrazine ligands. A range of different inhibitors of key clearance pathways were evaluated with limited success. However, co-administration of 14 with ALN resulted in a 75% decrease in gall bladder uptake of 14 (216 ± 75.9 to 33.6 ± 3.93 %ID/g). Pretargeting studies of 14 with TCO-ALN in the presence of excess ALN revealed that ALN did not hinder the uptake of TCO-ALN in the bone, with all organs and tissues having the same uptake with TCO-ALN or TCO-ALN + ALN (knee: 20.1 ± 4.91 and 14.9 ± 2.43 %ID/g, respectively). There was also a concomitant decrease in gall bladder uptake (91.5 ± 17.1 to 28.8 ± 2.63 %ID/g).
Further work on improving the distribution of the tetrazine ligands involved investigating the effect of the chelate. The core chelate found in 14 without the tetrazine moiety (compound 11a) was labeled with 99mTc to produce 11b in a 31% radiochemical yield. Biodistribution studies of 11b and 14 at 6 hours post injection demonstrated that the imidazole-based 99mTc-chelate was a major factor in the rapid and significant uptake and retention in the liver and gallbladder. A new triazole based chelate with optimal clearance from Kluba and coworkers was synthesized in 45% yield and successfully labeled with 99mTc (compound 23a). Biodistribution studies were performed where at 6 hours post injection, 23a had five times lower uptake in all non-target organs compared to 11b. The synthesis of a tetrazine derivative of 23a (compound 32) unfortunately demonstrated high hepatobiliary uptake compared to the original triazole chelate (gall bladder: 228 ± 251 and 8.77 ± 0.73 %ID/g, large intestine: 85.5 ± 83.5 and 6.88 ± 0.30 %ID/g, respectively). This particular derivative had a lipophilic linker as a result of the synthetic challenges faced during the preparation of a more hydrophilic triazole-tetrazine derivative.
In addition to pretargeting applications, the 99mTc-tetrazine was used as a reagent to create multimodal imaging agents. Nanoscale gas vesicle (GV) ultrasound contrast agents were functionalized with TCO via an amide coupling to lysine residues. TCO-GVs were then radiolabeled by adding compound 6 where the desired product, a new multimodal probe, was obtained in 59% radiochemical yield. SPECT imaging and biodistribution studies in mice were completed where the labeled GV’s showed uptake in the gall bladder (120 ± 29.1 %ID/g), liver (16.8 ± 7.50 %ID/g), lungs (3.26 ± 1.53 %ID/g), small intestines (14.5 ± 5.30 %ID/g), and spleen (5.47 ± 2.71 %ID/g) at 120 min post injection. In addition to radiolabelling, the TCO-GVs were also functionalized with a near IR-tetrazine dye to produce a multimodal ultrasound/photoacoustic (US/PA) imaging agent in a 68% yield. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25023 |
| Date | January 2019 |
| Creators | Bilton, Holly A |
| Contributors | Valliant, John F, Chemistry and Chemical Biology |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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