Luke Wharton

We report a detailed study on how the substitution of carboxylate groups of macropa for amides affects the coordination of Pb(II) and Bi(III). We describe a new synthesis of macropam, improving on previously reported yields. Pb(II) and Bi(III) complexes of macropa, macropapam, and macropam were prepared in situ and characterized. Crystal structures were obtained using XRD which were analyzed using eccentricity measurements and NBO analysis, showing that the lone pair is more active in Bi(III) complexes than in Pb(II) analogues and this activity decreases upon incorporation of amides. NMR and X-ray crystallography of [Bi(macropa)]⁺ evidenced a certain degree of fluxionality. This led to a detailed computational study in which the energies of the transition states for the switching of the X-CH₂-CH₂-X units of the macrocycle between λ and δ conformations were calculated. The relatively low energies of the transition states suggest that this process takes place in solution on the μs time scale. Radiolabeling with [²⁰³Pb]Pb(II) and [²¹³Bi]Bi(III) and stability assays of the [²⁰³Pb]Pb(II)-labeled complexes showed that the incorporation of amides worsens radiochemical performance. Overall, the incorporation of amides is detrimental for radiopharmaceutical applications, highlighting that as borderline acids, Pb(II) and Bi(III) at times prefer harder (negatively charged) donor atoms.

As an alternative to macrocyclic chelators, a family of high-denticity, acyclic chelating agents based on picolinate arms has been devised with the aim of finding versatile chelating agents for ²²⁵Ac, ¹⁶¹Tb, and ¹⁷⁷Lu. This family comprises six symmetrical four-arm decadentate chelators that differ in the nature of the backbone spacer, ethylene (H₄tpaen), o-phenylene (H₄tpaopd), 2,3-naphtylene (H₄tpaond), m-xylylene (H₄tpamxd), p-xylylene (H₄tpapxd), and 2-hydroxypropylene (H₄tpadapo), and two dissymmetrical octadentate ligands, H₃tripaen and H₄asyoctapa, structurally derived from H₄tpaen by the loss of one arm (the first) and the replacement of two vicinal picolinate pendent arms by acetate groups (the second). The coordination chemistry of each chelating ligand with nonradioactive Lu³⁺, Tb³⁺, and La³⁺ (the latter as a surrogate for [²²⁵Ac]Ac³⁺) has been explored to rationalize the findings in radiolabeling and human serum stability studies. With an RCY of 96% at 10^-7 M, which is directly comparable to the gold standard macrocyclic chelators H₂macropa and H₄crown, H₄tpaond is an excellent candidate for the development of radiopharmaceuticals based on ²²⁵Ac. In addition, both H₄tpaen and H₄tpaopd are also an opportunity for the ²²⁵Ac/¹⁵⁵Tb theranostic pair. Meanwhile, H₄asyoctapa appears to be a great opportunity, not only for developing terbium radiopharmaceuticals, but also for the promising ¹⁷⁷Lu/¹⁵⁵Tb pair.

Fibroblast activation protein (FAP) is overexpressed in a variety of cancers, making it an attractive target for bifunctional chelator-based radiopharmaceuticals. This study initially aimed to assess the effect of chelator structure on the biodistribution of ²⁰³Pb/²¹²Pb-labeled FAP inhibitor (FAPI) bioconjugates. However, suboptimal in vivo biodistribution and imaging results suggested the bioconjugate was unstable. RadioHPLC analysis of urine samples suggest the thiourea bond, formed during conjugation between an amine on the biomolecule, and an isothiocyanate-functionalized chelator, is unstable in vivo, resulting in detachment of the radiometal-chelator complex from the targeting vector, resulting in poor tumor accumulation. To determine whether this instability was specific to the FAPI system, a peptide-based (Cyclic melanocyte stimulating hormone, CycMSH) bioconjugate targeting the melanocortin-1 receptor was synthesized using the same thiourea linkage. Identical metabolites were observed, supporting the hypothesis that thiourea bonds are unstable in vivo with this theranostic isotope pair. Subsequently, the effect of bioconjugation chemistry, specifically thiourea and amide bonds, on the stability and biodistribution of ²⁰³Pb/²¹²Pb-labeled bioconjugates was assessed. Modifying the bioconjugation linker to be an amide bond, formed by utilizing a chelate containing an active ester instead of an isothiocyanate, led to significantly improved in vitro and in vivo stability, as demonstrated by radioHPLC and biodistribution and imaging studies in both models. These findings highlight the importance of the choice of bioconjugation chemistry in the development of lead-based radiopharmaceuticals and emphasize the importance of selecting stable linkages to ensure optimal radiometal retention and tumor targeting.

The element-equivalent theranostic pair lead-203 (²⁰³Pb, 100% EC, t_1/2 = 51.92 h) and lead-212 (²¹²Pb, 100% β^- to α emitting ²¹²Bi, t_1/2 = 10.64 h) enable both diagnostic imaging and targeted therapy of disease within a single chemical framework through the emission of γ-rays and α-particles, respectively. In efforts to further advance the clinical potential of this theranostic pair, we report the synthesis and evaluation of two N¹,N⁷ (N-trans)-symmetrical cyclen-based chelators─DO2A-2Py and DCMC-2Py─designed to form thermodynamically stable and kinetically inert Pb²⁺ complexes. Protonation and stability constants determined by pH-dependent ¹H NMR titrations revealed that [Pb(DO2A-2Py)] and [Pb(DCMC-2Py)]²⁺ form complexes with high thermodynamic stability (logβ_{[Pb(DO2A-2Py)]} = 22.05 ± 0.05 and logβ_{[Pb(DCMC-2Py)]}²⁺ = 18.62 ± 0.05). Structural analysis by single crystal X-ray diffractometry revealed eight-coordinate Pb²⁺ complexes with the expected trans arrangement. Quantitative radiolabeling of [²⁰³Pb]Pb²⁺ was achieved for both DO2A-2Py and DCMC-2Py under mild, highly dilute reaction conditions (pH 7, 10 min reaction time). The resulting complexes exhibited remarkable kinetic inertness in serum, and in the presence of excess Pb²⁺ or EDTA, over 52 h, remaining >96% ([²⁰³Pb]Pb²⁺-DO2A-2Py) and >94% ([²⁰³Pb]Pb²⁺-DCMC-2Py) intact. Together, these findings reveal DO2A-2Py and DCMC-2Py as highly promising chelators for [^203/212Pb]Pb²⁺ radiopharmaceuticals.

Targeted alpha therapy (TAT) has shown high promise for the effective treatment of advanced stage cancers. Of the proposed radionuclides for TAT, Thorium-227 represents an interesting candidate given its relatively long half-life, 18.7 days, and the cascade of short-lived, high-potency, alpha-emitting daughter progeny in its decay scheme. However, to date few chelators exist which can effectively and stably bind [²²⁷Th]Th⁴⁺ at molar activities high enough for TAT. To address this challenge, this study investigated various chelating ligands for coordination of [²²⁷Th]Th⁴⁺. H₄noneunpaX was identified as a promising chelator, demonstrating radiolabeling with [²²⁷Th]Th⁴⁺ at concentrations of 10^-6 M (A_m = 272 kBq/nmol). The coordination characteristics of [Th(noneunpaX)] have been investigated through ¹H NMR spectroscopy, mass spectrometry, and DFT calculations. In this study, we also investigate for the first time the pairing of Th-227 with a peptide-based bioconjugate and evaluate the in vivo biodistribution characteristics. [²²⁷Th]Th-nonenupaX-Ahx-Tyr³-TATE was prepared under mild conditions (ambient temperature, 30 min) and evaluated in NRG mice bearing AR42J xenografts as a model for pancreatic neuroendocrine tumors. The ²²⁷Th-labeled radiopeptide showed high uptake in tumors (25.8±6.2 %IA/g at 3 h p.i.) and low uptake in non-targeted organs. Although some release of Th-227 was noted in serum stability studies this was not observed in vivo. This ligand architecture serves as an interesting framework for future optimization, which will involve improvements to the overall stability by enhancing the rigidity of the backbone and assessing other pendent donor groups with a stronger affinity toward [²²⁷Th]Th⁴⁺. Overall, this study demonstrated for the first time the viability of using peptide-based targeting to effectively deliver Th-227 to tumor sites.

The effects of replacing nitrogen with sulfur atoms in the 18-membered macrocycle of the H₂macropa chelator on the binding affinity and stability of "intermediate" (radio)metal [²⁰³Pb]Pb²⁺ and [²¹³Bi]Bi³⁺ complexes are investigated. The 1,4,10,13-tetraoxo-7,16-diazacyclooctadecane backbone was replaced with derivatives containing sulfur in the 1,4- or the 1,4,10,13-positions to yield the novel chelators H₂S₂macropa (N₄O₄S₂) and H₂S₄macropa (N₄O₂S₄), respectively. Trends on the ^nat/203Pb- and ^nat/213Bi-complex stability constants, coordination chemistry, radiolabeling, and kinetic inertness were assessed via potentiometric titrations, UV-vis spectroscopy, NMR spectroscopy, X-ray crystallography and density functional theory (DFT) calculations. ¹H-²⁰⁷Pb NMR spectroscopy confirmed the involvement of backbone S and/or O donors in the metal coordination sphere. Overall, the trend demonstrated that increasing the softness of the donor atoms within the ligand backbone decreased the thermodynamic stability and kinetic inertness of both the Pb²⁺ and Bi³⁺ complexes. Conversely, DFT calculations with mock compounds dimethyl ether (DME) and dimethyl sulfide (DMS) demonstrated enhanced affinity of the S atom to both Pb²⁺ and Bi³⁺ with DMS compared to DME evinced by large ΔG° values for both Pb²⁺ and Bi³⁺ complexes. The decreased stability of Pb/Bi-S_xmacropa (x = 0, 2, 4) upon increased sulfur atom incorporation may be a result of the increased steric strain within the macrocyclic backbone upon sulfur atom introduction. Nonetheless, [²⁰³Pb]Pb²⁺ and [²¹³Bi]Bi³⁺ labeling (pH = 7, 30 min reaction time; 10^-4-10^-8 M chelator) resulted in both S₂macropa^2- and macropa^2- attaining similarly high radiolabeling efficiency. Meanwhile, S₄macropa^2- only possessed the ability to complex [²¹³Bi]Bi³⁺. Both [²⁰³Pb][Pb(macropa)] and [²⁰³Pb][Pb(S₂macropa)] remained greater than 97% intact when challenged against human serum over 72 h. The results of this study reveal the effects of incorporating sulfur donor atoms into macrocyclic chelators for [²⁰³Pb]Pb²⁺ and [²¹³Bi]Bi³⁺ radiopharmaceuticals.

Objective. ²²⁵Ac radiopharmaceuticals have tremendous potential for targeted alpha therapy, however, ²²⁵Ac (t _1/2 = 9.9 d) lacks direct gamma emissions for in vivo imaging. ²²⁶Ac (t _1/2 = 29.4 h) is a promising element-equivalent matched diagnostic radionuclide for preclinical evaluation of ²²⁵Ac radiopharmaceuticals. ²²⁶Ac has two gamma emissions (158 keV and 230 keV) suitable for SPECT imaging. This work is the first feasibility study for in vivo quantitative ²²⁶Ac SPECT imaging and validation of activity estimation. Approach. ²²⁶Ac was produced at TRIUMF (Vancouver, Canada) with its Isotope Separator and Accelerator (ISAC) facility. [²²⁶Ac]Ac³⁺ was radiolabelled with the bioconjugate crown-TATE developed for therapeutic targeting of neuroendocrine tumours. Mice with AR42J tumour xenografts were injected with either 2 MBq of [²²⁶Ac]Ac-crown-TATE or 4 MBq of free [²²⁶Ac]Ac³⁺ activity and were scanned at 1, 2.5, 5, and 24 h post injection in a preclinical microSPECT/CT. Quantitative SPECT images were reconstructed from the 158 keV and 230 keV photopeaks with attenuation, background, and scatter corrections. Image-based ²²⁶Ac activity measurements were assessed from volumes of interest within tumours and organs of interest. Imaging data was compared with ex vivo biodistribution measured via gamma counter. Main results. We present, to the best of our knowledge, the first ever in vivo quantitative SPECT images of ²²⁶Ac activity distributions. Time-activity curves derived from SPECT images quantify the in vivo biodistribution of [²²⁶Ac]Ac-crown-TATE and free [²²⁶Ac]Ac³⁺ activity. Image-based activity measurements in the tumours and organs of interest corresponded well with ex vivo biodistribution measurements. Significance. Here in, we established the feasibility of in vivo ²²⁶Ac quantitative SPECT imaging for accurate measurement of actinium biodistribution in a preclinical model. This imaging method could facilitate more efficient development of novel actinium labelled compounds by providing accurate quantitative in vivo pharmacokinetic information essential for estimating toxicities, dosimetry, and therapeutic potency.

Superior bifunctional chelating ligands, which can sequester both α-emitting radionuclides (²²⁵Ac, ²¹³Bi) and their diagnostic companions (¹⁵⁵Tb, ¹¹¹In), remain a formidable challenge to translating targeted alpha therapy, with complementary diagnostic imaging, to the clinic. H₄noneupaX, a chelating ligand with an unusual diametrically opposed arrangement of pendant donor groups, has been developed to this end. H₄noneunpaX preferentially complexes Ln³⁺ and An³⁺ ions, forming thermodynamically stable (pLa = 17.8, pLu = 21.3) and kinetically inert complexes─single isomeric species by nuclear magnetic resonance and density functional theory. Metal binding versatility demonstrated in radiolabeling [¹¹¹In]In³⁺, [¹⁵⁵Tb]Tb³⁺, [¹⁷⁷Lu]Lu³⁺, and [²²⁵Ac]Ac³⁺ achieved high molar activities under mild conditions. Efficient, scalable synthesis enabled in vivo evaluation of bifunctional H₄noneunpaX conjugated to two octreotate peptides targeting neuroendocrine tumors. Single photon emission computed tomography/CT and biodistribution studies of ¹⁵⁵Tb-radiotracers in AR42J tumor-bearing mice showed excellent image contrast, good tumor uptake, and high in vivo stability. H₄noneunpaX shows significant potential for theranostic applications involving ²²⁵Ac/¹⁵⁵Tb or ¹⁷⁷Lu/¹⁵⁵Tb.

^44gSc presents a particular interest for application in nuclear medicine for positron emission tomography (PET) due to its favorable nuclear decay properties (t_1/2 = 3.97 h, E_max = 1.47 MeV, branching ratio 94.3% β⁺). Its nuclear isomer ^44mSc (t_1/2 = 58.61 h) decays by isomeric transition (IT) into ^44gSc, accompanied by ≈12% of conversion electron emission, which can cause a partial release of the daughter ^44gSc from the chelate complex. A 13 MeV cyclotron at TRIUMF was used to produce both ^44mSc and ^44gSc via the ^natCa(p,n)^44m,gSc reaction. A ^44mSc/^44gSc generator was designed by using a Strata C-18E cartridge. After several tested systems, a successful separation method was developed using DOTATOC as a chelator, a Strata C-18E cartridge as a generator column, and an elution solution of 0.1 M NH₄-α-HIB. The yield of the generator with the daughter ^44gSc release was equal to 9.8 ± 1.0% (or ≈80% per portion of conversion). This result shows the important role of after-effects in the design of radionuclide generators. Nuclear cross-section calculations were applied using the TALYS code to allow for the determination of the most promising alternative routes for ^44mSc production, which will enable the development of a full-scale ^44mSc/^44gSc radionuclide generator based on after-effects.

Terbium radioisotopes (¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb) offer a unique class of radionuclides which encompass all four medicinally relevant nuclear decay modalities (α, β⁺, γ, β⁻/e⁻), and show high potential for the development of element-matched theranostic radiopharmaceuticals. The goal of this study was to design, synthesise, and evaluate the suitability of crown-TATE as a new peptide-conjugate for radiolabelling of [¹⁵⁵Tb]Tb³⁺ and [¹⁶¹Tb]Tb³⁺, and to assess the imaging and pharmacokinetic properties of each radiotracer in tumour-bearing mice. [¹⁵⁵Tb]Tb-crown-TATE and [¹⁶¹Tb]Tb-crown-TATE were prepared efficiently under mild conditions, and exhibited excellent stability in human serum (>99.5% RCP over 7 days). Longitudinal SPECT/CT images were acquired for ¹⁵⁵Tb- and ¹⁶¹Tb- labelled crown-TATE in male NRG mice bearing AR42J tumours. The radiotracers, [¹⁵⁵Tb]Tb-crown-TATE and [¹⁶¹Tb]Tb-crown-TATE, showed high tumour targeting (32.6 and 30.0 %ID/g, respectively) and minimal retention in non-target organs at 2.5 h post-administration. Biodistribution studies confirmed the SPECT/CT results, showing high tumour uptake (38.7 ± 8.0 %ID/g and 38.5 ± 3.5 %ID/g, respectively) and favourable tumour-to-background ratios. Blocking studies further confirmed SSTR2-specific tumour accumulation. Overall, these findings suggest that crown-TATE has great potential for element-matched molecular imaging and radionuclide therapy using ¹⁵⁵Tb and ¹⁶¹Tb.