Rearmed Bifunctional Chelating Ligand for 225Ac/155Tb Precision-Guided Theranostic Radiopharmaceuticals─H4noneunpaX.

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.

Preclinical Evaluation of [155/161Tb]Tb-Crown-TATE-A Novel SPECT Imaging Theranostic Agent Targeting Neuroendocrine Tumours.

Terbium radioisotopes (149Tb, 152Tb, 155Tb, 161Tb) offer a unique class of radionuclides which encompass all four medicinally relevant nuclear decay modalities (α, ß+, γ, ß-/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.

Toward Bifunctional Chelators for Thallium-201 for Use in Nuclear Medicine.

Auger electron therapy exploits the cytotoxicity of low-energy electrons emitted during radioactive decay that travel very short distances (typically <1 µm). 201Tl, with a half-life of 73 h, emits ∼37 Auger and other secondary electrons per decay and can be tracked in vivo as its gamma emissions enable SPECT imaging. Despite the useful nuclear properties of 201Tl, satisfactory bifunctional chelators to incorporate it into bioconjugates for molecular targeting have not been developed. H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa are multidentate N- and O-donor chelators that have previously been shown to have high affinity for 111In, 177Lu, and 89Zr. Herein, we report the synthesis and serum stability of [nat/201Tl]Tl3+ complexes with H4pypa, H5decapa, H4neunpa-NH2, and H4noneunpa. All ligands quickly and efficiently formed complexes with [201Tl]Tl3+ that gave simple single-peak radiochromatograms and showed greatly improved serum stability compared to DOTA and DTPA. [natTl]Tl-pypa was further characterized using nuclear magnetic resonance spectroscopy (NMR), mass spectroscopy (MS), and X-ray crystallography, showing evidence of the proton-dependent presence of a nine-coordinate complex and an eight-coordinate complex with a pendant carboxylic acid group. A prostate-specific membrane antigen (PSMA)-targeting bioconjugate of H4pypa was synthesized and radiolabeled. The uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive and PSMA-negative prostate cancer cells was evaluated in vitro and showed evidence of bioreductive release of 201Tl and cellular uptake characteristic of unchelated [201Tl]TlCl. SPECT/CT imaging was used to probe the in vivo biodistribution and stability of [201Tl]Tl-pypa-PSMA. In healthy animals, [201Tl]Tl-pypa-PSMA did not show the myocardial uptake that is characteristic of unchelated 201Tl. In mice bearing DU145 PSMA-positive and PSMA-negative prostate cancer xenografts, the uptake of [201Tl]Tl-pypa-PSMA in DU145 PSMA-positive tumors was higher than that in DU145 PSMA-negative tumors but insufficient for useful tumor targeting. We conclude that H4pypa and related ligands represent an advance compared to conventional radiometal chelators such as DOTA and DTPA for Tl3+ chelation but do not resist dissociation for long periods in the biological environment due to vulnerability to reduction of Tl3+ and subsequent release of Tl+. However, this is the first report describing the incorporation of [201Tl]Tl3+ into a chelator-peptide bioconjugate and represents a significant advance in the field of 201Tl-based radiopharmaceuticals. The design of the next generation of chelators must include features to mitigate this susceptibility to bioreduction, which does not arise for other trivalent heavy radiometals.

H2ampa─Versatile Chelator for [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3.

A new decadentate chelator, H2ampa, was designed to be a potential radiopharmaceutical chelator component. The chelator involves both amide and picolinate functional groups on a large non-macrocyclic, ether-bridged backbone. With its large scaffold, H2ampa was paired with [nat/203Pb]Pb2+, [nat/213Bi]Bi3+, and natLa3+/[225Ac]Ac3+ ions. Nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry were used to study the non-radioactive metal complexes. A single crystal of [Bi(ampa)](NO3) was obtained; its asymmetric, 10-coordinate complex structure was revealed by X-ray diffraction. Optimal conformations of the metal complexes were assessed by density functional theory studies to provide further structural information. Solution studies providing thermodynamic insights into metal complex formation revealed H2ampa coordinated Bi3+, Pb2+, and La3+ ions to obtain pM values of 26, 14.8, and 15.1, respectively. Preliminary concentration-dependent radiolabeling experiments were carried out between H2ampa and three different radiometals to evaluate their compatibility for radiopharmaceutical applications. The chelator radiolabeled [203Pb]Pb2+, [213Bi]Bi3+, and [225Ac]Ac3+ in short reaction times (7-30 min), at dilute concentrations, and under mild conditions. Thus, H2ampa was proven to be a versatile chelator able to well coordinate a small range of radiometals frequently considered to be alpha therapeutic candidates.

Getting a lead on Pb2+-amide chelators for 203/212Pb radiopharmaceuticals.

Amide-based chelators DTPAm, EGTAm and ampam were synthesized to investigate which chelator most ideally coordinates [nat/203Pb]Pb2+ ions for potential radiopharmaceutical applications. 1H NMR spectroscopy was used to study each metal-ligand complex in the solution state. The 1H NMR spectrum of [Pb(DTPAm)]2+ revealed minimal isomerization and fluxional behaviour compared to [Pb(EGTAm)]2+ and [Pb(ampam)]2+, both of which showed fewer spectral changes indicative of less static behaviour. The solid-state coordination properties of each complex were also examined from single crystal structures that were studied by X-ray diffraction (XRD). In the solid-state, octadentate DTPAm coordinated Pb2+ to form an eight-coordinate hemidirected complex; octadentate EGTAm coordinated Pb2+ forming a ten-coordinate holodirected complex with a bidentate NO3- ion also coordinated to the metal centre; decadentate ampam completely encapsulated the Pb2+ ion to form a ten-coordinate holodirected complex with a C2 axis of symmetry. Potentiometric titrations were carried out to assess the thermodynamic stability of each metal-ligand complex. The pM values obtained for [Pb(DTPAm)]2+, [Pb(EGTAm)]2+ and [Pb(ampam)]2+ were 9.7, 7.2 and 10.2, respectively. The affinity of each chelator for Pb2+ ions was tested by [203Pb]Pb2+ radiolabeling studies to evaluate their prospects as chelators for [203/212Pb]Pb2+-based radiopharmaceuticals. DTPAm radiolabeled [203Pb]Pb2+ ions achieving molar activities as high as 3.5 MBq µmol-1 within 15 minutes, at 25 °C, whereas EGTAm and ampam produced lower molar activities of 0.25 MBq µmol-1 within 30 minutes, at 37 °C. EGTAm and ampam were therefore deemed unsuitable for [203/212Pb]Pb2+-based radiopharmaceutical applications, while DTPAm warrants further studies.

H2CHXhox: Rigid Cyclohexane-Reinforced Nonmacrocyclic Chelating Ligand for [nat/67/68Ga]Ga3.

A rigid chiral acyclic chelator H2CHXhox was synthesized and evaluated for Ga3+-based radiopharmaceutical applications; it was compared to the previously reported hexadentate H2hox to determine the effect of a backbone reinforced from adding a chiral 1S,2S-trans-cyclohexane on metal complex stability, kinetic inertness, and in vivo pharmacokinetics. NMR spectroscopy and theoretical calculation revealed that [Ga(CHXhox)]+ showed a very similar coordination geometry to that of [Ga(hox)]+, and only one isomer in solution was observed by NMR spectroscopy. Solution studies showed that the modification results in a significant improvement in the exceptionally high thermodynamic stability of [Ga(hox)]+ with a 1.56 log unit increase in stability constant (logKML = 35.91(1)). More importantly, H2CHXhox showed very fast Ga3+ complexation at physiological pH 7.4, and acid-assisted Ga3+ complex dissociation kinetic studies (pH 1) in comparison with H2hox revealed a 50-fold increase of the dissociation half-life time from 73 min to 58 h. Fluorescence microscopy imaging study confirmed its cellular uptake and accumulation in endoplasmic reticulum and mitochondria. MTT studies indicated a quite low cytotoxicity of [Ga(CHXhox)]+ over a large concentration range. Dynamic PET imaging studies showed no accumulation in muscle, lungs, bone, and brain, suggesting no release of free Ga3+ ions. [68Ga][Ga(CHXhox)]+ is cleared from the mouse via hepatobiliary and renal pathways. Compared to [68Ga][Ga(hox)]+, the increased lipophilicity of [68Ga][Ga(CHXhox)]+ enhanced heart and liver uptake and decreased kidney clearance. [67Ga][Ga(CHXhox)]+ SPECT/CT imaging and biodistribution study revealed good clearance from liver to gallbladder after 90 min and finally into feces after 5 h. No decomposition or transchelation was observed over the 5 h study. These results confirmed H2CHXhox to be an obvious improvement over H2hox and an excellent candidate in this new "ox" family for the development of radiopharmaceutical compounds.

Experimental and computational studies of Criegee intermediate reactions with NH3 and CH3NH2.

Ammonia and amines are emitted into the troposphere by various natural and anthropogenic sources, where they have a significant role in aerosol formation. Here, we explore the significance of their removal by reaction with Criegee intermediates, which are produced in the troposphere by ozonolysis of alkenes. Rate coefficients for the reactions of two representative Criegee intermediates, formaldehyde oxide (CH2OO) and acetone oxide ((CH3)2COO) with NH3 and CH3NH2 were measured using cavity ring-down spectroscopy. Temperature-dependent rate coefficients, k(CH2OO + NH3) = (3.1 ± 0.5) × 10-20T2 exp(1011 ± 48/T) cm3 s-1 and k(CH2OO + CH3NH2) = (5 ± 2) × 10-19T2 exp(1384 ± 96/T) cm3 s-1 were obtained in the 240 to 320 K range. Both the reactions of CH2OO were found to be independent of pressure in the 10 to 100 Torr (N2) range, and average rate coefficients k(CH2OO + NH3) = (8.4 ± 1.2) × 10-14 cm3 s-1 and k(CH2OO + CH3NH2) = (5.6 ± 0.4) × 10-12 cm3 s-1 were deduced at 293 K. An upper limit of ≤2.7 × 10-15 cm3 s-1 was estimated for the rate coefficient of the (CH3)2COO + NH3 reaction. Complementary measurements were performed with mass spectrometry using synchrotron radiation photoionization giving k(CH2OO + CH3NH2) = (4.3 ± 0.5) × 10-12 cm3 s-1 at 298 K and 4 Torr (He). Photoionization mass spectra indicated production of NH2CH2OOH and CH3N(H)CH2OOH functionalized organic hydroperoxide adducts from CH2OO + NH3 and CH2OO + CH3NH2 reactions, respectively. Ab initio calculations performed at the CCSD(T)(F12*)/cc-pVQZ-F12//CCSD(T)(F12*)/cc-pVDZ-F12 level of theory predicted pre-reactive complex formation, consistent with previous studies. Master equation simulations of the experimental data using the ab initio computed structures identified submerged barrier heights of -2.1 ± 0.1 kJ mol-1 and -22.4 ± 0.2 kJ mol-1 for the CH2OO + NH3 and CH2OO + CH3NH2 reactions, respectively. The reactions of NH3 and CH3NH2 with CH2OO are not expected to compete with its removal by reaction with (H2O)2 in the troposphere. Similarly, losses of NH3 and CH3NH2 by reaction with Criegee intermediates will be insignificant compared with reactions with OH radicals.