Site-Specific 68Ga Radiolabeling of Trastuzumab Fab via Methionine for ImmunoPET Imaging.

Bioconjugates of antibodies and their derivatives radiolabeled with ß+-emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [68Ga]Ga3+, to yield the novel PET radiotracers, [68Ga]Ga-DFO-WT and [68Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [68Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [68Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [68Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [68Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody-chelator conjugates with favorable pharmacokinetics for PET imaging.

Probing Unexpected Reactivity in Radiometal Chemistry: Indium-111-Mediated Hydrolysis of Hybrid Cyclen-Hydroxypyridinone Ligands.

Chelators based on hydroxypyridinones have utility in incorporating radioactive metal ions into diagnostic and therapeutic agents used in nuclear medicine. Over the course of our hydroxypyridinone studies, we have prepared two novel chelators, consisting of a cyclen (1,4,7,10-tetraazacyclododecane) ring bearing two pendant hydroxypyridinone groups, appended via methylene acetamide motifs at either the 1,4-positions (L1) or 1,7-positions (L2) of the cyclen ring. In radiolabeling reactions of L1 or L2 with the γ-emitting radioisotope, [111In]In3+, we have observed radiometal-mediated hydrolysis of a single amide group of either L1 or L2. The reaction of either [111In]In3+ or [natIn]In3+ with either L1 or L2, in aqueous alkaline solutions at 80 °C, initially results in formation of [In(L1)]+ or [In(L2)]+, respectively. Over time, each of these species undergoes In3+-mediated hydrolysis of a single amide group to yield species in which In3+ remains coordinated to the resultant chelator, which consists of a cyclen ring bearing a single hydroxypyridinone group and a single carboxylate group. The reactivity toward hydrolysis is higher for the L1 complex compared to that for the L2 complex. Density functional theory calculations corroborate these experimental findings and importantly indicate that the activation energy required for the hydrolysis of L1 is significantly lower than that required for L2. This is the first reported example of a chelator undergoing radiometal-mediated hydrolysis to form a radiometalated complex. It is possible that metal-mediated amide bond cleavage is a source of instability in other radiotracers, particularly those in which radiometal complexation occurs in aqueous, basic solutions at high temperatures. This study highlights the importance of appropriate characterization of radiolabeled products.

Technetium Nitrido Complexes of Tetradentate Thiosemicarbazones: Kit-Based Radiolabeling, Characterization, and In Vivo Evaluation.

Bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators have demonstrated utility in nuclear medicine. In particular, the 64Cu2+ complexes have been extensively developed for hypoxia imaging and molecular imaging of peptide and protein markers of disease. However, the chemistry and application of bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators in combination with 99mTc, the most widely used radionuclide in nuclear medicine, is underexplored. Herein, a series of bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators were radiolabeled with nitrido-technetium-99m in an optimized one-pot synthesis from [99mTc]TcO4-. Optimization of the radiochemical syntheses allowed for production of the complexes in >90% radiochemical conversion with apparent molar activities of 3.3-5 GBq/µmol. Competition experiments demonstrated the excellent stability of the complexes. The nitrido-technetium-99 complexes were synthesized, and the chemical identities were investigated using mass spectrometry, spectroscopy, and density functional theory calculations. Complexation of nitrido-rhenium(V) was achieved with the N4-dialkylated bis(thiosemicarbazones). Planar imaging and ex vivo biodistribution studies of the five 99mTc complexes were conducted on healthy BALB/c mice to determine in vivo behavior. The lipophilic nature of the complexes resulted in uptake of 1.6-5.7% ID g-1 in the brain at 2 min postinjection and retention of 0.4-1.7% ID g-1 at 15 min postinjection. The stability of the complexes and the biodistribution data demonstrate that these chelators are ideal platforms for future production of radiopharmaceutical candidates.

Diphosphine Bioconjugates via Pt(0)-Catalyzed Hydrophosphination. A Versatile Chelator Platform for Technetium-99m and Rhenium-188 Radiolabeling of Biomolecules.

The ability to append targeting biomolecules to chelators that efficiently coordinate to the diagnostic imaging radionuclide, 99mTc, and the therapeutic radionuclide, 188Re, can potentially enable receptor-targeted "theranostic" treatment of disease. Here we show that Pt(0)-catalyzed hydrophosphination reactions are well-suited to the derivatization of diphosphines with biomolecular moieties enabling the efficient synthesis of ligands of the type Ph2PCH2CH2P(CH2CH2-Glc)2 (L, where Glc = a glucose moiety) using the readily accessible Ph2PCH2CH2PH2 and acryl derivatives. It is shown that hydrophosphination of an acrylate derivative of a deprotected glucose can be carried out in aqueous media. Furthermore, the resulting glucose-chelator conjugates can be radiolabeled with either 99mTc(V) or 188Re(V) in high radiochemical yields (>95%), to furnish separable mixtures of cis- and trans-[M(O)2L2]+ (M = Tc, Re). Single photon emission computed tomography (SPECT) imaging and ex vivo biodistribution in healthy mice show that each isomer possesses favorable pharmacokinetic properties, with rapid clearance from blood circulation via a renal pathway. Both cis-[99mTc(O)2L2]+ and trans-[99mTc(O)2L2]+ exhibit high stability in serum. This new class of functionalized diphosphine chelators has the potential to provide access to receptor-targeted dual diagnostic/therapeutic pairs of radiopharmaceutical agents, for molecular 99mTc SPECT imaging and 188Re systemic radiotherapy.

Versatile Diphosphine Chelators for Radiolabeling Peptides with 99mTc and 64Cu.

We have developed a diphosphine (DP) platform for radiolabeling peptides with 99mTc and 64Cu for molecular SPECT and PET imaging, respectively. Two diphosphines, 2,3-bis(diphenylphosphino)maleic anhydride (DPPh) and 2,3-bis(di-p-tolylphosphino)maleic anhydride (DPTol), were each reacted with a Prostate Specific Membrane Antigen-targeted dipeptide (PSMAt) to yield the bioconjugates DPPh-PSMAt and DPTol-PSMAt, as well as an integrin-targeted cyclic peptide, RGD, to yield the bioconjugates DPPh-RGD and DPTol-RGD. Each of these DP-PSMAt conjugates formed geometric cis/trans-[MO2(DPX-PSMAt)2]+ (M = 99mTc, 99gTc, natRe; X = Ph, Tol) complexes when reacted with [MO2]+ motifs. Furthermore, both DPPh-PSMAt and DPTol-PSMAt could be formulated into kits containing reducing agent and buffer components, enabling preparation of the new radiotracers cis/trans-[99mTcO2(DPPh-PSMAt)2]+ and cis/trans-[99mTcO2(DPTol-PSMAt)2]+ from aqueous 99mTcO4- in 81% and 88% radiochemical yield (RCY), respectively, in 5 min at 100 °C. The consistently higher RCYs observed for cis/trans-[99mTcO2(DPTol-PSMAt)2]+ are attributed to the increased reactivity of DPTol-PSMAt over DPPh-PSMAt. Both cis/trans-[99mTcO2(DPPh-PSMAt)2]+ and cis/trans-[99mTcO2(DPTol-PSMAt)2]+ exhibited high metabolic stability, and in vivo SPECT imaging in healthy mice revealed that both new radiotracers cleared rapidly from circulation, via a renal pathway. These new diphosphine bioconjugates also furnished [64Cu(DPX-PSMAt)2]+ (X = Ph, Tol) complexes rapidly, in a high RCY (>95%), under mild conditions. In summary, the new DP platform is versatile: it enables straightforward functionalization of targeting peptides with a diphosphine chelator, and the resulting bioconjugates can be simply radiolabeled with both the SPECT and PET radionuclides, 99mTc and 64Cu, in high RCYs. Furthermore, the DP platform is amenable to derivatization to either increase the chelator reactivity with metallic radioisotopes or, alternatively, modify the radiotracer hydrophilicity. Functionalized diphosphine chelators thus have the potential to provide access to new molecular radiotracers for receptor-targeted imaging.

In Vivo Trafficking of the Anticancer Drug Tris(8-Quinolinolato) Gallium (III) (KP46) by Gallium-68/67 PET/SPECT Imaging.

KP46 (tris(hydroxyquinolinato)gallium(III)) is an experimental, orally administered anticancer drug. Its absorption, delivery to tumours, and mode of action are poorly understood. We aimed to gain insight into these issues using gallium-67 and gallium-68 as radiotracers with SPECT and PET imaging in mice. [67Ga]KP46 and [68Ga]KP46, compared with [68Ga]gallium acetate, were used for logP measurements, in vitro cell uptake studies in A375 melanoma cells, and in vivo imaging in mice bearing A375 tumour xenografts up to 48 h after intravenous (tracer level) and oral (tracer and bulk) administration. 68Ga was more efficiently accumulated in A375 cells in vitro when presented as [68Ga]KP46 than as [68Ga]gallium acetate, but the reverse was observed when intravenously administered in vivo. After oral administration of [68/67Ga]KP46, absorption of 68Ga and 67Ga from the GI tract and delivery to tumours were poor, with the majority excreted in faeces. By 48 h, low but measurable amounts were accumulated in tumours. The distribution in tissues of absorbed radiogallium and octanol extraction of tissues suggested trafficking as free gallium rather than as KP46. We conclude that KP46 likely acts as a slow releaser of gallium ions which are inefficiently absorbed from the GI tract and trafficked to tissues, including tumour and bone.

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.

N-Centered Tripodal Phosphine Re(V) and Tc(V) Oxo Complexes: Revisiting a [3 + 2] Mixed-Ligand Approach.

N-Triphos derivatives (NP3R, R = alkyl, aryl) and asymmetric variants (NP2RXR', R' = alkyl, aryl, X = OH, NR2, NRR') are an underexplored class of tuneable, tripodal ligands in relation to the coordination chemistry of Re and Tc for biomedical applications. Mixed-ligand approaches are a flexible synthetic route to obtain Tc complexes of differing core structures and physicochemical properties. Reaction of the NP3Ph ligand with the Re(V) oxo precursor [ReOCl3(PPh3)2] generated the bidentate complex [ReOCl3(κ2-NP2PhOHAr)], which possesses an unusual AA'BB'XX' spin system with a characteristic second-order NMR lineshape that is sensitive to the bi- or tridentate nature of the coordinating diphosphine unit. The use of the asymmetric NP2PhOHAr ligand resulted in the formation of both bidentate and tridentate products depending on the presence of base. The tridentate Re(V) complex [ReOCl2(κ3-NP2PhOAr)] has provided the basis of a new reactive "metal-fragment" for further functionalization in [3 + 2] mixed-ligand complexes. The synthesis of [3 + 2] complexes with catechol-based π-donors could also be achieved under one-pot, single-step conditions from Re(V) oxo precursors. Analogous complexes can also be synthesized from suitable 99Tc(V) precursors, and these complexes have been shown to exhibit highly similar structural properties through spectroscopic and chromatographic analysis. However, a tendency for the {MVO}3+ core to undergo hydrolysis to the {MVO2}+ core has been observed both in the case of M = Re and markedly for M = 99Tc complexes. It is likely that controlling this pathway will be critical to the generation of further stable Tc(V) derivatives.

A General Design Strategy Enabling the Synthesis of Hydrolysis-Resistant, Water-Stable Titanium(IV) Complexes.

Despite its prevalence in the environment, the chemistry of the Ti4+ ion has long been relegated to organic solutions or hydrolyzed TiO2 polymorphs. A knowledge gap in stabilizing molecular Ti4+ species in aqueous environments has prevented the use of this ion for various applications such as radioimaging, design of water-compatible metal-organic frameworks (MOFs), and aqueous-phase catalysis applications. Herein, we show a thorough thermodynamic screening of bidentate chelators with Ti4+ in aqueous solution, as well as computational and structural analyses of key compounds. In addition, the hexadentate analogues of catechol (benzene-1,2-diol) and deferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone), TREN-CAM and THPMe respectively, were assessed for chelation of the 45 Ti isotope (t1/2 =3.08 h, ß+ =85 %, Eß+ =439 keV) towards positron emission tomography (PET) imaging applications. Both were found to have excellent capacity for kit-formulation, and [45 Ti]Ti-TREN-CAM was found to have remarkable stability in vivo.

New Bifunctional Chelators Incorporating Dibromomaleimide Groups for Radiolabeling of Antibodies with Positron Emission Tomography Imaging Radioisotopes.

Positron Emission Tomography (PET) imaging with antibody-based contrast agents frequently uses the radioisotopes [64Cu]Cu2+ and [89Zr]Zr4+. The macrobicyclic chelator commonly known as sarcophagine (sar) is ideal for labeling receptor-targeted biomolecules with [64Cu]Cu2+. The siderophore chelator, desferrioxamine-B (dfo), has been widely used to incorporate [89Zr]Zr4+ into antibodies. Here, we describe new bifunctional chelators of sar and dfo: these chelators have been functionalized with dibromomaleimides (dbm), that enable site-specific and highly stable attachment of molecular cargoes to reduced, solvent-accessible, interstrand native disulfide groups. The new sar-dbm and dfo-dbm derivatives can be easily conjugated with the IgG antibody trastuzumab via reaction with reduced interstrand disulfide groups to give site-specifically modified dithiomaleamic acid (dtm) conjugates, sar-dtm-trastuzumab and dfo-dtm-trastuzumab, in which interstrand disulfides are rebridged covalently with a small molecule linker. Both sar- and dfo-dtm-trastuzumab conjugates have been radiolabeled with [64Cu]Cu2+ and [89Zr]Zr4+, respectively, in near quantitative radiochemical yield (>99%). Serum stability studies, in vivo PET imaging, and biodistribution analyses using these radiolabeled immunoconjugates demonstrate that both [64Cu]Cu-sar-dtm-trastuzumab and [89Zr]Zr-dfo-dtm-trastuzumab possess high stability in biological milieu. Dibromomaleimide technology can be easily applied to enable stable, site-specific attachment of radiolabeled chelators, such as sar and dfo, to native interstrand disulfide regions of antibodies, enabling tracking of antibodies with PET imaging.