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.

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.

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.

Non-invasive radionuclide imaging of trace metal trafficking in health and disease: "PET metallomics".

Several specific metallic elements must be present in the human body to maintain health and function. Maintaining the correct quantity (from trace to bulk) and location at the cell and tissue level is essential. The study of the biological role of metals has become known as metallomics. While quantities of metals in cells and tissues can be readily measured in biopsy and autopsy samples by destructive analytical techniques, their trafficking and its role in health and disease are poorly understood. Molecular imaging with radionuclides - positron emission tomography (PET) and single photon emission computed tomography (SPECT) - is emerging as a means to non-invasively study the acute trafficking of essential metals between organs, non-invasively and in real time, in health and disease. PET scanners are increasingly widely available in hospitals, and methods for producing radionuclides of some of the key essential metals are developing fast. This review summarises recent developments in radionuclide imaging technology that permit such investigations, describes the radiological and physicochemical properties of key radioisotopes of essential trace metals and useful analogues, and introduces current and potential future applications in preclinical and clinical investigations to study the biology of essential trace metals in health and disease.

To chelate thallium(I) - synthesis and evaluation of Kryptofix-based chelators for 201Tl.

While best known for its toxic properties, thallium has also been explored for applications in nuclear diagnostics and medicine. Indeed, [201Tl]TlCl has been used extensively for nuclear imaging in the past before it was superceded by other radionuclides such as 99mTc. One reason for this loss of interest is the severe lack of suitable organic chelators able to effectively coordinate ionic forms of Tl and deliver it to specific diseased tissue by means of attached biological vectors. Herein, we describe the synthesis and characterisation of a series of Kryptofix 222-based chelators that can be radiolabelled with 201Tl(I) in high radiochemical yields at ambient temperature. We demonstrate that from these simple chelators, targeted derivatives are readily accessible and describe the synthesis and preliminary biological evaluation of a PSMA-targeted 201Tl-labelled Kryptofix 222-peptide conjugate. While the Kryptofix system is demonstrably capable of binding the thallium cation, no PSMA-mediated cell-uptake could be detected with the PSMA conjugate, suggesting that this targeting moiety may not be ideal for use in conjunction with 201Tl.

Quantifying the Movement Characteristics of Australian Football League Women's Competition.

ABSTRACT: Thornton, HR, Armstrong, CR, Gamble, T, Rigby, A, Johnston, RD, and Duthie, GM. Quantifying the movement characteristics of Australian Football League Women's competition. J Strength Cond Res 36(12): 3415-3421, 2022-The purpose is to provide an overview of the externally measured movement characteristics of Australian Football League Women's (AFLW) competition, and the variability in this between matches. A range of movement variables were measured from 28 athletes who wore 10-Hz global positioning system devices during games and were summarized according to positional groups. The variance in each athlete's match loads for each round were expressed using standardized scores ( z -scores), and the change compared with the first game played was calculated and expressed as a standard deviation. Furthermore, using the raw export, moving means (1-10 minutes) of speed (m·min -1 ) and acceleration (m·s -2 ) were calculated. Following log transformation of the maximal means, intercept and slopes were calculated. Linear mixed models identified differences between positional groups for match loads, and intercept and slopes. Effects were described using standardized effect sizes (ESs) and magnitude-based decisions. There were no substantial and unclear differences between positional groups for match loads (ES range; ±confidence limits = 0.10-0.80; ±0.30-4.30) and for intercept and slopes (ES range; 0.04-0.44; ±0.52-2.11). Large within-athlete variation in match demands between rounds was observed ( z -score up to -1.8 SD for distance), and the maximal means for speed and acceleration demonstrate the extensive physical demands of AFLW competition. These data describe the intense and variable physical demands of AFLW competition, and further provide novel information regarding the maximal mean intensities and intercept and slopes, which should assist practitioners in planning and prescribing training in preparation for competition.

Targeted Auger electron-emitter therapy: Radiochemical approaches for thallium-201 radiopharmaceuticals.

INTRODUCTION: Thallium-201 is a radionuclide that has previously been used clinically for myocardial perfusion scintigraphy. Although in this role it has now been largely replaced by technetium-99 m radiopharmaceuticals, thallium-201 remains attractive in the context of molecular radionuclide therapy for cancer micrometastases or single circulating tumour cells. This is due to its Auger electron (AE) emissions, which are amongst the highest in total energy and number per decay for AE-emitters. Currently, chemical platforms to achieve this potential through developing thallium-201-labelled targeted radiopharmaceuticals are not available. Here, we describe convenient methods to oxidise [201Tl]Tl(I) to chelatable [201Tl]Tl(III) and identify challenges in stable chelation of thallium to support future synthesis of effective [201Tl]-labelled radiopharmaceuticals. METHODS: A plasmid pBR322 assay was carried out to determine the DNA damaging properties of [201Tl]Tl(III). A range of oxidising agents (ozone, oxygen, hydrogen peroxide, chloramine-T, iodogen, iodobeads, trichloroisocyanuric acid) and conditions (acidity, temperature) were assessed using thin layer chromatography. Chelators EDTA, DTPA and DOTA were investigated for their [201Tl]Tl(III) radiolabelling efficacy and complex stability. RESULTS: Isolated plasmid studies demonstrated that [201Tl]Tl(III) can induce single and double-stranded DNA breaks. Iodo-beads, iodogen and trichloroisocyanuric acid enabled more than 95% conversion from [201Tl]Tl(I) to [201Tl]Tl(III) under conditions compatible with future biomolecule radiolabelling (mild pH, room temperature and post-oxidation removal of oxidising agent). Although chelation of [201Tl]Tl(III) was possible with EDTA, DTPA and DOTA, only radiolabeled DOTA showed good stability in serum. CONCLUSIONS: Decay of [201Tl]Tl(III) in proximity to DNA causes DNA damage. Iodobeads provide a simple, mild method to convert thallium-201 from a 1+ to 3+ oxidation state and [201Tl]Tl(III) can be chelated by DOTA with moderate stability. Of the well-established chelators evaluated, DOTA is most promising for future molecular radionuclide therapy using thallium-201; nevertheless, a new generation of chelating agents offering resistance to reduction and dissociation of [201Tl]Tl(III) complexes is required.

Protocols for Dual Tracer PET/SPECT Preclinical Imaging.

BACKGROUND: Multi-tracer PET/SPECT imaging enables different modality tracers to be present simultaneously, allowing multiple physiological processes to be imaged in the same subject, within a short time-frame. Fluorine-18 and technetium-99m, two commonly used PET and SPECT radionuclides, respectively, possess different emission profiles, offering the potential for imaging one in the presence of the other. However, the impact of the presence of each radionuclide on scanning the other could be significant and lead to confounding results. Here we use combinations of 18F and 99mTc to explore the challenges posed by dual tracer PET/SPECT imaging, and investigate potential practical ways to overcome them. METHODS: Mixed-radionuclide 18F/99mTc phantom PET and SPECT imaging experiments were carried out to determine the crossover effects of each radionuclide on the scans using Mediso nanoScan PET/CT and SPECT/CT small animal scanners. RESULTS: PET scan image quality and quantification were adversely affected by 99mTc activities higher than 100 MBq due to a high singles rate increasing dead-time of the detectors. Below 100 MBq 99mTc, PET scanner quantification accuracy was preserved. SPECT scan image quality and quantification were adversely affected by the presence of 18F due to Compton scattering of 511 keV photons leading to over-estimation of 99mTc activity and increased noise. However, 99mTc:18F activity ratios of > 70:1 were found to mitigate this effect completely on the SPECT. A method for correcting for Compton scatter was also explored. CONCLUSION: Suitable combinations of injection sequence and imaging sequence can be devised to meet specific experimental multi-tracer imaging needs, with only minor or insignificant effects of each radionuclide on the scan of the other.

Preparing for an Australian Football League Women's League Season.

The aims were to investigate the externally measured weekly loads, and the distribution intensity relative to the 1-min maximal mean (MM) intensity of matches. Athletes (n = 28) wore 10 Hz GNSS devices during training and matches. For the descriptive analysis, a range of movement variables were collected, including total distance, high-speed distance, very high-speed distance, acceleration, and acceleration load. Using raw GNSS files, 1-min moving averages were calculated for speed (m·min-1) and acceleration (m·s-2), and were multiplied by time, specifying total distance (m), and by body mass to quantify impulse (kN·s-1). The distribution of distance and impulse accumulated at varied intensities relative to MMs was calculated, with percentages ranging from zero to 110%. Drills were categorized as either; warm-ups, skill drills, games (i.e., small-sided games), conditioning and matches. Linear mixed models determined if the distribution of intensity within each threshold (>50%) varied between drill types and matches, and if the distribution within drill types varied across the season. Effects were described using standardized effect sizes (ES) and 90% confidence limits (CL). Compared to matches, a higher proportion of distance was accumulated at 50% of the MM within warm-ups and conditioning (ES range 0.86-1.14). During matches a higher proportion of distance was accumulated at 60% of MM when compared to warms ups, skill drills and conditioning (0.73-1.87). Similarly, greater proportion of distance was accumulated between 70 and 100% MM in matches compared to skill drills and warm-ups (1.05-3.93). For impulse, matches had a higher proportion between 60 and 80% of the MM compared to conditioning drills (0.91-3.23). There were no other substantial differences in the proportion of impulse between matches and drill types. When comparing phases, during competition there was a higher proportion of distance accumulated at 50% MM than general preparation (1.08). A higher proportion of distance was covered at higher intensities within matches compared to drills. The proportion of impulse was higher between 60 and 80% MM within matches compared to conditioning. Practitioners can therefore ensure athletes are not only exposed to the intensities common within competition, but also the volume accumulated is comparable, which may have positive performance outcomes, but is also extremely important in the return to play process.

Greater Association of Relative Thresholds Than Absolute Thresholds With Noncontact Lower-Body Injury in Professional Australian Rules Footballers: Implications for Sprint Monitoring.

Sprint capacity is an important attribute for team-sport athletes, yet the most appropriate method to analyze it is unclear. PURPOSE: To examine the relationship between sprint workloads using relative versus absolute thresholds and lower-body soft-tissue and bone-stress injury incidence in professional Australian rules football. METHODS: Fifty-three professional Australian rules football athletes' noncontact soft-tissue and bone-stress lower-body injuries (N = 62) were recorded, and sprint workloads were quantified over ∼18 months using the global positioning system. Sprint volume (m) and exposures (n) were determined using 2 methods: absolute (>24.9 km·h-1) and relative (≥75%, ≥80%, ≥85%, ≥90%, ≥95% of maximal velocity). Relationships between threshold methods and injury incidence were assessed using logistic generalized additive models. Incidence rate ratios and model performances' area under the curve were reported. RESULTS: Mean (SD) maximal velocity for the group was 31.5 (1.4), range 28.6 to 34.9 km·h-1. In comparing relative and absolute thresholds, 75% maximal velocity equated to ~1.5 km·h-1 below the absolute speed threshold, while 80% and 85% maximal velocity were 0.1 and 1.7 km·h-1 above the absolute speed threshold, respectively. Model area under the curve ranged from 0.48 to 0.61. Very low and very high cumulative sprint loads ≥80% across a 4-week period, when measured relatively, resulted in higher incidence rate ratios (2.54-3.29), than absolute thresholds (1.18-1.58). DISCUSSION: Monitoring sprinting volume relative to an athlete's maximal velocity should be incorporated into athlete monitoring systems. Specifically, quantifying the distance covered at >80% maximal velocity will ensure greater accuracy in determining sprint workloads and associated injury risk.