[68Ga]Ga-Schizokinen, a Potential Radiotracer for Selective Bacterial Infection Imaging.

Gallium-68-labeled siderophores as radiotracers have gained interest for the development of in situ infection-specific imaging diagnostics. Here, we report radiolabeling, in vitro screening, and in vivo pharmacokinetics (PK) of gallium-68-labeled schizokinen ([68Ga]Ga-SKN) as a new potential radiotracer for imaging bacterial infections. We radiolabeled SKN with ≥95% radiochemical purity. Our in vitro studies demonstrated its hydrophilic characteristics, neutral pH stability, and short-term stability in human serum and toward transchelation. In vitro uptake of [68Ga]Ga-SKN by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis, but no uptake by Candida glabrata, C. albicans, or Aspergillus fumigatus, demonstrated its specificity to bacterial species. Whole-body [68Ga]Ga-SKN positron emission tomography (PET) combined with computerized tomography (CT) in healthy mice showed rapid renal excretion with no or minimal organ uptake. The subsequent ex vivo biodistribution resembled this fast PK with rapid renal excretion with minimal blood retention and no major organ uptake and showed some dissociation of the tracer in the urine after 60 min postinjection. These findings warrant further evaluation of [68Ga]Ga-SKN as a bacteria-specific radiotracer for infection imaging.

Efficient bone marrow irradiation and low uptake by non-haematological organs with an yttrium-90-anti-CD66 antibody prior to haematopoietic stem cell transplantation.

We report the results of a Phase I radiation dose escalation study using an yttrium-90 (90Y) labelled anti-CD66 monoclonal antibody given with standard conditioning regimen for patients receiving haematopoietic stem cell transplants for myeloid leukaemia or myeloma. The 90Y-labelled anti-CD66 was infused prior to standard conditioning. In total, 30 patients entered the trial and 29 received 90Y-labelled mAb, at infused radiation activity levels of 5, 10, 25, or 37.5 megaBequerel (MBq)/kg lean body weight. A prerequisite for receiving the 90Y-labelled mAb was favourable dosimetry determined by single-photon emission computerised tomography (SPECT) dosimetry following administration of indium-111 (111In) anti-CD66. Estimated absorbed radiation doses delivered to the red marrow demonstrated a linear relationship with the infused activity of 90Y-labelled mAb. At the highest activity level of 37.5 MBq/kg, mean estimated radiation doses for red marrow, liver, spleen, kidneys and lungs were 24.6 ± 5.6 Gy, 5.8 ± 2.7 Gy, 19.1 ± 8.0 Gy, 2.1 ± 1.1 and 2.2 ± 0.9, respectively. All patients engrafted, treatment-related mortality 1-year post-transplant was zero. Toxicities were no greater than those anticipated for similar conditioning regimens without targeted radiation. The ability to substantially intensify conditioning prior to haematopoietic stem cell transplantation without increasing toxicity warrants further testing to determine efficacy. clinicaltrials.gov identifier: NCT01521611.

Multiple-Dose Dalbavancin Regimens as the Predominant Treatment of Deep-Seated or Endovascular Infections: A Scoping Review.

Off-label use of dalbavancin for deep-seated and endovascular infections has been increasing. We performed a scoping review to evaluate the evidence for use of multiple-dose dalbavancin regimens as the predominant therapy for these indications. Predominant therapy was defined as use of dalbavancin without other concurrent antibiotics for more than half of the total treatment duration. Fifteen publications were identified; 2 were small, open-label randomized controlled trials and the remainder were retrospective observational studies or case reports. A total of 144 cases from these publications met eligibility criteria for inclusion in this review. Types of infections included osteoarticular infections, catheter-related or complicated bloodstream infections, and infective endocarditis. Overall, the evidence for use of multiple-dose regimens of dalbavancin for deep-seated and endovascular infections is limited by a paucity of data from controlled trials, heterogeneity of dosing regimens, and a lack of standardized clinical outcomes.

In vivo trafficking of a tumor-targeting IgE antibody: molecular imaging demonstrates rapid hepatobiliary clearance compared to IgG counterpart.

IgE antibodies elicit powerful immune responses, recruiting effector cells to tumors more efficiently and with greater cytotoxicity than IgG antibodies. Consequently, IgE antibodies are a promising alternative to conventional IgG-based therapies in oncology (AllergoOncology). As the pharmacokinetics of IgE antibodies are less well understood, we used molecular imaging in mice to compare the distribution and elimination of IgE and IgG antibodies targeting the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4). Anti-CSPG4 IgE and IgG1 antibodies with human Fc domains were radiolabeled with 111In. CSPG4-expressing A375 human melanoma xenografts implanted in NOD-scid IL2rg-/- mice were also engrafted with human immune cells by intravenous administration. 111In-anti-CSPG4 antibodies were administered intravenously. Their distribution was determined by single-photon emission computed tomography (SPECT) and ex vivo gamma-counting over 120 h. SPECT imaging was conducted from 0 to 60 min after antibody administration to precisely measure the early phase of IgE distribution. 111In-labeled anti-CSPG4 IgG and IgE showed serum stability in vitro of >92% after 5 days. In A375 xenograft-bearing mice, anti-CSPG4 IgE showed much faster blood clearance and higher accumulation in the liver compared to anti-CSPG4 IgG. However, tumor-to-blood and tumor-to-muscle ratios were similar between the antibody isotypes and higher compared with a non-tumor-targeting isotype control IgE. IgE excretion was much faster than IgG. In non-tumor-bearing animals, early SPECT imaging revealed a blood clearance half-life of 10 min for IgE. Using image-based quantification, we demonstrated that the blood clearance of IgE is much faster than that of IgG while the two isotypes showed comparable tumor-to-blood ratios.

An overview of nuclear medicine research in the UK and the landscape for clinical adoption.

BACKGROUND AND OBJECTIVES: Nuclear medicine contributes greatly to the clinical management of patients and experimental medicine. This report aims to (1) outline the current landscape of nuclear medicine research in the UK, including current facilities and recent or ongoing clinical studies and (2) provide information about the available pathways for clinical adoption and NHS funding (commissioning) of radiopharmaceuticals. METHODS: Evidence was obtained through database searches for UK-based nuclear medicine clinical studies and by conducting a questionnaire-based survey of UK radiopharmaceutical production facilities. A recent history of clinical commissioning, either through recommendations from the National Institute for Health and Care Excellence (NICE) or through NHS specialised services commissioning, was compiled from publicly available documents and policies. RESULTS: The collected data highlighted the UK's active nuclear medicine research community and recent investment in new facilities and upgrades. All commissioning routes favour radiopharmaceuticals that have marketing authorisation and since 2017 there has been a requirement to demonstrate both clinical and cost-effectiveness. Whilst radiopharmaceuticals for molecular radiotherapy are well suited to these commissioning pathways, diagnostic radiotracers have not historically been assessed in this manner. CONCLUSIONS: We hope that by collating this information we will provide stimulus for future discussion and consensus statements around this topic.

Optimal His-Tag Design for Efficient [99mTc(CO)3]+ and [188Re(CO)3]+ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays.

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab.

INTRODUCTION: Molecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. METHODS: THP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2-negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. RESULTS: Labelling efficiencies for [67Ga]Ga-THP-trastuzumab and [111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/µg (78-92 GBq/µmol). At 4 nM total antibody concentration and 200 × 103 cells/mL, [67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. CONCLUSIONS: [67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.

The effects of trace metal impurities on Ga-68-radiolabelling with a tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelator.

GMP-grade 68Ge/68Ga generators provide access to positron-emitting 68Ga, enabling preparation of Positron Emission Tomography (PET) tracers and PET imaging at sites that do not have access to cyclotron-produced radionuclides. Radiotracers based on tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelators enable simple one-step preparations of 68Ga PET radiopharmaceuticals from pre-fabricated kits without pre-processing of generator eluate or post-purification. However, trace metal impurities eluted along with 68Ga could compete for THP and reduce radiochemical yields (RCY). We have quantified trace metal impurities in 68Ga eluate from an Eckert & Ziegler (E&Z) generator using ICP-MS. The metals Al, Fe, natGa, Pb, Ti and natZn were present in generator eluate in significantly higher concentrations than in the starting eluent solution. Concentrations of Fe and natGa in eluate were in the range of 0.01-0.1 µM, Al, Zn and Pb in the range of 0.1-1 µM, and Ti in the range of 0.9-1.5 µM. To assess the ability of THP to chelate 68Ga in the presence of such metal ions, radiolabelling reactions were undertaken in which selected metal ions were added to make them equimolar with THP, or higher. Al3+, Fe3+, natGa3+ and Ti4+ reduced RCY at concentrations equimolar with THP and higher, but at lower concentrations they did not affect RCY. Pb2+, Zn2+, Ni2+ and Cr3+ had no effect on RCY (even under conditions in which each metal ion was present in 100-fold molar excess over THP). The multi-sample ICP-MS analysis reported here is (to date) the most comprehensive and robust quantification of metal impurities in the widely used E&Z 68Ga generator. 68Ga from an E&Z generator enables near-quantitative radiolabelling of THP at chelator concentrations as low as 5 µM (lower than other common gallium chelators) without pre-processing. The combination of Al3+, Fe3+, natGa3+ and Ti4+ in unprocessed 68Ga eluate is likely to decrease RCY of 68Ga radiolabelling if a lower amount of THP chelator is used, and future kit design should take this into account. To increase specific activities by using even lower THP concentrations, purification of 68Ga from trace metal ions will likely be required.

Visualization of Tumor-Immune Interaction - Target-Specific Imaging of S100A8/A9 Reveals Pre-Metastatic Niche Establishment.

Background Systemic cancer spread is preceded by the establishment of a permissive microenvironment in the target tissue of metastasis - the premetastatic niche. As crucial players in establishment of the pre-metastatic niche, myeloid derived suppressor cells (MDSC) release S100A8/A9, an exosomal protein that contributes to metastasis, angiogenesis, and immune suppression. We report the application of antibody-based single-photon emission computed tomography (SPECT) for detection of S100A8/A9 in vivo as an imaging marker for pre-metastatic tissue priming. Methods A syngeneic model system for invasive breast cancer with (4T1.2) or without (67NR) the tendency to form lung metastasis was established in BALB/c mice. A SPECT-probe has been generated and tested for visualization of S100A9 release. Tumor-associated changes in numbers and fuction of immune cells in pre-metastatic tissue were evaluated by flow cytometry and confocal microscopy. Results S100A8/A9 imaging reflected MDSC abundance and the establishment of an immunosuppressive environment in pre-metastatic lung tissue (activity 4T1.2 vs. healthy control: 0.95 vs. 0.45 %ID; p<0.001). The S100A8/A9 imaging signal in the pre-metastatic lung correlated with the subsequent metastatic tumor burden in the same organ (r2=0.788; p<0.0001). CCL2 blockade and the consecutive inhibition of premetastatic niche establishment was clearly depicted by S100A9-SPECT (lung activity untreated vs. treated: 2 vs, 1.4 %ID). Conclusion We report S100A8/A9 as a potent imaging biomarker for tumor-mediated immune remodeling with potential applications in basic research and clinical oncology.

Evaluation of Radiopharmaceutical Adverse Reaction Reports to the British Nuclear Medicine Society from 2007 to 2016.

This study sought to answer the calls that have been made for the European Association of Nuclear Medicine to restore its surveys of reported adverse reactions. We assessed all reports of adverse reactions to radiopharmaceuticals that were submitted to the British Nuclear Medicine Society (BNMS) online database (Radiopharmaceutical Adverse Events and Product Defects) from January 2007 to December 2016. Methods: This investigation was a pharmacovigilance-based, nonexperimental, cross-sectional study aimed at finding the prevalence of, and association between, radiopharmaceuticals and adverse reactions. Results: During the study period, there were 204 reports of radiopharmaceutical adverse reactions, of which 13 were considered invalid, primarily because of incomplete entries or because a causal relationship between the radiopharmaceutical and the adverse reaction could not be determined. Tetrofosmin (34 reports) and oxidronate (32 reports) had the highest prevalence, followed by medronate (21 reports) and then sestamibi and nanocolloid (14 reports each). Rash (84 reports), itching (46 reports), and vomiting (30 reports) were the 3 most frequently occurring adverse reactions. Most reports (96.8%) were for diagnostic radiopharmaceuticals. Conclusion: The prevalence of adverse reactions to radiopharmaceuticals reported in the BNMS database remains low, with a frequency of 3.1 reports per 100,000 administrations in 2013 and 2.5 per 100,000 administrations in 2015. In our review spanning 10 years, we did not find any particular concern about the use of radiopharmaceuticals.

Variants on chromosome 4q21 near PKD2 and SIBLINGs are associated with dental caries.

A recent genome-wide association study (GWAS) for dental caries nominated the chromosomal region 4q21 near ABCG2, PKD2 and the SIBLING (small integrin-binding ligand N-linked glycoprotein) gene family. In this investigation, we followed up and fine-mapped this region using a tag-SNP (single-nucleotide polymorphism) approach in 13 age- and race-stratified samples from 6 independent studies (N=4089). Participants were assessed for dental caries via intraoral examination and 49 tag-SNPs were genotyped capturing much of the variation in the 4q21 locus. Linear models were used to test for genetic association, while adjusting for sex, age and components of ancestry. SNPs in and near PKD2 showed significant evidence of association in individual samples of black adults (rs17013735, P-value=0.0009) and white adults (rs11938025; P-value=0.0005; rs2725270, P-value=0.003). Meta-analyses across black adult samples recapitulated the association with rs17013735 (P-value=0.003), which occurs at low frequency in non-African populations, possibly explaining the race specificity of the effect. In addition to race-specific associations, we also observed evidence of gene-by-fluoride exposure interaction effects in white adults for SNP rs2725233 upstream of PKD2 (P=0.002). Our results show evidence of regional replication, though no single variant clearly accounted for the original GWAS signal. Therefore, while we interpret our results as strengthening the hypothesis that chromosome 4q21 may impact dental caries, additional work is needed.

Exploring Subclinical Phenotypic Features in Twin Pairs Discordant for Cleft Lip and Palate.

OBJECTIVE: Monozygotic twins of an individual with an orofacial cleft have a significantly elevated risk for orofacial cleft compared with the general population, but still the concordance rate for orofacial cleft in monozygotic twins is about 40% to 50%. The goal of this study was to determine whether unaffected cotwins have an increased frequency of orbicularis oris muscle defects, a subclinical form of orofacial cleft. The presence of such defects may reduce the overall rate of discordance. METHOD: A total of 63 discordant monozygotic and dizygotic twin pairs, 262 unaffected nontwin siblings, and 543 controls with no history of orofacial clefts were assessed for orbicularis oris defects by high-resolution ultrasound. Frequencies were compared by the Fisher exact test. RESULTS: Unaffected cotwins from discordant monozygotic pairs had a higher frequency of defects (12.5%) than the other test groups (6.38% to 6.99%), but the difference was not statistically significant (P = .74). CONCLUSIONS: In this study, orbicularis oris defects were not statistically significantly more common among the unaffected twins from orofacial cleft discordant twin pairs. The trends in the results warrant future studies with larger sample sizes and additional subclinical phenotypes.

In-Cell Activation of Organo-Osmium(II) Anticancer Complexes.

The family of iodido OsII arene phenylazopyridine complexes [Os(η6 -p-cym)(5-R1 -pyridylazo-4-R2 -phenyl))I]+ (where p-cym=para-cymene) exhibit potent sub-micromolar antiproliferative activity towards human cancer cells and are active in vivo. Their chemical behavior is distinct from that of cisplatin: they do not readily hydrolyze, nor bind to DNA bases. We report here a mechanism by which they are activated in cancer cells, involving release of the I- ligand in the presence of glutathione (GSH). The X-ray crystal structures of two active complexes are reported, 1-I (R1 =OEt, R2 =H) and 2-I (R1 =H, R2 =NMe2 ). They were labelled with the radionuclide 131 I (ß- /γ emitter, t1/2 8.02 d), and their activity in MCF-7 human breast cancer cells was studied. 1-[131 I] and 2-[131 I] exhibit good stability in both phosphate-buffered saline and blood serum. In contrast, once taken up by MCF-7 cells, the iodide ligand is rapidly pumped out. Intriguingly, GSH catalyzes their hydrolysis. The resulting hydroxido complexes can form thiolato and sulfenato adducts with GSH, and react with H2 O2 generating hydroxyl radicals. These findings shed new light on the mechanism of action of these organo-osmium complexes.

Ear Infection in Isolated Cleft Lip: Etiological Implications.

BACKGROUND AND HYPOTHESIS: Chronic ear infections are a common occurrence in children with orofacial clefts involving the secondary palate. Less is known about the middle ear status of individuals with isolated clefts of the lip, although several studies have reported elevated rates of ear infection in this group. The purpose of this retrospective study was to test the hypothesis that chronic ear infections occur more frequently in isolated cleft lip cases (n = 94) compared with controls (n = 183). METHODS: A questionnaire was used to obtain information on history of chronic ear infection. The association between ear infection status (present/absent) and cleft lip status (cleft lip case/control) was tested using both chi-square and logistic regression. RESULTS AND CONCLUSIONS: The reported occurrence of chronic ear infection was significantly greater in cleft lip cases (31%) compared with unaffected controls (11%). After adjusting for age and sex, having a cleft lip increased the odds of being positive for ear infection by a factor greater than 3 (odds ratio = 3.698; 95% confidence interval = 1.91 to 7.14). Within cleft lip cases, there was no difference in the occurrence of ear infection by defect laterality or by the type of clefting present in the family history. Although velopharyngeal insufficiency was present in 18.4% of our cleft lip sample, there was no statistical association between ear infection and abnormal speech patterns. These results may have potential implications both for the clinical management of isolated cleft lip cases and for understanding the etiology of orofacial clefting.