Association Between Years of Education and Amyloid Burden in Patients With Subjective Cognitive Decline, MCI, and Alzheimer Disease.

OBJECTIVES: Higher-educated patients with Alzheimer disease (AD) can harbor greater neuropathologic burden than those with less education despite similar symptom severity. In this study, we assessed whether this observation is also present in potential preclinical AD stages, namely in individuals with subjective cognitive decline and clinical features increasing AD likelihood (SCD+). METHODS: Amyloid-PET information ([18F]Flutemetamol or [18F]Florbetaben) of individuals with SCD+, mild cognitive impairment (MCI), and AD were retrieved from the AMYPAD-DPMS cohort, a multicenter randomized controlled study. Group classification was based on the recommendations by the SCD-I and NIA-AA working groups. Amyloid PET images were acquired within 8 months after initial screening and processed with AMYPYPE. Amyloid load was based on global Centiloid (CL) values. Educational level was indexed by formal schooling and subsequent higher education in years. Using linear regression analysis, the main effect of education on CL values was tested across the entire cohort, followed by the assessment of an education-by-diagnostic-group interaction (covariates: age, sex, and recruiting memory clinic). To account for influences of non-AD pathology and comorbidities concerning the tested amyloid-education association, we compared white matter hyperintensity (WMH) severity, cardiovascular events, depression, and anxiety history between lower-educated and higher-educated groups within each diagnostic category using the Fisher exact test or χ2 test. Education groups were defined using a median split on education (Md = 13 years) in a subsample of the initial cohort, for whom this information was available. RESULTS: Across the cohort of 212 individuals with SCD+ (M(Age) = 69.17 years, F 42.45%), 258 individuals with MCI (M(Age) = 72.93, F 43.80%), and 195 individuals with dementia (M(Age) = 74.07, F 48.72%), no main effect of education (ß = 0.52, 95% CI -0.30 to 1.58), but a significant education-by-group interaction on CL values, was found (p = 0.024) using linear regression modeling. This interaction was driven by a negative association of education and CL values in the SCD+ group (ß = -0.11, 95% CI -4.85 to -0.21) and a positive association in the MCI group (ß = 0.15, 95% CI 0.79-5.22). No education-dependent differences in terms of WMH severity and comorbidities were found in the subsample (100 cases with SCD+, 97 cases with MCI, 72 cases with dementia). DISCUSSION: Education may represent a factor oppositely modulating subjective awareness in preclinical stages and objective severity of ongoing neuropathologic processes in clinical stages.

Software compatibility analysis for quantitative measures of [18F]flutemetamol amyloid PET burden in mild cognitive impairment.

RATIONALE: Amyloid-ß (Aß) pathology is one of the earliest detectable brain changes in Alzheimer's disease pathogenesis. In clinical practice, trained readers will visually categorise positron emission tomography (PET) scans as either Aß positive or negative. However, adjunct quantitative analysis is becoming more widely available, where regulatory approved software can currently generate metrics such as standardised uptake value ratios (SUVr) and individual Z-scores. Therefore, it is of direct value to the imaging community to assess the compatibility of commercially available software packages. In this collaborative project, the compatibility of amyloid PET quantification was investigated across four regulatory approved software packages. In doing so, the intention is to increase visibility and understanding of clinically relevant quantitative methods. METHODS: Composite SUVr using the pons as the reference region was generated from [18F]flutemetamol (GE Healthcare) PET in a retrospective cohort of 80 amnestic mild cognitive impairment (aMCI) patients (40 each male/female; mean age = 73 years, SD = 8.52). Based on previous autopsy validation work, an Aß positivity threshold of ≥ 0.6 SUVrpons was applied. Quantitative results from MIM Software's MIMneuro, Syntermed's NeuroQ, Hermes Medical Solutions' BRASS and GE Healthcare's CortexID were analysed using intraclass correlation coefficient (ICC), percentage agreement around the Aß positivity threshold and kappa scores. RESULTS: Using an Aß positivity threshold of ≥ 0.6 SUVrpons, 95% agreement was achieved across the four software packages. Two patients were narrowly classed as Aß negative by one software package but positive by the others, and two patients vice versa. All kappa scores around the same Aß positivity threshold, both combined (Fleiss') and individual software pairings (Cohen's), were ≥ 0.9 signifying "almost perfect" inter-rater reliability. Excellent reliability was found between composite SUVr measurements for all four software packages, with an average measure ICC of 0.97 and 95% confidence interval of 0.957-0.979. Correlation coefficient analysis between the two software packages reporting composite z-scores was strong (r2 = 0.98). CONCLUSION: Using an optimised cortical mask, regulatory approved software packages provided highly correlated and reliable quantification of [18F]flutemetamol amyloid PET with a ≥ 0.6 SUVrpons positivity threshold. In particular, this work could be of interest to physicians performing routine clinical imaging rather than researchers performing more bespoke image analysis. Similar analysis is encouraged using other reference regions as well as the Centiloid scale, when it has been implemented by more software packages.

Quantification of amyloid PET for future clinical use: a state-of-the-art review.

Amyloid-ß (Aß) pathology is one of the earliest detectable brain changes in Alzheimer's disease (AD) pathogenesis. The overall load and spatial distribution of brain Aß can be determined in vivo using positron emission tomography (PET), for which three fluorine-18 labelled radiotracers have been approved for clinical use. In clinical practice, trained readers will categorise scans as either Aß positive or negative, based on visual inspection. Diagnostic decisions are often based on these reads and patient selection for clinical trials is increasingly guided by amyloid status. However, tracer deposition in the grey matter as a function of amyloid load is an inherently continuous process, which is not sufficiently appreciated through binary cut-offs alone. State-of-the-art methods for amyloid PET quantification can generate tracer-independent measures of Aß burden. Recent research has shown the ability of these quantitative measures to highlight pathological changes at the earliest stages of the AD continuum and generate more sensitive thresholds, as well as improving diagnostic confidence around established binary cut-offs. With the recent FDA approval of aducanumab and more candidate drugs on the horizon, early identification of amyloid burden using quantitative measures is critical for enrolling appropriate subjects to help establish the optimal window for therapeutic intervention and secondary prevention. In addition, quantitative amyloid measurements are used for treatment response monitoring in clinical trials. In clinical settings, large multi-centre studies have shown that amyloid PET results change both diagnosis and patient management and that quantification can accurately predict rates of cognitive decline. Whether these changes in management reflect an improvement in clinical outcomes is yet to be determined and further validation work is required to establish the utility of quantification for supporting treatment endpoint decisions. In this state-of-the-art review, several tools and measures available for amyloid PET quantification are summarised and discussed. Use of these methods is growing both clinically and in the research domain. Concurrently, there is a duty of care to the wider dementia community to increase visibility and understanding of these methods.

Derivation and utility of an Aß-PET pathology accumulation index to estimate Aß load.

OBJECTIVE: To evaluate a novel ß-amyloid (Aß)-PET-based quantitative measure (Aß accumulation index [Aß index]), including the assessment of its ability to discriminate between participants based on Aß status using visual read, CSF Aß42/Aß40, and post-mortem neuritic plaque burden as standards of truth. METHODS: One thousand one hundred twenty-one participants (with and without cognitive impairment) were scanned with Aß-PET: Swedish BioFINDER, n = 392, [18F]flutemetamol; Alzheimer's Disease Neuroimaging Initiative (ADNI), n = 692, [18F]florbetapir; and a phase 3 end-of-life study, n = 100, [18F]flutemetamol. The relationships between Aß index and standardized uptake values ratios (SUVR) from Aß-PET were assessed. The diagnostic performances of Aß index and SUVR were compared with visual reads, CSF Aß42/Aß40, and Aß histopathology used as reference standards. RESULTS: Strong associations were observed between Aß index and SUVR (R 2: BioFINDER 0.951, ADNI 0.943, end-of-life, 0.916). Both measures performed equally well in differentiating Aß-positive from Aß-negative participants, with areas under the curve (AUCs) of 0.979 to 0.991 to detect abnormal visual reads, AUCs of 0.961 to 0.966 to detect abnormal CSF Aß42/Aß40, and AUCs of 0.820 to 0.823 to detect abnormal Aß histopathology. Both measures also showed a similar distribution across postmortem-based Aß phases (based on anti-Aß 4G8 antibodies). Compared to models using visual read alone, the addition of the Aß index resulted in a significant increase in AUC and a decrease in Akaike information criterion to detect abnormal Aß histopathology. CONCLUSION: The proposed Aß index showed a tight association to SUVR and carries an advantage over the latter in that it does not require the definition of regions of interest or the use of MRI. Aß index may thus prove simpler to implement in clinical settings and may also facilitate the comparison of findings using different Aß-PET tracers. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the Aß accumulation index accurately differentiates Aß-positive from Aß-negative participants compared to Aß-PET visual reads, CSF Aß42/Aß40, and Aß histopathology.

Centiloid scaling for quantification of brain amyloid with [18F]flutemetamol using multiple processing methods.

INTRODUCTION: A standardised method for quantifying ß-amyloid PET tracers would allow comparison across different tracers and different sites. The development of the Centiloid scale has aimed to achieve this, applying a common scale to better aid the diagnosis and prognosis of Alzheimer's disease (AD) and to monitor anti-amyloid therapeutic interventions. Here, we apply the Centiloid method to [18F]flutemetamol and [11C]PiB (PiB, Pittsburgh compound B) PET images and derive the scaling factor to express their binding in Centiloids. METHODS: Paired PiB and [18F]flutemetamol scans for 74 subjects, including 24 young healthy controls (37 ± 5 years), were analysed using the standard Centiloid method. The same subjects were also analysed using PMOD- and FSL-based pipelines as well as SPM8. Test-retest analysis of 10 AD subjects was also performed with each pipeline. RESULTS: The standard uptake value ratios (SUVR), determined using the standard SPM8 Centiloid process, showed a strong correlation between [18F]flutemetamol (Flute) and PiB binding (SUVR-Flute = 0.77 × SUVR-PiB + 0.22, R2 = 0.96). Application of the standard Centiloid process allowed the calculation of a direct conversion equation for SUVR-Flute to Centiloid units (CL) (CL = (121.42*SUVR-Flute) - 121.16). Analysis of the data via the two alternate Centiloid pipelines allowed us to derive standardised, SPM8-equivalent equations for both PMOD (CL = (115.24*SUVR-Flute) - 107.86) and FSL (CL = (120.32*SUVR-Flute) - 112.75) respectively. Test-retest analysis of 10 AD subjects showed an approximate 2% difference for each pipeline. CONCLUSIONS: [18F]flutemetamol data can now be expressed in Centiloid units, enhancing its utility in clinical and research applications for ß-amyloid imaging. The standard Centiloid method also demonstrates that [18F]flutemetamol has favourable performance compared with PiB and other ß-amyloid tracers. Test-retest difference averaged 2%, with no difference between image processing pipelines. Centiloid scaling is robust and can be implemented on a number of platforms.

Measurement of Gross Photosynthesis, Respiration in the Light, and Mesophyll Conductance Using H218O Labeling.

A fundamental challenge in plant physiology is independently determining the rates of gross O2 production by photosynthesis and O2 consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O2 production (NOP and GOP, respectively) by labeling ambient O2 with 18O while leaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas-exchange measurement. The petiole is immersed in water enriched to a δ18O of ∼9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers 18O from H2O to O2 GOP is calculated from the increase in δ18O of O2 as air passes through the cuvette. NOP is determined from the increase in O2/N2 Both terms are measured by isotope ratio mass spectrometry. CO2 assimilation and other standard gas-exchange parameters also were measured. Reproducible measurements are made on a single leaf for more than 15 h. We used this method to measure the light response curve of NOP and GOP in French bean (Phaseolus vulgaris) at 21% and 2% O2 We then used these data to examine the O2/CO2 ratio of net photosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study and understand leaf physiological traits.

c-Met PET Imaging Detects Early-Stage Locoregional Recurrence of Basal-Like Breast Cancer.

UNLABELLED: Locoregional recurrence of breast cancer poses significant clinical problems because of frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable receptors, such as c-Met, would be of potential benefit. The aim of this study was to assess (18)F-AH113804, a peptide-based molecular imaging agent with high affinity for human c-Met, for the detection of early-stage locoregional recurrence in a human basal-like breast cancer model, HCC1954. METHODS: HCC1954 tumor-bearing xenograft models were established, and (18)F-AH113804 was administered. Distribution of radioactivity was determined via PET at 60 min after radiotracer injection. PET and CT images were acquired 10 d after tumor inoculation, to establish baseline distribution and uptake, and then on selected days after surgical tumor resection. CT images and caliper were used to determine the tumor volume. Radiotracer uptake was assessed by (18)F-AH113804 PET imaging. c-Met expression was assessed by immunofluorescence imaging of tumor samples and correlated with (18)F-AH113804 PET imaging results. RESULTS: Baseline uptake of (18)F-AH113804, determined in tumor-bearing animals after 10 d, was approximately 2-fold higher in the tumor than in muscle tissue or the contralateral mammary fat pad. The tumor growth rate, determined from CT images, was comparable between the animals with recurrent tumors, with detection of tumors of low volume (<10 mm(3)) only possible by day 20 after tumor resection. (18)F-AH113804 PET detected local tumor recurrence as early as 6 d after surgery in the recurrent tumor-bearing animals and exhibited significantly higher (18)F-AH113804 uptake (in comparison to mammary fatty tissue), with a target-to-background (muscle) ratio of approximately 3:1 (P < 0.01). The c-Met expression of individual resected tumor samples, determined by immunofluorescence, correlated with the respective (18)F-AH113804 imaging signals (r = 0.82, P < 0.05). CONCLUSION: (18)F-AH113804 PET provides a new diagnostic tool for the detection of c-Met-expressing primary tumor and has potential utility for the detection of locoregional recurrence from an early stage.

Evaluation of a novel series of fluorine-18-labeled imidazobenzodiazepines as potential new positron emission tomography radioligands for the GABAA receptor.

INTRODUCTION: [(11)C]Flumazenil has been used to study the GABAA receptor in many preclinical and clinical studies, but the short half-life of carbon-11 means that this molecule is restricted to use by investigators with access to on-site cyclotron and radiosynthesis facilities. The radiosynthesis of [(18)F]flumazenil has been evaluated by several groups, but the radiochemical yield can be low and inconsistent. We previously reported a series of fluorine-18-labeled imidazobenzodiazepine-based ligands for the GABAA receptor, which had significantly improved radiosynthesis yields. Here we report the in vivo evaluation and comparison of the distribution, metabolism and specificity of the novel ligands in comparison with [(18)F]flumazenil. METHODS: In vivo biodistribution studies, at time points up to 90min post-injection, were performed in naïve rats to compare the performance of the novel compounds with particular attention paid to regional brain uptake and clearance. In vivo metabolism studies were carried out to determine the percentage of parent compound remaining in the plasma and brain at selected time points. Blocking studies were carried out, using pre-treatment of the test animals with either bretazenil or unlabeled fluorine-19 test compound, to determine the levels of specific and non-specific binding in selected brain regions. RESULTS: Two of the 12 new compounds were rejected due to poor biodistribution showing significant bone uptake. Some of the compounds showed insufficient whole brain uptake or limited evidence of differential binding to GABAA-rich brain regions to warrant further investigation. Four of the compounds were selected for in vivo metabolism and blocking studies. Overall, the studies indicated that two compounds 3 and 5 showed comparable or improved performance compared with [(18)F]flumazenil, with respect to distribution, metabolic profile and specific binding. CONCLUSIONS: These studies have demonstrated that compounds based on [(18)F]flumazenil, but with alterations to allow improved radiosynthesis, can be prepared which have ideal properties and warrant further evaluation as PET agents for the GABAA receptor. In particular, compounds 3 and 5 show very promising profiles with specific binding and in vivo stability comparable to flumazenil.

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain.

INTRODUCTION: Two 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABAA/benzodiazepine receptor by comparing them to [(11)C]flumazenil in rhesus monkey. METHODS: Radiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [(18)F]AH114726 and [(18)F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil. RESULTS: [(18)F]GEH120348 and [(18)F]AH114726 were obtained in 13-24% yields (end of synthesis) with high chemical (>95%) and radiochemical (>99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [(18)F]AH114726 and [(18)F]GEH120348 were determined in a non-human primate and directly compared with [(11)C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [(11)C]flumazenil and the known concentrations of GABAA/benzodiazepine receptors in the monkey brain. [(18)F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [(11)C]flumazenil. In contrast, [(18)F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil. CONCLUSION: The ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [(11)C]flumazenil, support that [(18)F]AH114726 is a suitable option for imaging the GABAA receptor.

The development of potential new fluorine-18 labelled radiotracers for imaging the GABA(A) receptor.

Positron emission tomography (PET) using the tracer [(11)C]Flumazenil has shown changes in the distribution and expression of the GABA(A) receptor in a range of neurological conditions and injury states. We aim to develop a fluorine-18 labelled PET agent with comparable properties to [(11)C]Flumazenil. In this study we make a direct comparison between the currently known fluorine-18 labelled GABA(A) radiotracers and novel imidazobenzodiazepine ligands. A focussed library of novel compound was designed and synthesised where the fluorine containing moiety and the position of attachment is varied. The in vitro affinity of twenty-two compounds for the GABA(A) receptor was measured. Compounds containing a fluoroalkyl amide or a longer chain ester group were eliminated due to low potency. The fluorine-18 radiochemistry of one compound from each structural type was assessed to confirm that an automated radiosynthesis in good yield was feasible. Eleven of the novel compounds assessed appeared suitable for in vivo assessment as PET tracers.

Recent decreases in fossil-fuel emissions of ethane and methane derived from firn air.

Methane and ethane are the most abundant hydrocarbons in the atmosphere and they affect both atmospheric chemistry and climate. Both gases are emitted from fossil fuels and biomass burning, whereas methane (CH(4)) alone has large sources from wetlands, agriculture, landfills and waste water. Here we use measurements in firn (perennial snowpack) air from Greenland and Antarctica to reconstruct the atmospheric variability of ethane (C(2)H(6)) during the twentieth century. Ethane levels rose from early in the century until the 1980s, when the trend reversed, with a period of decline over the next 20 years. We find that this variability was primarily driven by changes in ethane emissions from fossil fuels; these emissions peaked in the 1960s and 1970s at 14-16 teragrams per year (1 Tg = 10(12) g) and dropped to 8-10 Tg yr(-1) by the turn of the century. The reduction in fossil-fuel sources is probably related to changes in light hydrocarbon emissions associated with petroleum production and use. The ethane-based fossil-fuel emission history is strikingly different from bottom-up estimates of methane emissions from fossil-fuel use, and implies that the fossil-fuel source of methane started to decline in the 1980s and probably caused the late twentieth century slow-down in the growth rate of atmospheric methane.

Monitoring tumor response to antiangiogenic sunitinib therapy with 18F-fluciclatide, an 18F-labeled αVbeta3-integrin and αV beta5-integrin imaging agent.

UNLABELLED: Arginine-glycine-aspartate (RGD)-binding α(V)ß(3)-integrin and α(V)ß(5)-integrin play key roles in tumor angiogenesis. We examined an (18)F-labeled small peptide (fluciclatide [United States Adopted Name (ASAN)-approved, International Nonproprietary Name (INN)-proposed name], previously referred to as AH111585) containing an RGD sequence. Fluciclatide binds with a high (nM) affinity to α(V)ß(3)-integrin and α(V)ß(5)-integrin, which are highly expressed on tumors and the tumor neovasculature. In this study, (18)F-fluciclatide was used to examine the response of human glioblastoma xenografts to treatment with the antiangiogenic agent sunitinib. METHODS: U87-MG tumor uptake of (18)F-fluciclatide was determined by small-animal PET after longitudinal administration of the antiangiogenic agent sunitinib (a 2-wk dosing regimen). Tumor sizes were measured throughout the study, and tumor volumes were calculated. Tumor microvessel density (MVD) after therapy was also analyzed. RESULTS: Dynamic small-animal PET of (18)F-fluciclatide uptake after administration of the clinically relevant antiangiogenic agent sunitinib revealed a reduction in the tumor uptake of (18)F-fluciclatide compared with that in vehicle-treated controls over the 2-wk dosing regimen. Skeletal muscle, used as a reference tissue, showed equivalent (18)F-fluciclatide uptake in both therapy and control groups. A reduction in tumor MVD was also observed after treatment with the antiangiogenic agent. No significant changes in tumor volume were observed in the 2 groups. CONCLUSION: The data demonstrated that (18)F-fluciclatide detected changes in tumor uptake after acute antiangiogenic therapy markedly earlier than any significant volumetric changes were observable. These results suggest that this imaging agent may provide clinically important information for guiding patient care and monitoring the response to antiangiogenic therapy.

Use of 2-[(18)F]fluoroethylazide for the Staudinger ligation - Preparation and characterisation of GABA(A) receptor binding 4-quinolones.

The labelling reagent 2-[(18)F]fluoroethylazide was used in a traceless Staudinger ligation. This reaction was employed to obtain the GABA(A) receptor binding 6-benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (2-[(18)F]fluoroethyl) amide. The radiotracer was prepared with a non-decay corrected radiochemical yield of 7%, a radiochemical purity >95% and a specific radioactivity of 0.9 GBq/micromol. The compound showed low brain penetration in normal rats. A series of fluoroalkyl 4-quinolone analogues with nanomolar to sub-nanomolar affinity for the GABA(A) receptor has been prepared as well.

Polar firn air reveals large-scale impact of anthropogenic mercury emissions during the 1970s.

Mercury (Hg) is an extremely toxic pollutant, and its biogeochemical cycle has been perturbed by anthropogenic emissions during recent centuries. In the atmosphere, gaseous elemental mercury (GEM; Hg degrees ) is the predominant form of mercury (up to 95%). Here we report the evolution of atmospheric levels of GEM in mid- to high-northern latitudes inferred from the interstitial air of firn (perennial snowpack) at Summit, Greenland. GEM concentrations increased rapidly after World War II from approximately 1.5 ng m(-3) reaching a maximum of approximately 3 ng m(-3) around 1970 and decreased until stabilizing at approximately 1.7 ng m(-3) around 1995. This reconstruction reproduces real-time measurements available from the Arctic since 1995 and exhibits the same general trend observed in Europe since 1990. Anthropogenic emissions caused a two-fold rise in boreal atmospheric GEM concentrations before the 1970s, which likely contributed to higher deposition of mercury in both industrialized and remotes areas. Once deposited, this toxin becomes available for methylation and, subsequently, the contamination of ecosystems. Implementation of air pollution regulations, however, enabled a large-scale decline in atmospheric mercury levels during the 1980s. The results shown here suggest that potential increases in emissions in the coming decades could have a similar large-scale impact on atmospheric Hg levels.

99mTc-NC100692--a tracer for imaging vitronectin receptors associated with angiogenesis: a preclinical investigation.

INTRODUCTION: Technetium 99m (99mTc)-NC100692 is being developed as a marker of vitronectin receptor expression. The purpose of this study was to confirm the binding affinity [dissociation constant (Kd)] of 99mTc-NC100692 for a range of integrin receptors including alphavbeta3 and alphavbeta5 as well as to establish the biodistribution and metabolic stability of 99mTc-NC100692 in Wistar rats. METHODS: The Kd of 99mTc-NC100692 for a range of human integrin receptors was established in an in vitro saturation binding assay. The biodistribution and metabolic stability of 99mTc-NC100692 in normal Wistar rats was investigated. RESULTS: The Kd of 99mTc-NC100692 to alphavbeta3 and alphavbeta5 was less than 1 nM. It was not possible to saturate the binding of 99mTc-NC10092 towards alphaIIbbeta3, alpha5beta1, alpha3beta1 or alpha1beta1, and as a result, accurate Kd values could not be determined. The biodistribution of 99mTc-NC100692 in male and female Wistar rats showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention apart from the liver and kidneys. Kidney and liver retention was reduced in the presence of excess NC100692 ligand. In vivo, there was little systemic metabolism of 99mTc-NC100692. CONCLUSIONS: 99mTc-NC100692 has a high affinity for the vitronectin receptors that are associated with angiogenesis. 99mTc-NC100692 is metabolically stable in the systemic circulation of rats with a biodistribution that is favourable for imaging purposes. This evidence suggests that 99mTc-NC100692 might be a useful marker of vitronectin receptor expression in vivo.

The biodistribution of NC100668 and the effect of excess NC100668 on the biodistribution and kidney retention of (99m)Tc-NC100668 in the rat.

INTRODUCTION: (99m)Tc-NC100668 is being developed to aid the diagnosis of thromboemboli. The purpose of this study was to investigate if the presence of excess NC100668 interferes with the biodistribution and blood clot uptake of (99m)Tc-NC100668. The secondary aim was to investigate the causes underlying the kidney retention of (99m)Tc-NC100668. METHODS: The uptake of a (14)C-labelled analogue of NC100668, as well as (99m)Tc-NC100668, into plasma (in vitro) and blood (in vivo) clots was determined. The biodistribution of (99m)Tc-NC100668 at a range of NC100668 doses was studied in normal Wistar rats and those bearing experimentally induced deep venous thrombosis. The biodistribution of a negative control peptide and (99m)Tc-NC100668 plus L-lysine was studied in healthy male Wistar rats. RESULTS: The biodistribution as well as plasma clot uptake of [Asn-U-(14)C]NC100668 and (99m)Tc-NC100668 was similar. Apart from some reduction in kidney retention, the biodistribution and uptake of radioactivity into the blood clot were not significantly affected by the presence of up to 1000 times the clinical dose of NC100668. Kidney retention of radioactivity could be more effectively reduced by coadministration of 889 microg/kg NC100668 than 450 mg/kg L-lysine. A negative control peptide with no affinity for FXIIIa demonstrated very little kidney retention. CONCLUSIONS: The biodistribution and blood clot uptake of (99m)Tc-NC100668 and [Asn-U-(14)C]NC100668 are similar. With the exception of the kidneys, (99m)Tc-NC100668 biodistribution and blood clot uptake are unaffected by the presence of unlabelled NC100668. The kidney retention of radioactivity is probably due to transglutaminase activity and, to a lesser extent, nonspecific charge-mediated endocytosis.

99mTc-NC100668, an agent for imaging venous thromboembolism: The effect of anticoagulant or thrombolytic therapy on the uptake and retention of radioactivity in blood clots in vivo.

BACKGROUND: The purpose of this study was to evaluate the uptake of (99m)Tc-NC100668 into blood clots and elucidate the potential for medications commonly used to treat thromboembolism to interfere with the uptake and retention of (99m)Tc-NC100668. METHODS: (99m)Tc-NC100668 in vivo uptake and retention in a range of blood clot of various ages (up to 4 h. old) and in the presence of anticoagulants or thrombolytic therapies was measured in a rat model of deep vein thrombosis. RESULTS: (99m)Tc-NC100668 was rapidly absorbed into and retained by blood clots and was not significantly affected by the presence of unfractionated or low molecular weight heparin or thrombin inhibitor. Tissue plasminogen activator reduced the uptake of (99m)Tc-NC100668 into blood clot by a factor of 3 when adjusted to allow for changes in the weight of the blood clot. CONCLUSIONS: This study has demonstrated that the uptake and retention of (99m)Tc-NC100668 into blood clots in the rat model of deep vein thrombosis is rapid and maintained over at least a 4 h. post-injection period. It has been shown that (99m)Tc-NC100668 is retained in blood clots even in the presence of therapeutic doses of those anticoagulant and thrombolytic therapies typically used to treat pulmonary embolism and venous thrombosis.

(99m)Tc-NC100668, a new tracer for imaging venous thromboemboli: pre-clinical biodistribution and incorporation into plasma clots in vivo and in vitro.

PURPOSE: (99m)Tc-NC100668 is a new radiotracer being developed to aid the diagnosis of thromboembolism. The structure of NC100668 is similar to a region of human alpha(2)-antiplasmin, which is a substrate for factor XIIIa (FXIIIa). The purpose of this study was to confirm the uptake of (99m)Tc-NC100668 into forming plasma clot and to establish the biodistribution of (99m)Tc-NC100668 in Wistar rats. METHODS: The in vitro plasma clot uptake of (99m)Tc-NC100668 and other compounds with known affinities to FXIIIa was measured using a plasma clot assay. The biodistribution and blood clot uptake of radioactivity of (99m)Tc-NC100668 in normal Wistar rats and those bearing experimentally induced deep vein thrombi were investigated. RESULTS: The in vitro uptake of (99m)Tc-NC100668 was greater than that for [(14)C]dansyl cadaverine, a known substrate of FXIIIa in the plasma clot assay. The biodistribution of (99m)Tc-NC100668 in male and female Wistar rats up to 24 h p.i. showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention. In vivo the uptake and retention of (99m)Tc-NC100668 into the blood clot was greater than could be accounted for by non-specific accumulation of the radiotracer within the blood clot. CONCLUSION: (99m)Tc-NC100668 was retained by plasma clots in vitro and blood clots in vivo. No significant tissue retention which could interfere with the ability to image thrombi in vivo was observed. This evidence suggests that (99m)Tc-NC100668 might be useful in the detection of thromboembolism.

The in vivo and in vitro metabolic profile of 99mTc-NC100668, a new tracer for imaging venous thromboembolism: identification and biodistribution of the principal radiolabeled metabolite.

(99m)Tc-NC100668 [Acetyl-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr(3-iodo)-Thr-Leu-Leu-Lys-Gly-NC100194] is a radiopharmaceutical imaging agent being developed to aid the diagnosis of thromboembolism. The stability profile of (99m)Tc-NC100668 was investigated by high-performance liquid chromatography (HPLC) after in vitro exposure to blood and plasma obtained from rat and human, as well as to urine and bile obtained from rat. The metabolic profile of (99m)Tc-NC100668 exposed to human and rat hepatic S9 (a liver homogenate-rich cytochrome P450) was also studied. The profile of (99m)Tc-labeled species in plasma, urine, and bile was investigated following i.v. administration of (99m)Tc-NC100668 to rat. The major species observed in vitro and in vivo consisted of the (99m)Tc-chelator (NC100194) [N,N-Bis(N-(1,1-dimethyl-2-(hydroxylimino-)propyl)aminoethyl)aminoethylamine] attached to the C-terminal amino acid residue and referred to as (99m)Tc-complex of Gly-NC100194. The identity of the major metabolite was confirmed by cochromatography with an authentic standard and the genuine metabolite using a second HPLC method. The minor metabolites were sodium pertechnetate ((99m)Tc) and (99m)Tc-NC100194. In addition, a small number of other species were transiently observed in vitro; they were not investigated further. The biodistribution of the major metabolite was studied in male Wistar rats. The affinity of the major metabolite toward plasma clot was established using a plasma clot-forming assay. A minor uptake of (99m)Tc-complex of Gly-NC100194 in the plasma clot and a rapid removal from the body were noted. In conclusion, the metabolites of (99m)Tc-NC100668 are not anticipated to have a negative impact on the ability of the test substance to image blood clots.