Synthesis and preclinical evaluation of [11C]EAI045 as a PET tracer for imaging tumors expressing mutated epidermal growth factor receptor.

BACKGROUND: Mutations in the epidermal growth factor receptor (EGFR) kinase domain are common in non-small cell lung cancer. Conventional tyrosine kinase inhibitors target the mutation site in the ATP binding pocket, thereby inhibiting the receptor's function. However, subsequent treatment resistance mutations in the ATP binding site are common. The EGFR allosteric inhibitor, EAI045, is proposed to have an alternative mechanism of action, disrupting receptor signaling independent of the ATP-binding site. The antibody cetuximab is hypothesized to increase the number of accessible allosteric pockets for EAI045, thus increasing the potency of the inhibitor. This work aimed to gain further knowledge on pharmacokinetics, the EGFR mutation-targeting potential, and the influence of cetuximab on the uptake by radiolabeling EAI045 with carbon-11 and tritium. RESULTS: 2-(5-fluoro-2-hydroxyphenyl)-2-((2-iodobenzyl)amino)-N-(thiazol-2-yl)acetamide and 2-(5-fluoro-2-hydroxyphenyl)-N-(5-iodothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)acetamide were synthesized as precursors for the carbon-11 and tritium labeling of EAI045, respectively. [11C]EAI045 was synthesized using [11C]CO in a palladium-catalyzed ring closure in a 10 ± 1% radiochemical yield (decay corrected to end of [11C]CO2 production), > 97% radiochemical purity and 26 ± 1 GBq/µmol molar activity (determined at end of synthesis) in 51 min. [3H]EAI045 was synthesized by a tritium-halogen exchange in a 0.2% radiochemical yield, 98% radiochemical purity, and 763 kBq/nmol molar activity. The ability of [11C]EAI045 to differentiate between L858R/T790M mutated EGFR expressing H1975 xenografts and wild-type EGFR expressing A549 xenografts was evaluated in female nu/nu mice. The uptake was statistically significantly higher in H1975 xenografts compared to A549 xenografts (0.45 ± 0.07%ID/g vs. 0.31 ± 0.10%ID/g, P = 0.0166). The synergy in inhibition between EAI045 and cetuximab was evaluated in vivo and in vitro. While there was some indication that cetuximab influenced the uptake of [3H]EAI045 in vitro, this could not be confirmed in vivo when tumor-bearing mice were administered cetuximab (0.5 mg), 24 h prior to injection of [11C]EAI045. CONCLUSIONS: EAI045 was successfully labeled with tritium and carbon-11, and the in vivo results indicated [11C]EAI045 may be able to distinguish between mutated and non-mutated EGFR in non-small cell lung cancer mouse models. Cetuximab was hypothesized to increase EAI045 uptake; however, no significant effect was observed on the uptake of [11C]EAI045 in vivo or [3H]EAI045 in vitro in H1975 xenografts and cells.

Synthesis and Preclinical Evaluation of [Methylpiperazine-11C]brigatinib as a PET Tracer Targeting Both Mutated Epidermal Growth Factor Receptor and Anaplastic Lymphoma Kinase.

Brigatinib, a tyrosine kinase inhibitor (TKI) with specificity for gene rearranged anaplastic lymphoma kinase (ALK), such as the EML4-ALK, has shown a potential to inhibit mutated epidermal growth factor receptor (EGFR). In this study, N-desmethyl brigatinib was successfully synthesized as a precursor in five steps. Radiolabeling with [11C]methyl iodide produced [methylpiperazine-11C]brigatinib in a 10 ± 2% radiochemical yield, 91 ± 17 GBq/µmol molar activity, and ≥95% radiochemical purity in 49 ± 4 min. [Methylpiperazine-11C]brigatinib was evaluated in non-small cell lung cancer xenografted female nu/nu mice. An hour post-injection (p.i.), 87% of the total radioactivity in plasma originated from intact [methylpiperazine-11C]brigatinib. Significant differences in tumor uptake were observed between the endogenously EML4-ALK mutated H2228 and the control xenograft A549. The tumor-to-blood ratio in H2228 xenografts could be reduced by pretreatment with ALK inhibitor crizotinib. Tracer uptake in EGFR Del19 mutated HCC827 and EML4-ALK fusion A549 was not significantly different from uptake in A549 xenografts.

Synthesis and preclinical evaluation of two osimertinib isotopologues labeled with carbon-11 as PET tracers targeting the tyrosine kinase domain of the epidermal growth factor receptor.

INTRODUCTION: Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) that is able to inhibit the EGFR treatment resistance mutation T790M and primary EGFR mutations Del19 and L858R. The aim of the study was to evaluate the potential of carbon-11 labeled osimertinib to be used as a tracer for the PET imaging of tumors bearing the T790M mutation. METHODS: Osimertinib was labeled with carbon-11 at two positions, and the effect of the labeling position on the metabolism and biodistribution was studied in female nu/nu mice. The mutation status specificity of osimertinib was confirmed in vitro in a cell growth inhibition experiment, and the tumor-targeting potential of the carbon-11 isotopologues was evaluated using female nu/nu mice xenografted with NSCLC cell lines; the wild-type EGFR expressing A549, the primary Del19 EGFR mutated HCC827 and the resistance T790M/L858R mutated H1975. One of the osimertinib tracers was selected based on the results acquired and evaluated for tracer specificity and selectivity by assessment of tumor uptake in a PET study where HCC827 tumor-bearing mice were pretreated with osimertinib or afatinib. RESULTS: [Methylindole-11C]- and [dimethylamine-11C]osimertinib were synthesized by 11C-methylation of precursors AZ5104 and AZ7550, respectively. Rapid metabolism of both analogs of [11C]osimertinib was observed. Although the tumor uptake and retention of [methylindole-11C]- and [dimethylamine-11C]osimertinib in tumors were similar, the tumor-to-muscle ratios appeared to be higher for [methylindole-11C]osimertinib. The highest uptake, tumor-to-blood, and tumor-to-muscle ratio were observed in the Del19 EGFR mutated HCC827 tumors. However, the specificity and selectivity of [methylindole-11C]osimertinib PET could not be demonstrated in HCC827 tumors. The uptake of [methylindole-11C]osimertinib was not significantly higher in T790M resistance mutated H1975 xenografts compared to the negative control cell line A549. CONCLUSIONS: Osimertinib was successfully labeled at two positions with carbon-11, yielding two EGFR PET tracers, [methylindole-11C]osimertinib and [dimethylamine-11C]osimertinib. The preclinical evaluation demonstrated uptake and retention in three NSCLC xenografts; A549, HCC827, and H1975. The highest uptake was observed in the primary Del19 EGFR mutated HCC827. The ability of [methylindole-11C]osimertinib to distinguish between the T790M resistance mutated H1975 xenografts and the wild-type EGFR expressing A549 could not be confirmed in the ex vivo study.

Advancing 89Zr-immuno-PET in neuroscience with a bispecific anti-amyloid-beta monoclonal antibody - The choice of chelator is essential.

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different 89Zr-labeling strategies and direct radioiodination with 125I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89Zr. 125I was used as a substitute for 124I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89Zr or 2.5 MBq 125I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [89Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125I-analog (2.21 ±0.15 %ID/g). [89Zr]Zr-DFO-NCS-Adu-8D3 and [89Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89Zr-labeled controls (B12-8D3). Autoradiography of [89Zr]Zr-DFO*-NCS-Adu-8D3 and [125I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [89Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [89Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that 89Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89Zr.

Synthesis and preclinical evaluation of [11C]LR111 and [18F]EW-7197 as PET tracers of the activin-receptor like kinase-5.

The transforming growth factor ß (TGFß) pathway plays a complex role in cancer biology, being involved in both tumour suppression as well as promotion. Overactive TGFß signalling has been linked to multiple diseases, including cancer, pulmonary arterial hypertension, and fibrosis. One of the key meditators within this pathway is the TGFß type I receptor, also termed activin receptor-like kinase 5 (ALK5). ALK5 expression level is a key determinant of TGFß signalling intensity and duration, and perturbation has been linked to diseases. A validated ALK5 positron emission tomography (PET) tracer creates an opportunity, therefore, to study its role in human diseases. To develop ALK5 PET tracers, two small molecule ALK5 kinase inhibitors were selected as lead compounds, which were labelled with carbon-11 and fluorine-18, respectively. [11C]LR111 was synthesized with a yield of 17 ± 6%, a molar activity of 126 ± 79 GBq·µmol-1 and a purity of >95% (n = 44). [18F]EW-7197 was synthesized with a yield of 10 ± 5%, a molar activity of 183 ± 126 GBq·µmol-1 and a purity of >95% (n = 11). Metabolic stability was evaluated in vivo in mice, showing 39 ± 2% of intact [11C]LR111 and 21 ± 2% of intact [18F]EW-7197 in blood plasma at 45 min p.i. In vitro binding experiments were conducted in breast cancer MDA-MB-231 and lung cancer A431 cell lines. In addition, both tracers were used for PET imaging in MDA-MB-231 xenograft models. Selective uptake of [18F]EW-7197 and [11C]LR111 was observed in MDA-MB-231 cells, in the MDA-MB-231 tumour xenografts in vivo and in the autoradiograms. As [11C]LR111 and [18F]EW-7197 showed selectivity of binding to ALK5 in vivo and in vitro. Both tracers are thereby valuable tools for the detection of ALK5 activity.

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Synthesis and evaluation of [18F]cinacalcet for the imaging of parathyroid hyperplasia.

INTRODUCTION: Parathyroid hyperplasia is a disease characterized by overactive parathyroid glands secreting increased levels of parathyroid hormone. Surgical removal of the parathyroid glands is the standard treatment but requires precise pre-operative localization of the glands. However, currently available imaging modalities show limited sensitivity. Since positron emission tomography (PET) is a molecular imaging technique with high accuracy and sensitivity, our aim was to develop a new PET tracer for overactive parathyroid glands imaging by radiolabelling cinacalcet, a drug binding to the calcium-sensing receptor of the parathyroid glands. METHODS: [18F]Cinacalcet was synthesized by copper-catalysed [18F]trifluoromethylation of a boronic acid precursor using high molar activity [18F]fluoroform. Ex vivo biodistribution and metabolism were evaluated in 12 healthy male Wistar rats at 5, 15, 45 and 90 min. PET scans were performed at baseline and after blocking with NPS R-568. RESULTS: [18F]Cinacalcet was obtained in an overall radiosynthesis time of 1 h with a radiochemical purity of 98 ± 1%, a radiochemical yield of 8 ± 4% (overall, n = 7, corrected for decay) and a molar activity of 40 ± 11 GBq/µmol (n = 7, at EOS). The ex vivo biodistribution showed uptake in the thyroid and parathyroid glands as well as in other glands such as adrenals, salivary glands and pancreas. The tracer was rapidly cleared from the blood via liver and kidneys and showed fast metabolism. PET images confirmed uptake in the target organ. However, in a blocking study with NPS R-568 specific binding of [18F]cinacalcet to the CaSR could not be confirmed. CONCLUSIONS: [18F]Cinacalcet was successfully synthesized. First in vivo experiments in healthy rats showed uptake of the tracer in the target organ and fast metabolism, encouraging further in vivo evaluation of this tracer.

Performance of nanoScan PET/CT and PET/MR for quantitative imaging of 18F and 89Zr as compared with ex vivo biodistribution in tumor-bearing mice.

INTRODUCTION: The assessment of ex vivo biodistribution is the preferred method for quantification of radiotracers biodistribution in preclinical models, but is not in line with current ethics on animal research. PET imaging allows for noninvasive longitudinal evaluation of tracer distribution in the same animals, but systemic comparison with ex vivo biodistribution is lacking. Our aim was to evaluate the potential of preclinical PET imaging for accurate tracer quantification, especially in tumor models. METHODS: NEMA NU 4-2008 phantoms were filled with 11C, 68Ga, 18F, or 89Zr solutions and scanned in Mediso nanoPET/CT and PET/MR scanners until decay. N87 tumor-bearing mice were i.v. injected with either [18F]FDG (~ 14 MBq), kept 50 min under anesthesia followed by imaging for 20 min, or with [89Zr]Zr-DFO-NCS-trastuzumab (~ 5 MBq) and imaged 3 days post-injection for 45 min. After PET acquisition, animals were killed and organs of interest were collected and measured in a γ-counter to determine tracer uptake levels. PET data were reconstructed using TeraTomo reconstruction algorithm with attenuation and scatter correction and regions of interest were drawn using Vivoquant software. PET imaging and ex vivo biodistribution were compared using Bland-Altman plots. RESULTS: In phantoms, the highest recovery coefficient, thus the smallest partial volume effect, was obtained with 18F for both PET/CT and PET/MR. Recovery was slightly lower for 11C and 89Zr, while the lowest recovery was obtained with 68Ga in both scanners. In vivo, tumor uptake of the 18F- or 89Zr-labeled tracer proved to be similar irrespective whether quantified by either PET/CT and PET/MR or ex vivo biodistribution with average PET/ex vivo ratios of 0.8-0.9 and a deviation of 10% or less. Both methods appeared less congruent in the quantification of tracer uptake in healthy organs such as brain, kidney, and liver, and depended on the organ evaluated and the radionuclide used. CONCLUSIONS: Our study suggests that PET quantification of 18F- and 89Zr-labeled tracers is reliable for the evaluation of tumor uptake in preclinical models and a valuable alternative technique for ex vivo biodistribution. However, PET and ex vivo quantification require fully described experimental and analytical procedures for reliability and reproducibility.

Head-to-head comparison of DFO* and DFO chelators: selection of the best candidate for clinical 89Zr-immuno-PET.

PURPOSE: Almost all radiolabellings of antibodies with 89Zr currently employ the hexadentate chelator desferrioxamine (DFO). However, DFO can lead to unwanted uptake of 89Zr in bones due to instability of the resulting metal complex. DFO*-NCS and the squaramide ester of DFO, DFOSq, are novel analogues that gave more stable 89Zr complexes than DFO in pilot experiments. Here, we directly compare these linker-chelator systems to identify optimal immuno-PET reagents. METHODS: Cetuximab, trastuzumab and B12 (non-binding control antibody) were labelled with 89Zr via DFO*-NCS, DFOSq, DFO-NCS or DFO*Sq. Stability in vitro was compared at 37 °C in serum (7 days), in formulation solution (24 h ± chelator challenges) and in vivo with N87 and A431 tumour-bearing mice. Finally, to demonstrate the practical benefit of more stable complexation for the accurate detection of bone metastases, [89Zr]Zr-DFO*-NCS and [89Zr]Zr-DFO-NCS-labelled trastuzumab and B12 were evaluated in a bone metastasis mouse model where BT-474 breast cancer cells were injected intratibially. RESULTS: [89Zr]Zr-DFO*-NCS-trastuzumab and [89Zr]Zr-DFO*Sq-trastuzumab showed excellent stability in vitro, superior to their [89Zr]Zr-DFO counterparts under all conditions. While tumour uptake was similar for all conjugates, bone uptake was lower for DFO* conjugates. Lower bone uptake for DFO* conjugates was confirmed using a second xenograft model: A431 combined with cetuximab. Finally, in the intratibial BT-474 bone metastasis model, the DFO* conjugates provided superior detection of tumour-specific signal over the DFO conjugates. CONCLUSION: DFO*-mAb conjugates provide lower bone uptake than their DFO analogues; thus, DFO* is a superior candidate for preclinical and clinical 89Zr-immuno-PET.

Synthesis and Preclinical Evaluation of the First Carbon-11 Labeled PET Tracers Targeting Substance P1-7.

Two potent SP1-7 peptidomimetics have been successfully radiolabeled via [11C]CO2-fixation with excellent yields, purity, and molar activity. l-[11C]SP1-7-peptidomimetic exhibited promising ex vivo biodistribution profile. Metabolite analysis showed that l-[11C]SP1-7-peptidomimetic is stable in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic stability can be significantly improved by substituting l-Phe for d-Phe, preserving 70% more of intact tracer and resulting in better brain and spinal cord tracer retention. Positron emission tomography (PET) scanning confirmed moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for l- and d-[11C]SP1-7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment with natural peptide SP1-7 in spinal cord for l-[11C]SP1-7-peptidomimetic. On the contrary, blocking using cold analogues of l- and d-[11C]tracers did not reduce the tracers' brain and spinal cord exposure. In summary, PET scanning of l- and d-[11C]SP1-7-peptidomimetics confirms rapid blood-brain barrier and blood-spinal-cord barrier penetration. Therefore, further validation of these two tracers targeting SP1-7 is needed in order to define a new PET imaging target and select its most appropriate radiopharmaceutical.

High resolution combined molecular and structural optical imaging of colorectal cancer in a xenograft mouse model.

With the emergence of immunotherapies for cancer treatment, there is a rising clinical need to visualize the tumor microenvironment (TME) non-invasively in detail, which could be crucial to predict the efficacy of therapy. Nuclear imaging techniques enable whole-body imaging but lack the required spatial resolution. Conversely, near-infrared immunofluorescence (immuno-NIRF) is able to reveal tumor cells and/or other cell subsets in the TME by targeting the expression of a specific membrane receptor with fluorescently labeled monoclonal antibodies (mAb). Optical coherence tomography (OCT) provides three-dimensional morphological imaging of tissues without exogenous contrast agents. The combination of the two allows molecular and structural contrast at a resolution of ~15 µm, allowing for the specific location of a cell-type target with immuno-NIRF as well as revealing the three-dimensional architectural context with OCT. For the first time, combined immuno-NIRF and OCT of a tumor is demonstrated in situ in a xenograft mouse model of human colorectal cancer, targeted by a clinically-safe fluorescent mAb, revealing unprecedented details of the TME. A handheld scanner for ex vivo examination and an endoscope designed for imaging bronchioles in vivo are presented. This technique promises to complement nuclear imaging for diagnosing cancer invasiveness, precisely determining tumor margins, and studying the biodistribution of newly developed antibodies in high detail.

Cytolytic virus activation therapy and treatment monitoring for Epstein-Barr virus associated nasopharyngeal carcinoma in a mouse tumor model.

Undifferentiated nasopharyngeal carcinoma (NPC) is 100% associated with Epstein-Barr virus (EBV). Expression of viral proteins in the tumor cells is highly restricted. EBV reactivation by CytoLytic Virus Activation (CLVA) therapy triggers de novo expression of early viral kinases (PK and TK) and uses antiviral treatment to kill activated cells. The mechanism of tumor elimination by CLVA was analyzed in NPC mouse model using C666.1 cells. Valproic acid (VPA) was combined with gemcitabine (GCb) to stimulate EBV reactivation, followed by antiviral treatment with ganciclovir (GCV). A single cycle of CLVA treatment resulted in specific tumor cell killing as indicated by reduced tumor volume, loss of EBV-positive cells in situ, and paralleled by decreased EBV DNA levels in circulation, which was more pronounced than treatment with GCb alone. In vivo reactivation was confirmed by presence of lytic gene transcripts and proteins in tumors 6 days after GCb/VPA treatment. Virus reactivation was visualized by [124 I]-FIAU accumulation in tumors using PET-scan. This studied showed that CLVA therapy is a potent EBV-specific targeting approach for killing tumor cells. The [124 I]-FIAU appears valuable as PET tracer for studies on CLVA drug dosage and kinetics in vivo, and may find clinical application in treatment monitoring.

In-vivo monitoring of anti-folate therapy in arthritic rats using [18F]fluoro-PEG-folate and positron emission tomography.

BACKGROUND: Folate receptor ß (FRß) is involved in facilitating cellular uptake of folates and anti-folates (such as methotrexate (MTX)). In rheumatoid arthritis, FRß is expressed on synovial macrophages and recently has been explored as a biomarker for imaging in arthritic rats using the folate-based positron emission tomography (PET) tracer [18F]fluoro-PEG-folate. The purpose of this study was to examine whether this folate tracer can also be used to monitor therapeutic efficacy of MTX in arthritic rats. METHODS: Arthritic rats received either no treatment or MTX therapy (1 mg/kg, either 2× or 4×). Healthy rats did not receive any arthritic induction or therapy. [18F]fluoro-PEG-folate PET-CT scans (60 min) were performed before and after MTX therapy. Following PET, the ex-vivo tissue distribution of radioactivity was determined in excised knees and multiple tissues. Synovial macrophage infiltration in knee sections was quantified by immunohistochemistry using ED1 and ED2 antibodies. RESULTS: PET scans clearly visualized increased uptake of [18F]fluoro-PEG-folate in arthritic knees compared with contralateral knees. Significantly lower standard uptake values (1.5-fold, p < 0.01) were observed in arthritic knees of both MTX-treated groups after therapy, approximating the levels seen in healthy rats. Consistently, ex-vivo tissue distribution demonstrated a 2-4-fold lower tracer uptake in the arthritic knee of 2× and 4× MTX-treated rats, respectively, compared with control rats. These results were corroborated with significantly reduced (2-4-fold, p < 0.01) ED1-positive and ED2-positive synovial macrophages in arthritic knees of the MTX-treated rats compared with those of the control rats. CONCLUSION: This study in arthritic rats underscores the potential and usefulness of [18F]fluoro-PEG-folate PET as a therapeutic monitoring tool of MTX therapy and potentially other anti-folate treatment of arthritis.

Number and duration of echolocation click trains produced by a harbor porpoise (Phocoena phocoena) in relation to target and performance.

Echolocation effort (number and duration of echolocation click trains produced) by a harbor porpoise is described in relation to target presence, strength and distance, and performance of the detection task. The porpoise was presented with two target sizes at five distances (12-20 m), or no target, and had to indicate whether it could detect the target. Small, distant targets required long and multiple click trains. Multiple click trains mostly occurred when the small target was far away and not detected, and during target-absent trials in which the animal correctly responded. In target-absent trials, an incorrect response was linked to short click trains. Click train duration probably increased until the animal's certainty about the target's presence or absence exceeded a certain level, after which the porpoise responded.