Localization,mechanism and reduction of renal retention of technetium‐99m labeled epidermal growth factor receptor‐specific nanobody in mice

Background

Nanobodies are single‐domain antigen binding fragments derived from functional heavy‐chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti‐EGFR ^99mTc‐7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of ^99mTc‐7C12.

Methods

First we compared the renal uptake of ^99mTc‐7C12 in megalin‐deficient and megalin‐wild‐type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of ^99mTc‐7C12 was analyzed in CD‐1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts.

Results

Renal uptake of ^99mTc‐7C12 was 44.22 ± 3.46% lower in megalin‐deficient compared with megalin‐wild‐type mice. In CD‐1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 ± 2.99 and 36.22 ± 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 ± 2.09%. Gelofusine and lysine coadministration improved tumor uptake.

Conclusion

Megalin contributes to the renal accumulation of ^99mTc‐7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy. Copyright © 2010 John Wiley & Sons, Ltd.     

A radiolabeled nonapeptide probe targeting PC‐3 cells and bone metastases of prostate cancer in mice

Previous investigations showed that interleukin‐11 (IL‐11) and the IL‐11 receptor (IL‐11R) are correlated with regulation of tumor progression and may play significant roles in bone metastases. The nonapeptide structure c(CGRRAGGSC) is a phage‐display‐selected IL‐11 mimic that binds to IL‐11R. The aim of this study was to synthesize radiolabeled c(CGRRAGGSC) and to investigate the possible interaction between this radioactive probe and an IL‐11R‐positive bone metastasis model of PC‐3 prostate cancer. The molecular probe ^99mTc–DTPA–c(CGRRAGGSC) was radiolabeled with ^99mTc using the diethylenetriaminepentaacetic acid (DTPA) chelate. Counterstaining was performed with LSS670, a near‐infrared dye. The binding sites of the molecular probe in PC‐3 cells were observed under a fluorescence microscope. The binding characteristics of the labeled probe were analyzed using radioreceptor analysis. Single photon emission tomography imaging and biodistribution of the probe were investigated using xenografts of PC‐3 cells into tibias of nude mice. The labeled product, ^99mTc–DTPA–c(CGRRAGGSC), was obtained with high labeling efficiency, high radiochemical purity and good stability. The molecular probe was combined with the PC‐3 cell membrane and cytoplasm through fluorescence tracing. In the saturation and competitive inhibition experiments performed in vitro, the K_d value was 0.32 ± 0.02 n m and the B_max value was 754 ± 34 fmol mg^?1 pro. The probe exhibited a high tumor uptake in vivo. The radioactive molecular probe ^99mTc–DTPA–c(CGRRAGGSC) may be used as a specific molecular imaging agent for detecting IL‐11R overexpression in tumors and bone metastasis, such as prostate cancers. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of 177Lu‐nanobodies for radioimmunotherapy of HER2‐positive breast cancer: evaluation of different bifunctional chelators

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor‐to‐background‐ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2‐overexpressing minimal residual disease. The therapeutic radionuclide Lutetium‐177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators ‐ p‐SCN‐Bn‐DOTA, DOTA‐NHS‐ester, CHX‐A”‐DTPA or 1B4M‐DTPA ‐ in order to select the optimal chemical link between Lutetium‐177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator‐conjugation. High stability in time was observed, together with nanomolar affinities on HER2‐expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro‐CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M‐DTPA‐coupled conjugate was further evaluated in a high HER2‐expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor‐to‐background ratios. No specific binding was observed in a HER2‐negative model. In conclusion, the in‐house developed anti‐HER2 nanobody 2Rs15dHIS can be successfully labeled with ¹⁷⁷Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M‐DTPA‐based conjugate. Copyright © 2012 John Wiley & Sons, Ltd.     

In vivo near‐infrared fluorescence targeting of T cells: comparison of nanobodies and conventional monoclonal antibodies

The large size of conventional antibodies impedes tissue penetration and renal elimination, resulting in suboptimal in vivo targeting. Here we assess the utility of nanobodies and nanobody‐Fc‐fusion proteins as alternatives to monoclonal antibodies as theranostics, using T cell ADP–ribosyltransferase 2 (ART2) as a model antigen for specific targeting of lymph nodes. ART2‐specific monovalent nanobody s + 16a (17 kDa), a bivalent Fc‐fusion protein of s + 16a (s + 16‐mFc, 82 kDa), and conventional antibody Nika102 (150 kDa) were labeled with AlexaFluor680. In vitro binding and inhibitory properties of the three AF680 conjugates were assessed by flow cytometry. For in vivo imaging experiments, AF680 conjugates were intravenously injected in mice lacking (KO) or overexpressing (TG) ART2. We monitored circulating and excreted AF680 conjugates in plasma and urine and performed in vivo near‐infrared fluorescence imaging. Nanobody s + 16a⁶⁸⁰ and s + 16mFc⁶⁸⁰ labeled and inhibited ART2 on T cells in lymph nodes within 10 min. In contrast, mAb Nika102⁶⁸⁰ required 2 h for maximal labeling without inhibition of ART2. In vivo imaging revealed specific labeling of ART2‐positive lymph nodes but not of ART2‐negative lymph nodes with all AF680 conjugates. Even though bivalent s + 16mFc⁶⁸⁰ showed the highest labeling efficiency in vitro, the best lymph node imaging in vivo was achieved with monovalent nanobody s + 16a⁶⁸⁰, since renal elimination of unbound s + 16a⁶⁸⁰ significantly reduced background signals. Our results indicate that small single‐domain nanobodies are best suited for short‐term uses, such as noninvasive imaging, whereas larger nanobody‐Fc‐fusion proteins are better suited for long‐term uses, such as therapy of inflammation and tumors. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid hepatic clearance of 99mTc‐TMEOP: a new candidate for myocardial perfusion imaging

Background

^99mTc labeled radiotracers used in clinical practice lack the perfect characteristics for myocardial perfusion imaging. In particular, the high liver uptake can interfere in the interpretation of the inferior myocardial wall. Within the tricarbonyl approach, we used tris(pyrazolyl)methane ^99mTc organometallic complexes as a lead structure. Herein we present the production, in vivo and in vitro metabolic studies in rats and the first in vivo biodistribution in rats for tri‐methoxy‐tris‐pyrazolyl‐^99mTc‐(CO)₃ (^99mTc‐TMEOP), compared with ^99mTc‐sestamibi and ^99mTc‐tetrofosmin.

Methods

The chemical identity of ^99mTc‐TMEOP was characterized by RP‐HPLC. The octanol–water partition coefficient was determined under physiological conditions. In vitro stability and protein binding were determined using RP‐HPLC. In vivo stability was determined in blood, heart, liver and kidney homogenates, intestine and urine using RP‐HPLC. In vivo biodistribution was determined using dynamic planar acquisitions. Pinhole gated SPECT images were performed in other animals. Cardiac, liver and lung uptake were expressed as differential uptake ratios by drawing regions of interest in the organs of interest and around the total body. Heart–liver and heart–lung ratios were derived. Cardiac uptake was also expressed as percentage of injected activity. SPECT images were processed to determine the heart–liver ratio on SPECT images, to compare functional parameters between different tracers and to visualize homogeneous intracardiac tracer distribution.

Results

^99mTc‐TMEOP is a moderately lipophilic cation, is stable and does not undergo any transformation in vitro. ^99mTc‐TMEOP also shows a high in vivo stability. In vivo imaging shows liver kinetics faster than those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin. Cardiac uptake and functional analysis of pinhole gated SPECT data are comparable to those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin.

Conclusion

Although ^99mTc‐TMEOP shows a cardiac uptake between those of ^99mTc‐sestamibi and ^99mTc‐tetrofosmin, a better heart–liver ratio is obtained due to the faster liver washout. These results suggest possible faster cardiac perfusion imaging using ^99mTc‐TMEOP without liver activity interference. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular imaging of tumors with nanobodies and antibodies: Timing and dosage are crucial factors for improved in vivo detection

The utility of nanobodies and conventional antibodies for in vivo imaging is well known, but optimum dosing and timing schedules for one versus the other have not been established. We aimed to improve specific tumor imaging in vivo with nanobodies and conventional antibodies using near‐infrared fluorescence (NIRF) imaging. We used ARTC2 expressed on lymphoma cells as a model target antigen. ARTC2‐specific nanobody s+16a and conventional antibody Nika102 were labeled with NIRF‐dye AF680. In vivo NIRF‐imaging of ARTC2‐positive and ARTC2‐negative xenografts was performed over 24 h post‐injection of 5, 10, 25, or 50 µg of each conjugate. Specific target‐binding and tissue‐penetration were verified by NIRF imaging ex vivo, flow cytometry and fluorescence microscopy. NIRF‐imaging of s+16a⁶⁸⁰ in vivo revealed a six times faster tumor accumulation than of Nika102⁶⁸⁰. Using 50 µg of s+16a⁶⁸⁰ increased the specific signals of ARTC2‐positive tumors without increasing background signals, allowing a tumor‐to‐background (T/B) ratio of 12.4 ± 4.2 within 6 h post‐injection. Fifty micrograms of Nika102⁶⁸⁰ increased specific signals of ARTC2‐positive tumors but also of ARTC2‐negative tumors and background, thereby limiting the T/B ratio to 6.1 ± 2.0. Ten micrograms of Nika102⁶⁸⁰ only slightly reduced specific tumor signals but dramatically reduced background signals. Ex vivo analyses confirmed a faster and deeper tumor penetration with s+16a⁶⁸⁰. Using nanobody s+16a allowed same‐day imaging with a high T/B ratio, whereas antibody Nika102 gave optimal imaging results only 24 h post injection. Nanobody s+16a required a high dose, whereas antibody Nika102 had the best T/B‐ratio at a low dose. Therefore, timing and dosage should be addressed when comparing nanobodies and conventional antibodies for molecular imaging purposes. Copyright © 2015 John Wiley & Sons, Ltd.     

Correlation Between Epidermal Growth Factor Receptor-Specific Nanobody Uptake and Tumor Burden: A Tool for Noninvasive Monitoring of Tumor Response to Therapy

Purpose  

Nanobodies represent an interesting class of probes for the generic development of molecular imaging agents. We studied the relationship between tumor uptake of the epidermal growth factor receptor (EGFR)-specific nanobody ^99mTc-7C12 and tumor burden and evaluated the possibility of using this probe to monitor tumor response to erlotinib.     

TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation

Summary. Background: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co‐administration of t‐PA and a TAFIa inhibitor enhances t‐PA treatment. Objective: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t‐PA induced clot lysis. Results: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A¹⁴⁷‐C³⁰⁵‐I³²⁵‐I³²⁹‐Y³³³‐Q³³⁵), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16‐fold molar excess of nanobody over TAFIa, IC₅₀’s range between 0.38‐ and > 16‐fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t‐PA concentration. The nanobodies were found to bind in the active‐site region of TAFIa and their time‐dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition. Conclusion: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.     

The effect of MRI contrast agents on hepatic and splenic uptake in the rabbit during 99mTc‐MDP bone scintigraphy

The objective of this study was to investigate the effects of Omniscan® and Magnevist® on ^99mTc‐MDP uptake in rabbits during ^99mTc‐MDP bone scintigraphy. In Experiment Group 1, 30 healthy adult rabbits were randomized into six subgroups (n = 5); each subgroup experienced a different time interval between injections (30 min, 60 min, 120 min, 240 min, 360 min, 24 h). All six subgroups were injected first with Omniscan®, then with ^99mTc‐MDP. After 7 days, the same six subgroups were injected with normal saline followed by ^99mTc‐MDP at the same time intervals. In Experiment Group 2, 20 healthy adult rabbits were allocated randomly to four subgroups (n = 5); each subgroup experienced a different time interval between injections (30 min, 60 min, 120 min, 240 min). All four subgroups were injected first with Magnevist®, then with ^99mTc‐MDP. After 7 days, the same four subgroups were injected with normal saline followed by ^99mTc‐MDP. In all experiments, whole‐body skeletal imaging was performed. Liver, spleen, and background were delineated to determine the target‐to‐background (T/B) ratio. Diffusely increased intake of the imaging agent was seen in the liver and spleen when the injection‐time interval between Omniscan® and ^99mTc‐MDP varied from 30 min to 240 min and when the time interval between Magnevist® and ^99mTc‐MDP was 30 min–60 min. The imaging findings are consistent with the results of L/B and S/B ratios in each experiment group. Both Omniscan® and Magnevist® have an effect on ^99mTc‐MDP uptake during bone scanning; the main effect is diffusely increased hepatic and splenic activity. Copyright © 2015 John Wiley & Sons, Ltd.     

The washout rate of a subcutaneous 99mTc-HSA depot in lower extremity lymphoedema

Purpose: Lymphoscintigraphy is currently the leading diagnostic modality of lower extremity lymphoedema but has been criticized for being unreliable. Washout rate constants have been investigated and proven to be of diagnostic value in several studies of breast‐cancer‐related lymphoedema; however, the applicability in lower extremity lymphoedema needs further evaluation. The aim of the study was to verify if washout of ^99mTc‐Human Serum Albumin (^99mTc‐HSA) is a reliable diagnostic tool in lower extremity lymphoedema. Methods: Twenty healthy volunteers and eight patients (11 legs) with lymphoscintigraphy verified lower extremity lymphoedema participated in the study. A depot consisting of 0·1 ml 10 MBq/ml ^99mTc‐HSA was injected subcutaneously into the dorsum of each foot. The depot washout rate was measured using a portable scintillation detector system and time‐activity curves were generated. After 30 min of supine rest and 10 min of standardized ergometric exercise, measurements were recorded for 20 min. Following correction for physical decay of ^99mTc, the depot washout rate constant was calculated using linear regression analysis. Finally depot half‐life was calculated from the washout rate constant. Results: Median half‐life for healthy volunteers was 9·4 h (range 2·5–28·3 h). Median half‐life for lymphoedema patients was 10·7 h (range 1·5–35·1 h). No statistical significant difference could be detected between healthy volunteers and lymphoedema patients (P = 0·78). Conclusions: The washout rate of a subcutaneous ^99mTc‐HSA depot is not a reliable diagnostic tool in examination of lower extremity lymphoedema. Additional examinations revealed in vivo instability of the utilized ^99mTc‐HSA as the likely reason.     

A novel Tc‐99 m and fluorescence labeled peptide as a multimodal imaging agent for targeting angiogenesis in a murine tumor model

The serine–aspartic acid–valine (SDV) peptide binds specifically to integrin α_Vβ₃. In the present study, we successfully developed a TAMRA–GHEG–ECG–SDV peptide labeled with both Tc‐99 m and TAMRA to target the integrin α_Vβ₃ of tumor cells; furthermore, we evaluated the diagnostic performance of Tc‐99 m TAMRA–GHEG–ECG–SDV as a dual‐modality imaging agent for tumor of the murine model. TAMRA–GHEG–ECG–SDV was synthesized using Fmoc solid‐phase peptide synthesis. Radiolabeling of TAMRA–GHEG–ECG–SDV with Tc‐99 m was done using ligand exchange methods. Labeling stability and cytotoxicity studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed in murine models with HT‐1080 and HT‐29 tumors. A tumor tissue slide was prepared and analyzed using confocal microscopy. After radiolabeling procedures with Tc‐99 m, the Tc‐99 m TAMRA–GHEG–ECG–SDV complexes were prepared in high yield (>99%). In the gamma camera imaging study, a substantial uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV into HT‐1080 tumor (integrin α_Vβ₃ positive) and low uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV into HT‐29 tumor (integrin α_Vβ₃ negative) were demonstrated. A competition study revealed that HT‐1080 tumor uptake was effectively blocked by the co‐injection of an excess concentration of SDV. Specific uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV was confirmed by biodistribution, ex vivo imaging and confocal microscopy studies. Our in vivo and in vitro studies revealed substantial uptake of Tc‐99 m TAMRA–GHEG–ECG–SDV in the integrin α_Vβ₃‐positive tumor. Tc‐99 m TAMRA–GHEG–ECG–SDV could be a good candidate for a dual‐modality imaging agent targeting tumor angiogenesis. Copyright © 2016 John Wiley & Sons, Ltd.     

Targeting human prostate cancer with 111In‐labeled D2B IgG,F(ab′)2 and Fab fragments in nude mice with PSMA‐expressing xenografts

D2B is a new monoclonal antibody directed against an extracellular domain of prostate‐specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab′)₂ and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with ¹¹¹In‐D2B IgG, ¹¹¹In‐capromab pendetide, ¹¹¹In‐D2B F(ab′)₂ and ¹¹¹In‐D2B Fab fragments in mice with PSMA‐expressing LNCaP and PSMA‐negative PC3 tumors at several time points after injection. All ¹¹¹In‐labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of ¹¹¹In‐D2B IgG and ¹¹¹In‐capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of ¹¹¹In‐D2B F(ab′)₂ and ¹¹¹In‐D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor‐to‐blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for ¹¹¹In‐D2B IgG, ¹¹¹In‐F(ab′)₂, ¹¹¹In‐Fab and ¹¹¹In‐capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab′)₂ and Fab fragments for targeting PSMA‐expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.     

Technetium-99m-labelled HL91 and technetium-99m-labelled MIBI SPECT imaging for the detection of ischaemic viable myocardium: a preliminary study

Purpose: The assessment of myocardial viability has become an important aspect of the diagnostic and prognostic work‐up of patients with coronary artery disease. Technetium‐99m labelled sestamibi (^99mTc‐MIBI) myocardial perfusion imaging may underestimate the viability of ischaemic myocardium. Technetium‐99m labelled 4,9‐diaza‐3,3,10,10‐tetramethyldodecan‐2,11‐dione dioxime (^99mTc‐HL91) is a hypoxia‐avid agent which can identify acutely ischaemic viable myocardium in a canine model using a standard gamma camera. The aim of this study was to evaluate uptake character of ischaemic viable myocardium and diagnostic performance of single‐photon emission computed tomography (SPECT) imaging by ^99mTc‐HL91 and ^99mTc‐MIBI in detecting ischaemic viable myocardium in coronary heart disease. Methods: A total of 41 patients with coronary artery disease were recruited from March 2008 to May 2009. For detecting ischaemic viable myocardium, SPECT imaging by ^99mTc‐HL91 and ^99mTc‐MIBI were performed in all patients before coronary revascularization. Six patients with single ischaemic myocardial segment received a 2‐day SEPCT/CT imaging protocol and the uptake of ^99mTc‐HL91 in ischaemic myocardium was quantitatively analysed. The remaining 35 patients received a 1‐day ^99mTc‐HL91 and ^99mTc‐MIBI SPECT imaging protocol. Resting ^99mTc‐MIBI myocardial perfusion imaging in 3–18 months after revascularization was used as the standard methodology to evaluate the myocardial viability. Results: In 41 patients, 66 ischaemic myocardial segments were proven to be viable and 12 to be necrotic by resting ^99mTc‐MIBI myocardial perfusion imaging after coronary revascularization. Furthermore, 60 viable segments with negative uptake of ^99mTc‐MIBI showed positive uptake of ^99mTc‐HL91. The remaining six viable segments and 12 necrotic segments showed both negative uptake of ^99mTc‐HL91 and ^99mTc‐MIBI. The sensitivity, specificity, accuracy, Younden Index, positive predictive value and negative predictive value for evaluating ischaemic viable myocardium were 90·9%, 100%, 92·3%, 90·9%, 100% and 66·7%, respectively. Ischaemic viable myocardium had the negative ^99mTc‐MIBI uptake and positive ^99mTc‐HL91 uptake, which demonstrated a mismatched uptake character. Quantitative analysis indicated the uptake of ^99mTc‐HL91 in viable myocardium was increasing in the first 1–3 h and remained stable at the 3–4 h after injection. Conclusion: Functional SPECT imaging with ^99mTc‐HL91 and ^99mTc‐MIBI can be used to detect the seriously ischaemic but viable myocardium with a mismatched uptake character. The uptake of ^99mTc‐HL91 in the viable myocardium reached a stable level at 3–4 h after injection.     

Quantitative imaging of myocardial infarct in rats with high resolution pinhole SPECT

Small animal imaging of cardiovascular disease using single photon emission tomography (SPECT) can be used to provide quantitative measurements of myocardial infarct. The purpose of this study was to demonstrate the accuracy of pinhole SPECT imaging with [^99mTc]sestamibi for estimation of infarct size in a rat model of coronary artery disease. Nine rats had their left anterior descending artery ligated to induce a region of myocardial infarct. These animals were injected with 37 MBq [^99mTc]sestamibi, and, 1 h later, scanned on a pinhole SPECT system for 30 min. The defect size measured with SPECT, which was dependent on a threshold applied to the short axis circumferential profiles, was compared against the gold standard triphenyltetrazolium chloride (TTC) staining. The size of the perfusion deficit measured using [^99mTc]sestamibi SPECT compared very favorably with the TTC staining result, for threshold values in the range 50–70%. The optimum threshold was approximately 70%, giving an excellent correlation (R ²=0.89, p<0.001). Estimation of infarct size by [^99mTc] sestamibi SPECT yielded an excellent agreement with TTC staining. In conclusion, measurement of myocardial infarct with SPECT can be used to study the rat heart in vivo, and provides a quantitative measure of myocardial viability.     

SPECT Imaging with <Superscript>99m</Superscript>Tc-Labeled EGFR-Specific Nanobody for <Emphasis Type="Italic">In Vivo</Emphasis> Monitoring of EGFR Expression

Purpose

Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody®, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice.

Methods

Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with ^99mTc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of ^99mTc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR.

Results

FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of ^99mTc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6?±?0.9%IA) compared to total liver uptake (18.9?±?0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2?±?0.5%IA/cm³) of ^99mTc-8B6 Nanobody compared to DU145 xenografts (1.8?±?0.3%IA/cm³, p?

Conclusion

The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with ^99mTc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

     

A comparison of single plasma sample methods to estimate renal clearance using 99mTc‐ethylenedicysteine and 99mTc‐mercaptoacetyltriglycine

Several single sample methods for determination of ^99mTc‐mercaptoacetyltriglycine (MAG3) clearance are being used clinically. Kabasakal et al. proposed a similar formula for ^99mTc‐ethylenedicysteine (EC). This study was performed to compare his method with Bubeck et al. formula for ^99mTc‐MAG3 already in use. Twenty‐eight subjects divided in two groups were registered which included 22 patients with various renal diseases (group‐I) and six normal volunteers (group II). All subjects were studied twice using both the radiopharmaceuticals. The images and renogram parameters, that is T_MAX and T_1/2 of both the agents, were similar in all the subjects. The clearance of the ^99mTc‐EC was however considerably higher than ^99mTc‐MAG3 in both the groups (mean ± SEM =279 ± 14 ml min^?1/1·73 m² versus 177 ± 15 ml min^?1/1·73 m² in group‐I and 377 ± 11·90 ml min^?1/1·73 m² versus 238 ± 8·23 ml min^?1/1·73 m² in group II). This difference was more pronounced in cases with reduced renal functions. Among the Effective Renal Plasma Flow (ERPF) values determined from EC and MAG3 clearances in six normal volunteers, four cases only in MAG3 had ERPF below the lower limit. This study has demonstrated superiority of single sample method for ^99 mTc‐EC clearance over its analogous method for ^99mTc‐MAG3.     

pH-Sensitive,Long-Circulating Liposomes as an Alternative Tool to Deliver Doxorubicin into Tumors: a Feasibility Animal Study

Purpose

Therapeutic agents used in chemotherapy have low specificity leading to undesired severe side effects. Hence, the development of drug delivery systems that improve drug specificity, such as liposome moieties, is an alternative to overcome chemotherapy limitations and increase antitumor efficacy. In this study, the biodistribution profile evaluation of pH-sensitive long-circulating liposomes (SpHL) containing [^99mTc]DOX in 4T1 tumor-bearing BALB/c mice is described.

Procedures

[^99mTc]DOX was radiolabeled by direct method. Liposomes were prepared and characterized. [^99mTc]DOX was encapsulated into liposomes by freezing and thawing. Circulation time for SpHL-[^99mTc]DOX was determined by measuring the blood activity from healthy animals. Biodistribution studies were carried out in tumor-bearing mice at 1, 4, and 24 h after injection.

Results

Blood levels of the SpHL-[^99mTc]DOX declined in a biphasic manner, with an α half-life of 14.1 min and β half-life of 129.0 min. High uptake was achieved in the liver and spleen, due to the macrophages captured. Moreover, tumor uptake was higher than control tissue, resulting in high tumor-to-muscle ratios, indicating higher specificity for the tumor area.

Conclusion

[^99mTc]DOX was successfully encapsulated in liposomes. Biodistribution indicated high tumor-to-muscle ratios in breast tumor-bearing BALB/c mice. In summary, these results showed the higher accumulation of SpHL-[^99mTc]DOX in the tumor area, suggesting selective delivery of doxorubicin into tumor.

     

Tobacco mosaic virus for the targeted delivery of drugs to cells expressing prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics. To improve the efficacy of nanoparticle formulations for the imaging and/or eradication of prostate cancer, we synthesized the PSMA-binding glutamic acid derivative DUPA and conjugated it to the external surface of tobacco mosaic virus (TMV) particles. DUPA-targeted TMV was subsequently loaded with the antineoplastic agent mitoxantrone (MTO) or conjugated internally with the fluorescent dye cyanine 5 (Cy5). We found that TMV particles could be efficiently decorated with DUPA and loaded with MTO or Cy5 while maintaining structural integrity. DUPA-targeted TMV particles were able to bind more efficiently to the surface of PSMA⁺ LNCaP cells compared to non-targeted TMV; but there was little difference in binding efficiency between targeted and untargeted TMV when we tested PSMA⁻ PC3 cells (both cell lines are prostate cancer cell lines). DUPA-targeted TMV particles were internalized by LNCaP cells enabling drug delivery. Finally, we loaded the DUPA-targeted TMV particles and untargeted control particles with MTO to test their cytotoxicity against LNCaP cells in vitro. The cytotoxicity of the TMV-MTO particles (IC₅₀ = 10.2 nM) did not differ significantly from that of soluble MTO at an equivalent dose (IC₅₀ = 12.5 nM) but the targeted particles (TMV-DUPA-MTO) were much more potent (IC₅₀ = 2.80 nM). The threefold increase in cytotoxicity conferred by the DUPA ligand suggests that MTO-loaded, DUPA-coated TMV particles are promising as a therapeutic strategy for PSMA⁺ prostate cancer and should be advanced to preclinical testing in mouse models of prostate cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound protein that is preferentially expressed in the prostate gland and induced in many prostate cancers, making it an important target for new diagnostics and therapeutics.     

Correlation between serum calcium levels and dual-phase (99m)Tc-sestamibi parathyroid scintigraphy in primary hyperparathyroidism

Aim: The goal of the study is to correlate serum calcium levels with the results of dual‐phase ^99mTc‐sestamibi parathyroid scintigraphy to find the best cut‐off level of the serum calcium that correlates with a positive presurgery. Methods: In 111 patients, serum calcium and plasma parathormone (PTH) levels were compared with the results of the ^99mTc‐MIBI scintigraphy and with this data determined the level of calcium above which the ^99mTc‐MIBI scintigraphy was likely to be positive and below which the study was likely to be negative. Results: In total, 11 men (18%) and 50 women (82%) had a positive ^99mTc‐MIBI study. Overall 67% of those patients with a positive ^99mTc‐MIBI study had a PTH >200 ng l^?1 compared to only 9% of those with a negative ^99mTc‐MIBI scintigraphy; however, for those with a positive study on an early ^99mTc‐MIBI scintigraphy, this rose to 85%. Overall a serum calcium of >2·70 mmol l^?1 was found in 82% of patients with a positive ^99mTc‐MIBI study but only 14% of those with a negative ^99mTc‐MIBI study, this is rose to 97% of patients with a parathyroid adenoma identified on early images. It is also shown that patients whose serum total calcium <2·51 mmol l^?1 rarely have positive ^99mTc‐MIBI scintigraphy. Conclusion: ^99mTc‐MIBI parathyroid scintigraphy is most likely to yield identification and localization of a parathyroid adenoma when both PTH and calcium are elevated; however, although there is no lower limit of PTH which can predict a negative study, we cannot recommend ^99mTc‐MIBI parathyroid scintigraphy if the serum calcium is <2·51 mmol l^?1.     

Pulmonary clearance of [99mTc]DTPA and [99mTc]albumin in smokers

We measured the pulmonary clearance of [^99mTc]DTPA and [^99mTc]albumin for 3 h in 10 non-smokers and 10 healthy smokers. Seven of the non-smokers had a monoexponential clearance of [^99mTc]DTPA with a mean half-life of 66±18 min. The other three had a biexponential clearance of [^99mTc]DTPA with a fraction of radioactivity clearing rapidly (f_F) of 14±4%. Eight smokers had biexponential clearance of [^99mTc]DTPA. The half-life of the fast and slow clearance components was 1265 and 62611 min respectively. The f_F was 56±25%. Two of the smokers showed a monoexponential clearance of [^99mTc]DTPA with a half-life of 72 and 55 min. All non-smokers had monoexponential clearance curves for [^99mTc]albumin, compared with seven smokers. The half-life was 279±43 min in non-smokers and 236±64 min in smokers. The difference in half-life was not significant. In three smokers, the clearance curves of [^99mTc]albumin were significantly better described by a bi-exponential equation. The f_F was 22±9%. The effects of smoking on the clearance of [^99mTc]albumin appear to be qualitatively similar to those on the clearance of [^99mTc]DTPA. Clearance of [^99mTc]albumin seems less sensitive to the effects of smoking than clearance of [^99mTc]DTPA.