Effect of co-ligands on chemical and biological properties of 99mTc(III) complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl,F, SCN and N3; CDOH2 = cyclohexanedione dioxime)

Introduction^99mTc-Teboroxime ([^99mTcCl(CDO)(CDOH)₂BMe]) is a member of the BATO (boronic acid adducts of technetium dioximes) class of ^99mTc(III) complexes. This study sought to explore the impact of co-ligands on solution stability, heart uptake and myocardial retention of [^99mTc(L)(CDO)(CDOH)₂BMe] (^99mTc-Teboroxime: L = Cl; ^99mTc-Teboroxime(F): L = F; ^99mTc-Teboroxime(SCN): L = SCN; and ^99mTc-Teboroxime(N₃): L = N₃).MethodsRadiotracers ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared by reacting ^99mTc-Teboroxime with NaF, NaSCN and NaN₃, respectively. Biodistribution and imaging studies were carried out in Sprague–Dawley rats. Image quantification was performed to compare their heart retention and liver clearance kinetics.ResultsComplexes ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared in high yield with high radiochemical purity. All new radiotracers were stable for > 6 h in the kit matrix. In its HPLC chromatogram, ^99mTc-Teboroxime showed one peak at ~ 15.5 min, which was shorter than that of ^99mTc-Teboroxime(F) (~ 16.4 min). There were two peaks for ^99mTc-Teboroxime(SCN) at 16.5 and 18.3 min. ^99mTc-Teboroxime(N₃) appeared as a single peak at 18.4 min. Their heart retention and liver clearance curves were best fitted to the bi-exponential decay function. The half-times of fast/slow components were 1.6 ± 0.4/60.7 ± 8.9 min for ^99mTc-Teboroxime, 0.8 ± 0.2/101.7 ± 20.7 min for ^99mTc-Teboroxime(F), 1.2 ± 0.3/84.8 ± 16.6 min for ^99mTc-Teboroxime(SCN), and 2.9 ± 0.9/51.6 ± 5.0 min for ^99mTc-Teboroxime(N₃). The 2-min heart uptake followed the order of ^99mTc-Teboroxime (3.00 ± 0.37%ID/g) > ^99mTc-Teboroxime(N₃) (2.66 ± 0.01 %ID/g) ≈ ^99mTc-Sestamibi (2.55 ± 0.46 %ID/g) > ^99mTcN-MPO (2.38 ± 0.15 %ID/g). ^99mTc-Teboroxime remains the best in first-pass extraction. The best image acquisition window is 0–5 min for ^99mTc-Teboroximine and 0–15 min for ^99mTc-Teboroximine(N₃).ConclusionCo-ligands had significant impact on the heart uptake and myocardial retention of complexes [^99mTc(L)(CDO)(CDOH)₂BMe] (L = Cl, F, SCN and N₃). Future studies should be directed towards minimizing the liver uptake and radioactivity accumulation in the blood vessels while maintaining their high heart uptake.     

Apoptotic abscess imaging with 99mTc-HYNIC-rh-Annexin-V

Abscess formation causes systemic and localized up-regulation of neutrophil [polymorphonuclear leukocytes (PMNs)] signaling pathways. In the abscess, following bacterial ingestion or PMN activation by inflammatory mediators, PMN apoptosis is elevated and leads to the externalization of phosphatidylserine. Annexin-V (AnxV) has been shown to have high affinity to externalized phosphatidylserine. We hypothesized that ^99mTc-AnxV will target high densities of apoptotic PMNs and image abscesses. AnxV, conjugated with hydrazinenicaotinamide (HYNIC), was labeled with reduced ^99mTcO₄^? and its purity was determined by instant thin-layer chromatography. Apoptosis was induced in isolated human PMNs by incubation in 2% saline for 17 and 22 h at 37°C. PMNs were then incubated with ^99mTc-HYNIC-AnxV and associated ^99mTc was determined. Abscesses were induced in mice by intramuscular injection of bacteria or turpentine. Following intravenous administration of ^99mTc-HYNIC-AnxV, mice were imaged and tissue distribution studied at 4 and 24 h. Radiochemical purity of ^99mTc-HYNIC-AnxV was 84.9±8.11%. At 17 h, ^99mTc-HYNIC-AnxV bound to apoptotic PMNs was 71.6±0.01% and 48.6±0.01% for experimental and control cells, respectively (P=.002). At 22 h, experimental cells retained 74.9±0.02% and control cells retained 47.2±0.02% (P=.005). ^99mTc-HYNIC-AnxV associated with bacterial abscesses was 1.25±0.09 and 3.75±0.83 percent injected dose per gram (%ID/g) at 4 and 24 h compared to turpentine abscesses which was 1.02±0.16 and 0.72±0.17 %ID/g at 4 (P≤.05) and 24 h (P≤.01). ^99mTc-HYNIC-AnxV represents a minimally invasive and promising agent to image and potentially distinguish between infectious and inflammatory abscesses.     

In vivo comparison of DOTA based 68Ga-labelled bisphosphonates for bone imaging in non-tumour models

Bone metastases are a class of cancerous metastases that result from the invasion of a tumor into bone. The solid mass which forms inside the bone is often associated with a constant dull ache and severe spikes in pain, which greatly reduce the quality of life of the patient. Numerous ^99mTc-labeled bisphosphonate functionalised complexes are well established tracers for bone metastases imaging. The objective of this research was to evaluate the pharmacokinetics and behaviour of three DOTA based bisphosphonate functionalised ligands (BPAMD, BPAPD and BPPED), using both ⁶⁸Ga μ-PET in vivo imaging and ex vivo biodistribution studies in healthy Wistar rats. The compounds were labelled with ⁶⁸Ga in high yields using an ammonium acetate buffer, and subsequently purified using a cation exchange resin. High bone uptake values were observed for all ⁶⁸Ga-labelled bisphosphonates at 60 minutes p.i. The highest uptake was observed for [⁶⁸Ga]BPPED (2.6 ± 0.3% ID/g) which compares favourably with that of [^99mTc]MDP (2.7 ± 0.1 ID/g) and [¹⁸F]fluoride (2.4 ± 0.2% ID/g). The ⁶⁸Ga-labelled DOTA-bisphosphonates showed rapid clearance from the blood and renal system, as well as low binding to soft tissue, resulting in a high bone to blood ratio (9.9 at 60 minutes p.i. for [⁶⁸Ga]BPPED, for example). Although further studies are required to assess their performance in tumor models, the results obtained suggest that these ligands could be useful both in imaging (⁶⁸Ga) and therapeutic treatment (¹⁷⁷Lu) of bone metastases.     

Characterization of (99m)Tc-labeled cytokine ligands for inflammation imaging via TNF and IL-1 pathways

IntroductionTNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize ^99mTc-labeled forms of these ligands, ^99mTc-IL-1ra-Fc (IF), ^99mTc-TNFR2-Fc (TF), and ^99mTc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging.MethodsThe cytokine ligands were labeled with ^99mTc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice.ResultsRadiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3 h after injection were 2.76 ± 0.20 vs. 0.69 ± 0.12 for IF, 5.86 ± 0.40 vs. 2.86 ± 0.61 for TF, and 7.61 ± 0.86 vs. 1.99 ± 0.31 for TFI (P < 0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P < 0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1β and TNF-α in the inflamed ears.Conclusions^99mTc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.     

Comparison of two cross-bridged macrocyclic chelators for the evaluation of 64Cu-labeled-LLP2A,a peptidomimetic ligand targeting VLA-4-positive tumors

Integrin α₄β₁ (also called very late antigen-4 or VLA-4) plays an important role in tumor growth, angiogenesis and metastasis, and there has been increasing interest in targeting this receptor for cancer imaging and therapy. In this study, we conjugated a peptidomimetic ligand known to have good binding affinity for α₄β₁ integrin to a cross-bridged macrocyclic chelator with a methane phosphonic acid pendant arm, CB-TE1A1P. CB-TE1A1P-LLP2A was labeled with ⁶⁴Cu under mild conditions in high specific activity, in contrast to conjugates based on the “gold standard” di-acid cross-bridged chelator, CB-TE2A, which require high temperatures for efficient radiolabeling. Saturation binding assays demonstrated that ⁶⁴Cu-CB-TE1A1P-LLP2A had comparable binding affinity (1.2 nM vs 1.6 nM) but more binding sites (B_max = 471 fmol/mg) in B16F10 melanoma tumor cells than ⁶⁴Cu-CB-TE2A-LLP2A (B_max = 304 fmol/mg, p < 0.03). In biodistribution studies, ⁶⁴Cu-CB-TE1A1P-LLP2A had less renal retention but higher uptake in tumor (11.4 ± 2.3 %ID/g versus 3.1 ± 0.6 %ID/g, p < 0.001) and other receptor-rich tissues compared to⁶⁴Cu-CB-TE2A-LLP2A. At 2 h post-injection, ⁶⁴Cu-CB-TE1A1P-LLP2A also had significantly higher tumor:blood and tumor:muscle ratios than ⁶⁴Cu-CB-TE2A-LLP2A (CB-TE1A1P = 19.5 ± 3.0 and 13.0 ± 1.4, respectively, CB-TE2A = 4.2 ± 1.4 and 5.5 ± 0.9, respectively, p < 0.001). These data demonstrate that ⁶⁴Cu-CB-TE1A1P-LLP2A is an excellent PET radiopharmaceutical for the imaging of α₄β₁ positive tumors and also has potential for imaging other α₄β₁ positive cells such as those of the pre-metastatic niche.     

Radiosynthesis and biodistribution of 99mTc-rifampicin: A novel radiotracer for in-vivo infection imaging

^99mTc–rifampicin (^99mTc–RMP) a new radioantibiotic complex was synthesized specifically for the infection localization caused by methicillin-resistant Staphylococcus aureus (MRSA). The in-vitro radiochemical purity (RCP) yield, in-vivo biodistribution behavior in artificially infected rats (AIT) and scintigraphic accuracy in artificially infected rabbit (AIB) of the ^99mTc–RMP complex was investigated using different concentration of the RMP, sodium pertechnetate (Na^99mTcO₄), stannous chloride dihydrate (SnCl₂·2H₂O) at different pH ranges 5–6. The best RCP yield observed at 30, 60, 90 and 120 min after labeling was; 98.95±0.20, 98.15±0.24, 96.50±0.27 and 91.55±0.22%, respectively, using 1.5 mg RMP, 175 μL of SnCl₂·2H₂O (1 μg/μL in 0.01 N HCl), 3 mCi of Na^99mTcO₄, at pH 5.6. Initially in the infected muscle (INM) of the AIT the activity was lower but after 90 min it went up to 18.35±0.20% from 5.95±0.25%. The activity in the inflamed muscle (IMM), normal (NM) muscle, blood, liver and spleen was initially high that decreased with time. The ratios of the INM/NM and IMM/NM were 7.34±0.74 and 1.20±0.85, respectively. The whole body static (WBS) imaging of the MRSA infected rabbit confirmed the usefulness of the ^99mTc-RMP as a precise radiotracer for MRSA infection imaging. On the basis of in-vitro RCP, in-vivo biodistribution and scintigraphic precision, we recommend the ^99mTc–RMP complex prepared aseptically for in-vivo assessment of MRSA infection.     

Validation of curve-fitting method for blood retention of99mTc-GSA: Comparison with blood sampling method

We investigated a curve-fitting method for the rate of blood retention of^99mTc-galactosyl serum albumin (GSA) as a substitute for the blood sampling method. Seven healthy volunteers and 27 patients with liver disease underwent^99mTc-GSA scanning. After normalization of they-intercept as 100 percent, a biexponential regression curve for the precordial time-activity curve provided the percent injected dose (%ID) of^99mTc-GSA in the blood without blood sampling. The discrepancy between %ID obtained by the curve-fitting method and that by the multiple blood samples was minimal in normal volunteers 3.1 ± 2.1% (mean ± standard deviation, n = 77 sampling). Slightly greater discrepancy was observed in patients with liver disease (7.5 ± 6.1%, n = 135 sampling). The %ID at 15 min after injection obtained from the fitted curve was significantly greater in patients with liver cirrhosis than in the controls (53.2 ± 11.6%, n = 13; vs. 31.9 ± 2.8%, n = 7, p < 0.0001). There was a highly linear correlation between the %IDs of^99mTc-GSA and the plasma retention rate for indocyanine green (r = ? 0.869, p < 0.0001, n = 27). These results indicate that the curve-fitting method provides an accurate %ID of^99mTc-GSA and could be a substitute for the blood sampling method.     

64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

IntroductionWe synthesized and evaluated ⁶⁴Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin α_Vβ₃.MethodsTetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with ⁶⁴Cu at 40 °C for 1 h and purified using a PD-10 column. ⁶⁴Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/⁶⁴Cu-DOTA-liposomes or ⁶⁴Cu-DOTA-liposomes.ResultsHAEC binding studies exhibited that tetrac/⁶⁴Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while ⁶⁴Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/⁶⁴Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of ⁶⁴Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/⁶⁴Cu-DOTA-liposomes and ⁶⁴Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively.ConclusionThese results demonstrate that tetrac/⁶⁴Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to ⁶⁴Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/⁶⁴Cu-DOTA-liposomes.     

Validation of Tc-labeled “4+1” fatty acids for myocardial metabolism and flow imaging: Part 1: myocardial extraction and biodistribution

Introduction¹³C, ¹⁸F and ¹²³I fatty acids (FA) are used for myocardial imaging. Recently, our group showed that [^99mTc]-labeled “4+1” FA are extracted into the rat and guinea pig myocardium. The present study evaluates determinants of myocardial uptake and whole body biodistribution of these FA derivatives.MethodsStudies were performed with isolated perfused hearts of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) with a FAT/CD36 deficiency, as well as with heart type FA binding protein knockout mice (H-FABP)^?/? and H-FABP^+/+. Eight 4+1-^99mTc-FA were applied for 3 min followed by 1-min washout. A mathematical model was used to analyze FA dynamics and binding to proteins. Whole-body distribution was studied in rats with and without Tween 80. In vitro fractionation studies with [^99mTc]-FA assessed red blood cell uptake as well as association with plasma lipoproteins very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL).ResultsMyocardial extraction was 19.0–33.0% of the infused dose in isolated WKY and 15.2–26.4% in SHR hearts. However, H-FABP^?/? showed a marked reduction of tracer extraction [2.8±0.6%ID (percent injected dose) vs. 17±2%ID P<.001]. Uptake in red blood cells (<1.2%ID) and incorporation into lipoproteins were negligible. Incubation of ^99mTc-FA with albumin reduced ventricular extraction (P<.001) into the range of established iodinated FA tracers. polyoxyethylene(20) sorbitan monooleate improved the heart-to-liver ratio in the biodistribution studies.ConclusionsMyocardial uptake of [^99mTc]-FA 4+1 derivatives is dependent on H-FABP. These substances may therefore provide a new tool to specifically assess regional myocardial changes of H-FABP.     

Evaluation of 18F-labeled BODIPY dye as potential PET agents for myocardial perfusion imaging

IntroductionDespite the great potential of positron emission tomography/computed tomography (PET/CT) in cardiovascular disease imaging, one of the major limitations is the availability of PET probes with desirable half-lives and reasonable cost. In this report, we hypothesized that lipophilic cationic BODIPY dye could be selectively accumulated in cardiac muscle, possibly for the development of novel PET myocardial perfusion imaging (MPI) probes.MethodsA ¹⁸F-labeled BODIPY dye ([¹⁸F]1) was synthesized efficiently through a fluoride exchange reaction catalyzed by the Lewis acid tin chloride (SnCl₄). The compound was first evaluated by a cellular uptake assay in vitro, followed by biodistribution and microPET imaging studies in vivo.Results[¹⁸F]1 was obtained in more than 90% labeling yield, with > 98% radiochemical purity. The HEK-293 cellular uptake assay showed that the preferential uptake of [¹⁸F]1 could be related to the cell membrane potential. The biodistribution data demonstrated high levels of [¹⁸F]1 accumulation in the heart. In the biodistribution study in mice, the radioactivity uptake in the heart, blood, liver and lung was 3.01 ± 0.44, 0.39 ± 0.09, 0.69 ± 0.07, 1.71 ± 0.27%ID/g, respectively, at 3 h post-injection (p.i.). The heart-to-lung and heart-to-liver ratios are 1.76 ± 0.14 and 4.37 ± 0.51 at 3 h p.i., respectively. Volume-of-interest analysis of the microPET images correlated well with the biodistribution studies in mice. The heart was clearly visualized in normal rats, with 0.72 ± 0.18, 0.69 ± 0.18, 0.67 ± 0.20 and 0.59 ± 0.17%ID/g uptake at 0.5, 1, 2 and 4 h p.i., respectively.Conclusions¹⁸F-labeled BODIPY dye showed good heart uptake and heart-to-blood and heart-to-lung contrast. A ¹⁸F-labeled BODIPY dyes may represent a new category of cationic PET agents for myocardial perfusion imaging.     

The preparation and biological characterization of a new HL91-derivative for hypoxic imaging on stroke mice

Aim^99mTc-HL91 (Prognox, GE-Healthcare) was the first nonnitro-aryl-based radiotracer for evaluating hypoxic fraction in neoplasm, stroke and myocardium infarction regions. However, the high hydrophilicity of ^99mTc-HL91 might hamper its penetration into cells. In this study, we prepared a new ligand 4,4,11,11-tetramethyl- 5,10-diazatetradecane- 3,12-dionedioxime (HL91-ET) with higher lipophilicity but structurally similar compared with that of HL91. The chemical and biological characterizations of ^99mTc-HL91-ET as a scintigraphic probe for hypoxia were performed with a stroke-bearing mouse model.Materials and MethodsHL91-ET was synthesized and formulated with stannous chloride and buffer to afford kits. After mixing with ^99mTc-pertechnetate, ^99mTc-HL91-ET can be prepared in high yield and high radiochemical purity (both >96%). The partition coefficient of ^99mTc-HL91-ET was determined in n-octanol/PBS system. Cellular uptake assays under normoxic and hypoxic conditions were performed in an oxygen-controlled CO₂ incubator. Brain stroke in the mouse model was induced by the electrocautery of the middle cerebral artery. After intravenous injection of ^99mTc-HL91-ET into the Balb/c mouse suffering brain stroke, small-animal SPECT images were acquired at designated time points and autoradiography of the brain slides was conducted. Parallel studies of ^99mTc-HL91 were also conducted at the same conditions for comparison.ResultsThe higher partition coefficient of ^99mTc-HL91-ET (0.294±0.007) indicated higher lipophlicity compared with that of ^99mTc-HL91 (0.089±0.005). The ^99mTc-HL91-ET preparation was stable at ambient temperature for 24 h. Cellular uptake assay showed that ^99mTc-HL91-ET was less selectively retained in hypoxic cells than ^99mTc-HL91. The target-to-normal brain ratios derived from the autoradiograms of the brains of stroke mice were 1.31±0.02 and 17.47±0.10 (n=3), respectively, at 2 h post injection of ^99mTc-HL91-ET and ^99mTc-HL91.ConclusionsThis study revealed that ^99mTc-HL91-ET, though with higher lipophilicity than ^99mTc-HL91, did not suggest better specific accumulation in hypoxic cells or tissues than ^99mTc-HL91. The uptake mechanism of ^99mTc-HL91 was at least not solely by passive diffusion. Lipophilicity should not be the major consideration in designing HL91-derivatives for hypoxia imaging.     

Labeling of ceftriaxone for infective inflammation imaging using 99mTc eluted from 99Mo/99mTc generator based on zirconium molybdate

Zirconium molybdate gel was prepared by mixing ⁹⁹Mo, produced from ⁹⁸Mo(n,γ) reaction and Zr solutions in nitrate media with excess H₂O₂, and used as the base material for ⁹⁹Mo/^99mTc generator. The prepared generator showed a good performance. ^99mTc eluted from the prepared generator passed the quality control tests with specifications meeting the requirements of European and US Pharmacopeias. The ^99mTc eluate was used for labeling of cephalosporin analogue, ceftriaxone, which was then assessed for infection imaging in a mouse model. ^99mTc-ceftriaxone was prepared at pH 9 with a radiochemical yield of 95±2% by adding ^99mTc to 30 mg ceftriaxone in the presence of 50 μg SnCl₂·2H₂O. Biodistribution studies in mice were carried out using experimentally induced infection in the left thigh using E. coli. Both thighs of the mice were dissected and counted to evaluate the ratio of bacterial infected thigh/contralateral thigh. ^99mTc-ceftriaxone showed high uptake in the infectious lesion (T/NT =5.6±0.6 at 4 h post injection). The abscess to normal muscle ratio indicated that ^99mTc-ceftriaxone could be used for infection imaging. Besides, in vitro studies showed that ^99mTc-ceftriaxone can differentiate between bacterial infection and sterile inflammation.     

Kit formulated asialoglycoprotein receptor targeting tracer based on copolymer for liver SPECT imaging

IntroductionSpecific targeting of galactose-carrying molecule to ASGP-R in normal hepatocytes has been demonstrated before. In this study, galactosyl polystyrene was synthesized from controllable ratio of functional monomers and radio-labelled with ^99mTc by formulated kit for SPECT imaging of hepatic function.Methodsp(VLA-co-VNI)(46:54) was synthesized by free-radical copolymerization initiated by AIBN, purified by dialysis, lyophilized to kit with Tricine and TPPTS as co-ligands for ^99mTc labeling. Radiotracer ^99mTc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) was prepared and evaluated by in vitro stability, in vivo metabolism, ex vivo biodistribution and microSPECT/CT imaging in normal KM mice. MicroSPECT/CT and microMRI imaging were also performed in C57BL/b6 mice with xenograft hepatic carcinoma for hepatic function evaluation.Results^99mTc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) was obtained in high radio chemical purity (RCP) (> 99%) by using instant kit without further purification and excellent in vitro and in vivo stability. The result of biodistribution showed that liver had high uptake (90.49 ± 10.68 ID%/g) at 30 min after injection and was blocked significantly by cold copolymer. MicroSPECT imaging in normal KM mice at 1 h and 4 h after injection showed good liver retention and targeting properties. Significant defect of activity was observed in the tumor site which was confirmed by MRI imaging.Conclusion^99mTc-p(VLA-co-VNI)(46:54)(Tricine)(TPPTS) with lower ratio of targeting moiety has no observable effect on the specific binding affinity and liver uptake. This makes it possible to introduce more imaging units for multi-modality imaging. Furthermore, the instant kit preparation of ^99mTc-labeling provides great potential for the evaluation of hepatocyte function in clinical application.     

In vivo imaging of tumour angiogenesis in mice with the α<Subscript>v</Subscript>β<Subscript>3</Subscript> integrin-targeted tracer <Superscript>99m</Superscript>Tc-RAFT-RGD

Purpose The molecular imaging of tumour neoangiogenesis currently represents a major field of research for the diagnostic and treatment strategy of solid tumours. Endothelial cells from tumour neovessels overexpress the α_vβ₃ integrin, which selectively binds to Arg-Gly-Asp (RGD)-containing peptides. We evaluated the potential of the novel radiotracer ^99mTc-RAFT-RGD for the non-invasive molecular imaging of α_vβ₃ integrin expression in mice models of tumour development. Methods ^99mTc-RAFT-RGD, ^99mTc-cRGD (specific control) and ^99mTc-RAFT-RAD (non-specific control) were injected intravenously to mice bearing B16F0 or TS/A-pc tumours. In vivo whole-body tomographic imaging and post-mortem biodistribution studies were performed 60 min following tracer injection. Adjacent tumour slices were used to compare the localisation of neovessels from immunostaining and the pattern of ^99mTc-RAFT-RGD uptake from autoradiographic ex vivo imaging. Results Biodistribution studies indicated that ^99mTc-RAFT-RGD tumour uptake was significantly higher than that of ^99mTc-RAFT-RAD in B16F0 (2.4±0.5 vs 1.0±0.1%ID/g, respectively) and in TS/A-pc tumours (2.7±0.8 vs 0.7±0.1%ID/g, respectively). Immunohistochemical and autoradiographic studies indicated that ^99mTc-RAFT-RGD intratumoural uptake preferentially occurred in angiogenic areas. Tomographic imaging allowed tumour visualisation following injection of ^99mTc-RAFT-RGD and ^99mTc-cRGD with similar tumour-to-contralateral muscle (T/CM) ratios in B16F0 and in TS/A-pc tumours whereas ^99mTc-RAFT-RAD T/CM ratios did not allow tumour imaging. In accordance with the higher level of α_vβ₃ integrin expression on TS/A-pc tumours than on B16F0 tumours as determined from western blot and immunoprecipitation analyses, the ^99mTc-RAFT-RGD T/CM ratio was significantly higher in TS/A-pc than in B16F0 tumours. Conclusion ^99mTc-RAFT-RGD allowed the in vivo imaging of α_vβ₃ integrin tumour expression.     

Design and biological evaluation of 99mTc-N2S2-Tat(49–57)-c(RGDyK): A hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins

The α(ν)β(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) (^99mTc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional ^99mTc-RGD peptide (^99mTc-EDDA/HYNIC-E-[c(RGDfK)]₂). Methods: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys¹² side chain of the Tat(49–57)-spacer-N₂S₂ peptide. ^99mTc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)β(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. Results: ^99mTc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for ^99mTc-Tat-RGD and 5.6% for ^99mTc-RGD. The IC₅₀ values were 6.7 nM (^99mTc-Tat-RGD) and 4.6 nM (^99mTc-RGD). Internalization in C6 cells was higher in ^99mTc-Tat-RGD (37.5%) than in ^99mTc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for ^99mTc-Tat-RGD (6.98% ± 1.34% ID/g at 3 h) than that of ^99mTc-RGD (3.72% ± 0.52% ID/g at 3 h) with specific recognition for α(v)β(3) integrins. Conclusions: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)β(3) integrins, the hybrid ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.     

Evaluation of renal function in low-dose cyclosporine-treated patients using technetium-99m diaminocyclohexane: a cationic tubular excretion agent

Technetium-99m diaminocyclohexane (DACH) is a new tubular agent excreted via a cationic transport mechanism, like cyclosporine-A (CsA). It is expected that ^99mTc-DACH will permit effective assessment of tubular function in CsA-treated patients. To establish the pharmacokinetic characteristics of ^99mTc-DACH and to ascertain whether this new agent is useful in CsA-treated patients, 11 healthy volunteers and 15 CsA-treated patients underwent renal imaging and clearance studies using ^99mTc-DACH and chromium-51 ethylene diamine tetra-acetic acid (EDTA). ^99mTc-DACH yielded satisfactory dynamic renal images in all participants. The mean plasma clearance of ^99mTc-DACH was significantly greater than that of ⁵¹Cr-EDTA in volunteers (109.4±19.7 ml/min versus 86.6±13.7 ml/min, P<0.05). However, the urinary excretion of ^99mTc-DACH at 90 min was significantly lower than that of ⁵¹Cr-EDTA (46.1%±9.3% versus 53.1%±8.6%, P<0.05), most probably due to its partial parenchymal retention. The elimination half-life of ^99mTc-DACH was significantly increased in CsA-treated patients in comparison to volunteers, and consequently the plasma clearance values were significantly suppressed in these patients, in contrast to ⁵¹Cr-EDTA and endogenous creatinine clearance values. In conclusion, our findings indicate that ^99mTc-DACH, as a sensitive marker of cationic tubular function, could be used to monitor renal haemodynamics in patients receiving CsA treatment. Received 19 June and in revised form 28 July 1998     

Radiotracer properties determined by high performance liquid chromatography: a potential tool for brain radiotracer discovery

IntroductionPreviously, development of novel brain radiotracers has largely relied on simple screening tools. Improved selection methods at the early stages of radiotracer discovery and an increased understanding of the relationships between in vitro physicochemical and in vivo radiotracer properties are needed. We investigated if high performance liquid chromatography (HPLC) methodologies could provide criteria for lead candidate selection by comparing HPLC measurements with radiotracer properties in humans.MethodsTen molecules, previously used as radiotracers in humans, were analysed to obtain the following measures: partition coefficient (Log P); permeability (P_m); percentage of plasma protein binding (%PPB); and membrane partition coefficient (K_m). Relationships between brain entry measurements (Log P, P_m and %PPB) and in vivo brain percentage injected dose (%ID); and K_m and specific binding in vivo (BP_ND) were investigated. Log P values obtained using in silico packages and flask methods were compared with Log P values obtained using HPLC.ResultsThe modelled associations with %ID were stronger for %PPB (r²=0.65) and P_m (r²=0.77) than for Log P (r²=0.47) while 86% of BP_ND variance was explained by K_m. Log P values were variable dependant on the methodology used.ConclusionsLog P should not be relied upon as a predictor of blood-brain barrier penetration during brain radiotracer discovery. HPLC measurements of permeability, %PPB and membrane interactions may be potentially useful in predicting in vivo performance and hence allow evaluation and ranking of compound libraries for the selection of lead radiotracer candidates at early stages of radiotracer discovery.     

The synthesis, magnetic purification and evaluation of 99mTc-labeled microbubbles

Introduction

Ultrasound (US) contrast agents based on microbubbles (MBs) are being investigated as platforms for drug and gene delivery. A methodology for determining the distribution and fate of modified MBs quantitatively in vivo can be achieved by tagging MBs directly with ^99mTc. This creates the opportunity to employ dual-modality imaging using both US and small animal SPECT along with quantitative ex vivo tissue counting to evaluate novel MB constructs.

Methods

A ^99mTc-labeled biotin derivative (^99mTcL1) was prepared and incubated with streptavidin-coated MBs. The ^99mTc-labeled bubbles were isolated using a streptavidin-coated magnetic-bead purification strategy that did not disrupt the MBs. A small animal scintigraphic/CT imaging study as well as a quantitative biodistribution study was completed using ^99mTcL1 and ^99mTc-labeled bubbles in healthy C57Bl-6 mice.

Results

The imaging and biodistribution data showed rapid accumulation and retention of ^99mTc-MBs in the liver (68.2±6.6 %ID/g at 4 min; 93.3±3.2 %ID/g at 60 min) and spleen (214.2±19.7 %ID/g at 4 min; 213.4±19.7 %ID/g at 60 min). In contrast, ^99mTcL1 accumulated in multiple organs including the small intestine (22.5±3.6 %ID/g at 4 min; 83.4±5.9 %ID/g at 60 min) and bladder (184.0±88.1 %ID/g at 4 min; 24.2±17.7 %ID/g at 60 min).

Conclusion

A convenient means to radiolabel and purify MBs was developed and the distribution of the labeled products determined. The result is a platform which can be used to assess the pharmacokinetics and fate of novel MB constructs both regionally using US and throughout the entire subject in a quantitative manner by employing small animal SPECT and tissue counting.     

Technetium-99m somatostatin analogues: effect of labelling methods and peptide sequence

In this paper the preclinical evaluation of the somatostatin analogue RC160 labelled with technetium-99m using bifunctional chelators (BFCs) based on the hydrazinonicotinamide (HYNIC) and N₃S system is described and a comparison made with [Tyr³]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG₃ and an N₃S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N’-diacetic adic (EDDA) as co-ligands for HYNIC conjugates. All conjugates and ^99mTc-labelled peptides showed preserved binding affinity for the somatostatin receptor (IC50, Kd<5 nM). The biodistribution was markedly dependent on the BFC and co-ligand used, with the amidothiol ligands showing a greater degree of hepatobiliary clearance, the HYNIC/tricine complex higher blood levels and the HYNIC/EDDA complex the highest level of renal excretion and lowest blood levels. All peptide conjugates showed receptor-mediated uptake in tumour xenografts, but tumour uptake was significantly lower for the ^99mTc-RC160 derivatives compared with ^99mTc-EDDA/HYNIC-[Tyr³]-octreotide (0.2%–3.5%ID/g vs 9.7%ID/g) and correlated well with the reduced internalisation rate for RC160 derivatives. Our results show that the selection of the labelling approach as well as the right choice of the peptide structure are crucial for labelling peptides with ^99mTc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with ^99mTc-EDDA/HYNIC-[Tyr³]-octreotide, ^99mTc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives. Received 26 January and in revised form 16 April 1999     

Comparative in vivo kinetics of some new 99mTc-labelled acetanilido iminodiacetates

The in vivo kinetics of five new ^99mTc-labelled acetanilido iminodiacetates, analogous to ^99mTc-p-butyl IDA, were studied in experimental animals by means of their distribution in mice and scintigrams of rabbits. The new compounds were specifically eliminated via the hepatobiliary system with various rates of hepatic extraction. Urinary excretion of the complexes was minimal.Abbreviations used in text IDA Iminodiacetic acid - HIDA 2,6 dimethyl acetanilido iminodiacetic acid - EHIDA 2,6 diethyl acetanilido iminodiacetic acid