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     

The use of technetium-99m labelled heat-damaged red cells for the quantitative measurement of splenic function

A comparison of the rates of clearance of ⁵¹Cr labelled and ^99mTc labelled heat-damaged red cells in 25 patients and 4 control subjects is reported. Very little correlation was found between the clearance half-times of the two types of labelled cells when the cells were labelled with ^99mTc prior to heat damaging. The correlation was improved when the labelling step occurred after the cells had been damaged. Urinary excretion measurements revealed that the rate of excretion of ^99mTc could be as much as nine times that of the ⁵¹Cr label. ^99mTc labelled heat-damaged red cells were found not to be sufficiently stable a preparation for use in quantitative clearance studies.     

Transplant renography: 99m-Tc-DTPA versus 99m-Tc-MAG3. a preliminary note

^99mTc-MAG3 has been proposed as a replacement for both ¹³¹I-hippuran and ^99mTc-DTPA on clinical grounds. We undertook a prospective preliminary study to ascertain whether ^99mTc-MAG3 works better than ^99mTc-DTPA in the follow up of renal transplant recipients. Seventeen patients (21 renograms each MAG3 and DTPA) were studied, together, with a reference group of 10 patients in whom MAG3 and Hippuran clearance rates were determined simultaneously. As expected, ^99mTc-MAG3 analog images were excellent and ^99mTcMAG3 clearance correlated very well with ¹³¹I-hippuran clearance (r=0.978). MAG3 values were 60% of hippuran values. However, when the corresponding renographic and perfusion findings were faced with different diagnostic challenges, such as post transplant renal failure and rejection, ^99mTc MAG3 did not differ from ^99mTc-DTPA in a significant way. ^99mTc-DTPA was superior to ^99mtc-MAG3 in one case of rejection.     

Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the <Superscript>99m</Superscript>Tc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence

Purpose Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Non-invasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule Z_HER2:342, which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2-expressing xenografts using radioiodinated Z_HER2:342. The goal of this study was to develop a method for ^99mTc labelling of Z_HER2:342 using the MAG3 chelator, which was incorporated into Z_HER2:342 using peptide synthesis, and evaluate the targeting properties of the labelled conjugate. Methods MAG3-Z_HER2:342 was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for ^99mTc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of ^99mTc-MAG3-Z_HER2:342 and ¹²⁵I-para-iodobenzoate -Z_HER2:342 was compared 6 h p.i. Results Synthetic MAG3-Z_HER2:342 possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with ^99mTc with an efficiency of about 75–80%. Labelled ^99mTc-MAG3-Z_HER2:342 retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. ^99mTc-MAG3-Z_HER2:342 showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression. Conclusion Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific ^99mTc labelling of the Z_HER2:342 Affibody molecule with preserved targeting capacity.     

Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide

IntroductionInterleukin-2 (IL-2) when radiolabelled with ^99mTc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of ^99mTc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.MethodsVarious molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The ^99mTc-labelling was optimized using various amounts of HYNIC–rhIL-2, ^99mTc, SnCl₂, tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.ResultsGenerally, the highest radiolabelling yields were achieved when the HYNIC–rhIL-2 conjugates of ca. 2–4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of ^99mTc-HYNIC–rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the ^99mTc-HYNIC–rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC–rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl₂. The monomer of ^99mTc-HYNIC–rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.ConclusionsOur study shows that rhIL-2 can be efficiently radiolabelled with ^99mTc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use ^99mTc-HYNIC(tricine,NA)-rhIL-2 within 1 h.     

Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms

IntroductionTumour-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, and it is recognised as having strong prognostic relevance as well as being a therapeutic target. The specific uPA inhibitor plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) specifically targets cell bound uPA and is internalised. Furthermore, preclinical studies have established the “proof-of-principle” of uPA-targeting by PAI-2-cytotoxin conjugates in human carcinoma models. However, these studies also suggest that PAI-2 is rapidly cleared via the renal system with low total dose reaching the tumour. In this study, a comparative single photon emission computed tomography (SPECT) and biodistribution (BD) analysis of different forms of PAI-2 labelled with the radioisotopes iodine-123 (¹²³I) and technetium-99m (^99mTc) was undertaken.MethodsThe pharmacokinetic (PK) properties and BD of wild-type, ΔCD-loop and PEGylated ΔCD-loop PAI-2 labelled with the commonly used diagnostic SPECT radioisotopes ^99mTc or ¹²³I were compared in mouse models of human prostate carcinoma. Whole body SPECT imaging was also performed.ResultsBoth wild-type and the shorter but active ΔCD-loop form of PAI-2 ¹²³I-labelled indirectly via conjugation to free amine groups (termed ¹²³I-Bn-PAI-2) exhibited low tumour uptake, rapid excretion and similar PK profiles. Preliminary studies with a short branched-chain PEGylated ¹²³I-Bn-PAI-2 ΔCD-loop indicated an increase in blood retention time and tumour uptake. All ¹²³I-Bn-labelled radiotracers were largely excreted through the kidneys. By comparison, both wild-type ¹²³I-PAI-2 (labelled directly via tyrosine residues) and ^99mTc-PAI-2 displayed different PK/BD patterns compared to ¹²³I-Bn-PAI-2, suggesting greater liver based catabolism and thus slower elimination. SPECT imaging mimicked the BD results of all radiotracers.ConclusionThe different labelling methods gave distinct PAI-2 BD and tumour uptake profiles, with radioiodination resulting in the best non-tumour organ clearance profiles. Preliminary analyses with short branched-chain PEGylated ¹²³I-Bn-PAI-2 ΔCD-loop suggest that further investigations with other PEGylation reagents are required to optimise this approach for tumour imaging. These findings impact on the use of PAI-2 for drug delivery and/or diagnostic development.     

Technetium-99m labelled hydrazinonicotinamido human non-specific polyclonal immunoglobulin G for detection of infectious foci: a comparison with two other technetium-labelled immunoglobulin preparations

Recently a new linker — hydrazinonicotinate (HYNIC) — was introduced for labelling of proteins and peptides with technetium-99m. HYNIC and other linkers have been used for labelling of human non-specific polyclonal immunoglobulin G (hIgG) with^99mTc for the detection of infections. In this study we compared the tissue distribution of three different^99mTc-hIgG preparations in groups of five Wistar rats with a focal intramuscular infection withStaphylococcus aureus. We compared^99mTc-HYNIC-hIgG with^99mTc-hIgG labelled via the so-called Schwarz method (reduction of disulphide bonds) and with the^99mTc-labelled commercially available Technescan-HIG. Unlike the HYNIC linker, in the two other labelling methods free sulph-hydryl groups are involved in the binding of^99mTc. High-performance liquid chromatography analysis of the labelled preparations and of plasma samples revealed aggregate or polymer formation in all three agents; this was least pronounced in the product labelled by means of the Schwarz method. The tested preparations did not show signs of degradation in vitro. The difference in linker chemistry was reflected in the tissue distribution. Thus the biodistribution of^99mTc-HYNIC-hIgG was significantly different from the distribution of the two other preparations: abscess (1.4%±0.2%ID/g), muscle, liver, spleen, plasma, lung, bone marrow, and small intestine concentrations were higher at 24 h p.i.; kidney uptake (1.19%±0.08%ID/g) was significantly lower. The abscess-to-plasma and the abscess-to-muscle ratios (0.5 and 11, respectively), however, were in the same range for the three preparations tested. Quantitative analysis of the scintigraphs revealed that the total body clearance of^99mTc-HYNIC-hIgG was significantly slower than for the other agents. The abscess uptake of^99mTc-HYNIC-hIgG as a percentage of the remaining body activity was significantly higher. Based on its high abscess uptake, its low uptake in the kidneys and the high percentage of its abscess uptake in relation to the remaining body activity, we conclude that^99mTc-HYNIC-hIgG seems superior to the two other preparations tested for the detection of infections.     

Pre-targeted immunodetection in glioma patients: tumour localization and single-photon emission tomography imaging of [99mTc]PnAO-biotin

The imaging of cerebral gliomas with radiolabelled monoclonal antibodies (MoAbs) has been previously reported. However, previous studies have been hampered by the drawback of a low tumour to non-tumour ratio. In order to overcome this problem we have developed a three-step pre-targeting method using the avidin-biotin system. The rationale of this technique consists in vivo labelling of biotinylated MoAbs targeted onto tumour deposits, when most of the unbound antibodies have been cleared from the bloodstream as avidin-bound complexes. The anti-tenascin MoAb BC2, specific for the majority of gliomas, was biotinylated and 1 mg was administered i.v. in 20 patients with histologically documented cerebral lesions. After 24–36 h, 5 mg avidin was injected i.v. followed 24 h later by a third i.v. injection of 0.2 mg PnAO-biotin labelled with 15–20 tnCi technetium-99m. No evidence of toxicity was observed. Whole-body biodistribution was measured at 20 min, 3 h and 5 h post-injection. [^99mTc]PnAO-bio-tin had a fast blood clearance and was primarily excreted through the biliary system. A dedicated single-photon emission tomography system was used to acquire brain tomographic images 1–2 h after the administration of [^99mTc]PnAO-biotin. Tumours were detected in 15/18 glioma patients with a tumour to non-tumour ratio of up 14:1. This three-step method, based on the sequential administration of anti-tenascin MoAb BC2, avidin and [^99mTc]PnAO-biotin, can support computed tomography or magnetic resonance imaging for the diagnosis and follow-up of patients with glioma. Further studies are required to evaluate the potential of this technique for therapeutic application.     

Isolation and labelling of human leukocytes with 99mTc

The role of the leukocyte isolation procedure on cell labelling with ^99mTc has been evaluated. Separation of leukocytes was performed by two procedures: (1) sedimentation on methyl cellulose, followed by discontinuous gradient centrifugation; (2) methyl cellulose sedimentation and hypotonic haemolysis of residual red blood cells. After washing the cells in saline and incubation with a stannous pyrophosphate agent, the leukocytes were labelled with 5–10 mCi ^99mTc. Procedure 1 gave a higher purity but lower recovery of polymorphonuclear leukocytes, and a minor contamination of red blood cells. ^99mTc labelling of cells was slightly more efficient with this method, probably due to the presence of red blood cells. Procedure 1 is recommended for in vitro studies on cell kinetics and procedure 2 is recommended for clinical use.     

99mTc/188Re-labelled lipid nanocapsules as promising radiotracers for imaging and therapy: formulation and biodistribution

Purpose This study focusses on a promising carrier system for imaging and therapeutic purposes using lipid nanocapsules. To assess their potential for clinical use, we labelled nanocapsules with ^99mTc and ¹⁸⁸Re and analysed some kinetic biodistribution parameters after intravenous injection in rats. Methods Lipophilic complexes [^99mTc/¹⁸⁸Re(S₃CPh)₂(S₂CPh)] (^99mTc/¹⁸⁸Re-SSS) were encapsulated within the nanoparticles during their manufacture with quantitative yield and satisfactory radiochemical purity. Rats were injected intravenously with 3.7 MBq ^99mTc/¹⁸⁸Re-labelled nanocapsules and sacrificed at 5, 15 and 30 min and 1, 2, 4, 8, 12, 16, 20 and 24 h. Results Dynamic scintigraphic acquisitions showed predominant hepatic uptake, and ex vivo counting indicated a long circulation time of labelled nanocapsules, with a half-life of 21±1 min for ^99mTc and 22±2 min for ¹⁸⁸Re. Very weak urinary elimination was observed, indicating good stability of ^99mTc and ¹⁸⁸Re labelling. Conclusion ^99mTc/¹⁸⁸Re-SSS nanocapsules can be obtained with high yield and satisfactory radiochemical purity. The biodistributions of ^99mTc/¹⁸⁸Re-labelled nanocapsules are close to those of classical PEG-coated particles and show good stability of ¹⁸⁸Re/^99mTc-SSS labelling.     

Technetium-99m labelling of the anti-tumour antibody PR1A3 by photoactivation

Irradiation of antibody with ultraviolet light leads to reduction of disulphide bonds. Thus irradiation can be used to generate free thiols prior to direct labelling of antibody with technetium-99m, and has a potential advantage over methods using chemical reducing agents such as mercaptoethanol or tin, in that no purification step is needed to remove excess reducing agent. We have used the photoactivation method developed by Sykes et al. to label the anti-tumour antibody PR1A3 with^99mTc. The antibody was irradiated at 300 nm using a Rayonet photochemical reactor with eight RMR3000 lamps. In a typical experiment, the antibody solution was injected into a nitrogen-filled borosilicate glass vial and purged with nitrogen. A degassed solution containing stannous fluoride and methylene diphosphonate was then added to the antibody and the vial was irradiated. Following the irradiation, [^99mTc]pertechnetate was injected into the vial and the reaction mixture was incubated for 30 min at room temperature before being analysed by size-exclusion high-pressure liquid chromatography and instant thin-layer chromatography. Labelling yields greater than 95% were obtained using antibody concentrations ranging from 0.5 mg/ml to 5 mg/ml. Irradiation times as short as 5 min and tin to antibody ratios in the range between 11 and 32 µg tin per mg antibody gave high labelling yields. Labelling yields greater than 95% were obtained after storage of the photoactivated antibody at –70° C for several weeks. The stability of the^99mTc-labelled photoactivated PR1A3 was similar to that of^99mTc-labelled mercaptoethanol-reduced PR1A3. The mean immunoreactive fraction was 77% for the photoactivation-labelled PR1A3, compared to 93% for PR1A3 labelled by mercaptoethanol reduction. Biodistribution studies were carried out using^99mTc-photoactivation-labelled PR1A3 or PR1A3 labelled by mercaptoethanol reduction in Balb/c mice and in nude mice with MKN-45 human tumour xenografts. There was no significant difference in tumour uptake between the mice that received photoactivated PR1A3 and those that received mercaptoethanol-reduced PR1A3. There was also no significant difference in uptake in most organs in Balb/c mice; however, the photoactivated antibody cleared more rapidly from the blood, and whole-body clearance was also faster for the photoactivated PR1A3. In conclusion, the photoactivation technique provides a very convenient one-pot method for labelling antibodies with^99mTc.     

Synthesis,radiochemistry and biological evaluation of a new somatostatin analogue (SDZ 219–387) labelled with technetium-99m

A new derivative of octreotide SDZ 219-387 [PnAO-(D)Phe¹-octreotide] was synthesized, which binds specifically and with high affinity to somatostatin receptors in vitro (pK= 9.79±0.16). This new somatostatin analogue chelates technetium-99m under mild labelling conditions in good yields. The resulting [^99mTc]SDZ 219–387 was stable up to 6 h after labelling and could be isolated in a pure radiochemical and chemical form by high-performance liquid chromatographic purification. The intravenous administration of purified [^99mTc]SDZ 219–387 revealed that the radioligand was rapidly cleared from circulation, and tumour uptake of 0.38% ID/g was observed at 1.5 h post injection. [^99mTc]SDZ 219–387 specifically interacted with somatostatin binding sites on the tumour. However, the radioligand is highly lipophilic and excreted mainly through the hepatobiliary system. As a consequence, [^99mTc]SDZ 219–387 exhibits increased background activity and therefore is not appropriate for the in vivo visualization of somatostatin receptor-positive tumours and/or their metastases in the abdomen. Correspondence to: T. Maina     

99mTc-rituximab radiolabelled by photo-activation: a new non-Hodgkin’s lymphoma imaging agent

Purpose Rituximab was the first chimeric monoclonal antibody to be approved for treatment of indolent B-cell non-Hodgkins lymphoma (NHL). It is directed against the CD20 antigen, which is expressed by 95% of B-cell NHLs. The aim of this study was to explore the possibility of radiolabelling rituximab with ^99mTc for use as an imaging agent in NHL for early detection, staging, remission assessment, monitoring for metastatic spread and tumour recurrence, and assessment of CD20 expression prior to (radio)immunotherapy.Methods Rituximab was purified from Mabthera solution (Roche), photo-activated at 302 nm by UV irradiation and radiolabelled with ^99mTc. The effectiveness of the labelling method was evaluated by determination of the number of free thiol groups per photoreduced antibody, radiochemical purity and in vitro stability of ^99mTc-rituximab.Results On average, 4.4 free thiol groups per photoreduced antibody were determined. Radiolabelling yields greater than 95% were routinely observed after storage of the photo-activated antibody at –80°C for 195 days. The direct binding assay showed preserved ability of ^99mTc-rituximab to bind to CD20, with an average immunoreactive fraction of 93.3%. The internalisation rate was proven to be low, with only 5.3% of bound ^99mTc-rituximab being internalised over 4 h at 37°C.Conclusion Our results demonstrate that ^99mTc-rituximab of high radiochemical purity and with preserved binding affinity for the antigen can be prepared by photoreduction and that the method shows good reproducibility. ^99mTc-rituximab will be further explored as an imaging agent applicable in NHL for the purposes mentioned above.     

Synthesis of 99mTc-HYNIC-interleukin-12, a new specific radiopharmaceutical for imaging T lymphocytes

Purpose Few radiopharmaceuticals have been described for the study of lymphocyte trafficking despite its high clinical relevance. The main difficulty resides in the identification of a suitable highly specific probe to target these cells. Interleukin-12 (IL12) is a heterodimeric cytokine which plays a key role in the development of Th₁ lymphocytes. The aims of the present study were to label IL12 with ^99mTc, to evaluate its ability to bind to activated T lymphocytes in vitro and to study its biodistribution in normal mice and mice affected by autoimmune colitis. Methods IL12 was derivatised with HYNIC-NHS and labelled with ^99mTc. An in vitro binding assay was performed on KIT225 cells, an IL12 receptor-positive cell line. ^99mTc-IL12 biodistribution in normal mice was studied. Targeting experiments were performed in Balb/c mice injected with KIT225 cells and in mice with chemically induced chronic colitis. Results ^99mTc-IL12 labelling efficiency ranged between 75% and 85%. Saturation binding analysis revealed a K _d of 2.09 nM. Results of biodistribution studies showed a predominant hepatic route of excretion. A significant degree of uptake in the spleen and thymus was also observed. In mice injected with KIT225 cells, ^99mTc-IL12-specific uptake in these cells increased over time. ^99mTc-IL12 also accumulated significantly in bowel of mice affected by TNBS-induced colitis showing T lymphocyte infiltration at histology, while accumulation in colon from control animals was negligible. Conclusion We conclude that this radiolabelled cytokine is a suitable candidate for specific in vivo imaging of T lymphocytes: a step forward in molecular imaging of immune-mediated processes.     

Studies on technetium-99m-labelled monophosphonates: 1,2-Epoxypropylphosphonic acid and its hydrolysed form

1,2-Epoxypropylphosphonic acid (fosfomycin) when labelled with ^99mTc at pH = 6.8 was described as a renal imaging agent. Under the same experimental conditions, except pH = 2.5, ^99mTc fosfomycin has shown bone uptake. The hydrolysed form of fosfomycin, the 1,2-dihydroxypropyl-1-phosphonic acid when labelled with ^99mTc in identical conditions, also has shown similar biological behaviour. Extraction and adsorption experiments conducted on both radiopharmaceuticals have shown that renal uptake preparations are Tc(V) complexes, meanwhile bone uptake preparations are Tc(IV) and/or Tc(III) complexes.     

Development and preclinical characterisation of <Superscript>99m</Superscript>Tc-labelled Affibody molecules with reduced renal uptake

Purpose  Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, ^99mTc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. Materials and methods  Anti-HER2 Z_HER2:342 Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with ^99mTc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, ^99mTc-maEEE-Z_HER2:342, in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, ^99mTc-maESE-Z_HER2:342, was compared with radioiodinated Z_HER2:342. Results  All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 ± 5, 68 ± 21 and 71 ± 10%IA/g, for^99mTc-maESE-Z_HER2:342, ^99mTc-maEES-Z_HER2:342 and ^99mTc-maSEE-Z_HER2:342, respectively, were significantly reduced in comparison with ^99mTc-maEEE-Z_HER2:342 (102 ± 13%IA/g). For ^99mTc-maESE-Z_HER2:342, a tumour uptake of 9.6 ± 1.8%IA/g and a tumour-to-blood ratio of 58 ± 6 were reached at 4 h p.i. Conclusions  A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of ^99mTc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.     

Technetium-99m labelled LDL as a tracer for quantitative LDL scintigraphy

The goal of the present study was to optimize technetium-99m labelling of low-density lipoprotein (LDL) and to investigate the in vitro and in vivo properties of the tracer to determine whether its application for quantitative scintigraphy of hepatic LDL receptor activity is feasible. LDL labelled with iodine-125 by the iodine monochloride method was used as a reference tracer. Comparison of different assessments of radiochemical purity [trichloro-acetic acid precipitation (%ppTCA), paper chromatography, size-exclusion chromatography and chloroform-methanol extraction] exhibited %ppTCA to be superior as a parameter of tracer quality. In spite of a high radiochemical purity immediately after labelling, modifications of ^99mTc labelling of LDL did not overcome the poor long-term stability of the tracer. Subsequent dialysis in phosphate buffer over about 3 h sufficiently increased the long-term stability in vitro and in vivo. The competitive recognition of dialysed ^99mTc-LDL and ¹²⁵I-LDL with native LDL by high-affinity binding sites was demonstrated in human hepatoma cells (HepG2) and human fibroblasts. Biodistribution data of simultaneously injected ^99mTc-LDL and ¹²⁵I-LDL in New Zealand White rabbits showed a high uptake of both tracers in tissues with high LDL receptor activity, yet ^99mTc-LDL uptake exceeded ¹²⁵I-LDL uptake by two- to sevenfold. In contrast to ¹²⁵I-LDL, ^99mTc-LDL showed a higher unspecific uptake into the bone marrow and the spleen, suggesting an additional uptake mechanism probably via the scavenger pathway. Curve deconvolution of plasma clearance in five female New Zealand White rabbits and five male hyperlipidaemic patients again showed a marginally different biokinetic behaviour of ^99mTc-LDL and ¹²⁵I-LDL. It is concluded that dialysis of ^99mTc-LDL substantially increases long-term stability, which is essential for quantification purposes, and that the dialysed tracer has retained its biological integrity as it is recognized by the LDL receptor in vitro. It remains to be determined to what extent the estimation of hepatic LDL receptor activity by quantitative ^99mTc-LDL scintigraphy is influenced by the different biokinetic behaviour of ^99mTc-LDL and ¹²⁵I-LDL.     

Evaluation of white cell scintigraphy using indium-111 and technetium-99m labelled leucocytes

Indium-111 oxine labelled leucocyte (¹¹¹In oxine leucocyte) scintigraphy is the test of choice in detecting occult infection and localising focal inflammation. ¹¹¹In oxine labelling is technically difficult and expensive and leucocyte labelling with technetium-99m stannous colloid (^99mTc Sn colloid) has been considered to be an alternative. Leucocytes from 40 cases referred for investigation of occult infection or localisation of inflammation were simultaneously labelled with ¹¹¹In oxine and ^99mTc Sn colloid with dual isotope acquisition performed at 1, 3 and 24 h. Twenty-four hour ^99mTc Sn colloid scans were corrected for ¹¹¹In downscatter. Each case was independently interpreted by two experienced observers. Twentyone patients demonstrated positive ¹¹¹In oxine leucocyte scans. Using ¹¹¹In oxine leucocyte scans as the gold standard, ^99mTc Sn colloid leucocyte scanning had an overall sensitivity of 86% and a specificity of 95%. Clinical follow-up verified that three patients had false negative ^99mTc Sn colloid leucocyte scans and one patient had a false positive. Further clinical evaluation of ^99mTc Sn colloid labelled leucocytes is required before they can become a reliable replacement for ¹¹¹In oxine leucocytes. Correspondence to: S. Boyd     

Studies of the myocardial uptake and excretion mechanisms of a novel 99mTc heart perfusion agent

Introduction^99mTc-TMEOP is a novel heart perfusion radiotracer exhibiting high initial and persistent heart uptake associated with rapid blood and liver clearance. This study aimed at determining the mechanisms of myocardial localization and fast liver clearance of ^99mTc-TMEOP.MethodsSubcellular distribution of ^99mTc-TMEOP was determined in excised rat heart tissue by differential centrifugation. The effect of cyclosporin A on the pharmacokinetic behaviour of ^99mTc-TMEOP was evaluated by both ex vivo biodistribution and in vivo planar imaging studies.ResultsSubcellular distribution studies showed that more than 73% of ^99mTc-TMEOP was associated with the mitochondrial fraction. Comparison with subcellular distribution of ^99mTc-sestamibi showed no significant difference in the mitochondrial accumulation between the two tracers. Biodistribution studies in the presence of cyclosporin A revealed an increase in kidneys and liver uptake of ^99mTc-TMEOP, suggesting the involvement of multidrug resistance transporters in determining its pharmacokinetic profile.ConclusionsThe heart uptake mechanism of ^99mTc-TMEOP is similar to that of the other reported monocationic ^99mTc cardiac agents and is associated with its accumulation in the mitochondria. Cyclosporin A studies indicate that the fast liver and kidney clearance kinetics is mediated by P-glycoprotein (Pgp), supporting the potential interest of this radiotracer for imaging Pgp function associated with multidrug-resistant tumours.     

Technetium-99m mercaptoalbumin as a potential substitute for technetium-99m labelled red blood cells

Technetium-99m labelled red blood cells (^99mTc-RBCs) are far superior to ^99mTc-labelled human serum albumin (^99mTc-HSA) for radionuclide ventriculography, but their labelling is more complex, time consuming and risk bearing (in vitro labelling) or suffers from interference by some medications (in vivo labelling). We have now modified HSA by the introduction of mercapto groups with the purpose of preparing stable and practical ^99mTc-mercaptoalbumin with long retention in the vascular system, that could replace ^99mTc-RBCs. HSA was incubated with N-succinimidyl S-acetylthioacetate (SATA) or N-succinimidyl 2,3-di(S-acetylthio) propionate (SATP) to introduce a chain containing one or two protected sulfhydryl groups on some of the lysine amino groups. After purification by size-exclusion chromatography (SEC), the mercapto groups were deprotected by incubation at alkaline pH or by treatment with hydroxylamine. The reaction products were used with or without SEC purification for direct or exchange labelling experiments with ^99mTc at neutral pH. SEC-HPLC was used to determine labelling yields and to isolate pure ^99mTc-mercaptoalbumin. Stable ^99mTc-mercaptoalbumin complexes could be formed in 90%–95% yield after coupling albumin with SATA or SATP in all molar ratios used followed by deacetylation in one of the mentioned conditions. The most favourable results were obtained after reaction of SATA or SATP with HSA in a 25: 1 ratio and deprotection with NH₂OH. The stability of the resulting ^99mTc-mercaptoacetyl-albumin (^99mTc-MAHSA) and ^99mTc-dimercaptopropionyl-albumin (^99mTcDMP-HSA) and their retention in vivo in plasma of mice and rabbits are clearly higher than that of conventional ^99mTc-HSA preparations. ^99mTc-DMP-HSA approaches the behaviour of ¹²⁵I-HSA quite well in both animal species. A preliminary study with ^99mTc-DMP-HSA in a volunteer showed a retention in the vascular compartment almost identical to that of ^99mTc-RBCs and clearly higher than that of a common ^99mTc-HSA preparation. The results indicate that these ^99mTc-mercaptoalbumins and especially ^99mTc-DMP-HSA are very promising as a practical alternative to ^99mTc-RBCs.K.A. Verbeke is a Research Assistant for the Belgian National Fund for Scientific Research