Positioning of 99mTc-chelators influences radiolabeling,stability and biodistribution of Affibody molecules

Affibody molecules represent a novel class of affinity proteins with a high potential as tracers for radionuclide molecular imaging. In this comparative structure–property study, a series of Affibody molecules with the ^99mTc-chelators maGGG, maSSS, or maESE attached to the ε-amine of the internally positioned K49 was prepared by peptide synthesis, for comparison to molecules with similar chelators positioned at the N-terminus. The conjugates were labeled with ^99mTc and evaluated in vitro and in vivo. It was found that both composition and position of the chelating moiety influence the label stability, biodistribution and targeting properties of HER2-binding Affibody molecules.     

99mTc/Re complexes based on flavone and aurone as SPECT probes for imaging cerebral β-amyloid plaques

Two ^99mTc/Re complexes based on flavone and aurone were tested as potential probes for imaging β-amyloid plaques using single photon emission computed tomography. Both ^99mTc-labeled derivatives showed higher affinity for Aβ(1–42) aggregates than did ^99mTc-BAT. In sections of brain tissue from an animal model of AD, the Re-flavone derivative 9 and Re-aurone derivative 19 intensely stained β-amyloid plaques. In biodistribution experiments using normal mice, ^99mTc-labeled flavone and aurone displayed similar radioactivity pharmacokinetics. With additional modifications to improve their brain uptake, ^99mTc complexes based on the flavone or aurone scaffold may serve as probes for imaging cerebral β-amyloid plaques.     

(99m)Tc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of (99m)Tc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z(HER2:342) with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of (99m)Tc-maGEG-Z(HER2:342) and (99m)Tc-maEEE-Z(HER2:342) was compared with (99m)Tc-maGGG-Z(HER2:342) in normal mice, and the tumor targeting properties of (99m)Tc-maEEE-Z(HER2:342) were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for (99m)Tc-labeling of Z(HER2:342) reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. (99m)Tc-maEEE-Z(HER2:342) showed a receptor-specific tumor uptake of 7.9 +/- 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with (99m)Tc-maEEE-Z(HER2:342) could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for (99m)Tc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making (99m)Tc-maEEE-Z(HER2:342) a promising tracer for clinical imaging of HER2 overexpression in tumors.     

Influence of Macrocyclic Chelators on the Targeting Properties of 68Ga-Labeled Synthetic Affibody Molecules: Comparison with 111In-Labeled Counterparts

Affibody molecules are a class of small (7 kDa) non-immunoglobulin scaffold-based affinity proteins, which have demonstrated substantial potential as probes for radionuclide molecular imaging. The use of positron emission tomography (PET) would further increase the resolution and quantification accuracy of Affibody-based imaging. The rapid in vivo kinetics of Affibody molecules permit the use of the generator-produced radionuclide ⁶⁸Ga (T_1/2 = 67.6 min). Earlier studies have demonstrated that the chemical nature of chelators has a substantial influence on the biodistribution properties of Affibody molecules. To determine an optimal labeling approach, the macrocyclic chelators 1,4,7,10-tetraazacylododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and 1-(1,3-carboxypropyl)-1,4,7- triazacyclononane-4,7-diacetic acid (NODAGA) were conjugated to the N-terminus of the synthetic Affibody molecule Z_HER2:S1 targeting HER2. Affibody molecules were labeled with ⁶⁸Ga, and their binding specificity and cellular processing were evaluated. The biodistribution of ⁶⁸Ga-DOTA-Z_HER2:S1, ⁶⁸Ga-NOTA-Z_HER2:S1 and ⁶⁸Ga-NODAGA-Z_HER2:S1, as well as that of their ¹¹¹In-labeled counterparts, was evaluated in BALB/C nu/nu mice bearing HER2-expressing SKOV3 xenografts. The tumor uptake for ⁶⁸Ga-DOTA-Z_HER2:S1 (17.9±0.7%IA/g) was significantly higher than for both ⁶⁸Ga-NODAGA-Z_HER2:S1 (16.13±0.67%IA/g) and ⁶⁸Ga-NOTA-Z_HER2:S1 (13±3%IA/g) at 2 h after injection. ⁶⁸Ga-NODAGA-Z_HER2:S1 had the highest tumor-to-blood ratio (60±10) in comparison with both ⁶⁸Ga-DOTA-Z_HER2:S1 (28±4) and ⁶⁸Ga-NOTA-Z_HER2:S1 (42±11). The tumor-to-liver ratio was also higher for ⁶⁸Ga-NODAGA-Z_HER2:S1 (7±2) than the DOTA and NOTA conjugates (5.5±0.6 vs.3.3±0.6). The influence of chelator on the biodistribution and targeting properties was less pronounced for ⁶⁸Ga than for ¹¹¹In. The results of this study demonstrate that macrocyclic chelators conjugated to the N-terminus have a substantial influence on the biodistribution of HER2-targeting Affibody molecules labeled with ⁶⁸Ga.This can be utilized to enhance the imaging contrast of PET imaging using Affibody molecules and improve the sensitivity of molecular imaging. The study demonstrated an appreciable difference of chelator influence for ⁶⁸Ga and ¹¹¹In.     

In vivo evaluation of cysteine-based chelators for attachment of 99mTc to tumor-targeting Affibody molecules

Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (ZHER2:342) with a dissociation constant (Kd) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled ZHER2:342 in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with 99mTc. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-ZHER2:342 and CGGG-ZHER2:342, respectively. Conjugates were directly labeled with 99mTc with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of 99mTc-CGG-ZHER2:342 was HER2-specific and a tumor-to-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with 99mTc-CGG-ZHER2:342 clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables 99mTc-labeling of Affibody molecules for imaging.     

Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.     

A new bisphosphonate-containing <Superscript>99m</Superscript>Tc(I) tricarbonyl complex potentially useful as bone-seeking agent: synthesis and biological evaluation

Aiming to develop new bone-seeking radiotracers based on the organometallic core fac-[^99mTc(CO)₃]⁺ with improved radiochemical and biological properties, we have prepared new conjugates with phosphonate pendant groups. The conjugates comprise a chelating unit for metal coordination, which corresponds to a pyrazolyl-containing backbone (pz) with a N,N,N donor-atom set, and a pendant diethyl phosphonate (pz-MPOEt), phosphonic acid (pz-MPOH) or a bisphosphonic acid (pz-BPOH) group for bone targeting. Reactions of the conjugates with the precursor [^99mTc(H₂O)₃(CO)₃]⁺ yielded (mote than 95%) the single and well-defined radioactive species [^99mTc(CO)₃(κ³-pz-MPOEt)]⁺ (1a), [^99mTc(CO)₃(κ³-pz-MPOH]⁺ (2a) and [^99mTc(CO)₃(κ³-pz-BPOH)]⁺ (3a), which were characterized by reversed-phase high-performance liquid chromatography . The corresponding Re surrogates (1–3), characterized by the usual analytical techniques, including X-ray diffraction analysis in the case of 1, allowed for macroscopic identification of the radioactive conjugates. These radioactive complexes revealed high stability both in vitro (phosphate-buffered saline solution and human plasma) and in vivo, without any measurable decomposition. Biodistribution studies of the complexes in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal–urinary pathway in the case of complex 3a. Despite presenting moderate bone uptake (3.04 ± 0.47% injected dose per gram of organ, 4 h after injection), the high stability presented by 3a and its adequate in vivo pharmacokinetics encourages the search for new ligands with the same chelating unit and different bisphosphonic acid pendant arms.     

HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [⁹⁹(m)Tc(CO)₃](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [??(m)Tc(CO)?](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)?(:)??? were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [??(m)Tc(CO)?](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)?(:)???-H? and (HE)?-Z(HER)?(:)???, respectively, could be efficiently purified using IMAC and stably labeled with [??(m)Tc(CO)?](+) and were subsequently compared with the parental H?-Z(HER)?(:)??? having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [??(m)Tc(CO)?](+)-Z(HER2:342)-H? compared to [??(m)Tc(CO)?](+)-H?-Z(HER)?(:)???, and more than 10-fold lower with [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)???. These differences translated into appreciably superior tumor-to-liver ratio for [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)??? compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.     

Evaluation of a maleimido derivative of CHX-A' DTPA for site-specific labeling of affibody molecules

Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule Z HER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A' DTPA was site-specifically conjugated at the C-terminal cysteine of Z HER2:2395-C, a variant of Z HER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with (111)In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of (111)In-CHX-A' DTPA-Z 2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of (111)In-CHX-A' DTPA-Z 2395-C was 17.3 +/- 4.8% IA/g and the tumor-to-blood ratio 86 +/- 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A' DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.     

Influence of DOTA Chelator Position on Biodistribution and Targeting Properties of (111)In-Labeled Synthetic Anti-HER2 Affibody Molecules

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.     

The synthesis of pteroyl-lys conjugates and its application as Technetium-99m labeled radiotracer for folate receptor-positive tumor targeting

Aiming to develop a new ^99mTc-labeled folate derivative for FR-positive tumor imaging, a simpler method has been established to synthesize the folate-drug conjugates with free α-carboxyl group. In this study, the conjugate pteroyl-lys-HYNIC was synthesized and labeled with ^99mTc using tricine and TPPTS as co-ligands. The radiochemical purity of the final complex ^99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS), 5 was high (>98%), and it remained stable in saline and plasma over 6 h after preparation. The biologic evaluation results showed that the ^99mTc labeled pteroyl-lys conjugate was able to specifically target the FR-positive tumor cells and tissues both in vitro and in vivo, highlighting its potential as an effective folate receptor targeted agent for tumor imaging.     

Design of an Optimized Scaffold for Affibody Molecules

Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z_HER2:342. Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to + 12 °C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z_HER2:2891-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K_D, of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 °C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor α, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor β) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.     

Radiolabeled affibody-albumin bioconjugates for HER2-positive cancer targeting

Affibody molecules have received significant attention in the fields of molecular imaging and drug development. However, Affibody scaffolds display an extremely high renal uptake, especially when modified with chelators and then labeled with radiometals. This unfavorable property may impact their use as radiotherapeutic agents in general and as imaging probes for the detection of tumors adjacent to kidneys in particular. Herein, we present a simple and generalizable strategy for reducing the renal uptake of Affibody molecules while maintaining their tumor uptake. Human serum albumin (HSA) was consecutively modified by 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS ester) and the bifunctional cross-linker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC). The HER2 Affibody analogue, Ac-Cys-Z(HER2:342), was covalently conjugated with HSA, and the resulting bioconjugate DOTA-HSA-Z(HER2:342) was further radiolabeled with ??Cu and 111In and evaluated in vitro and in vivo. Radiolabeled DOTA-HSA-Z(HER2:342) conjugates displayed a significant and specific cell uptake into SKOV3 cell cultures. Positron emission tomography (PET) investigations using ??Cu-DOTA-HSA-Z(HER2:342) were performed in SKOV3 tumor-bearing nude mice. High tumor uptake values (>14% ID/g at 24 and 48 h) and high liver accumulations but low kidney accumulations were observed. Biodistribution studies and single-photon emission computed tomography (SPECT) investigations using 111In-DOTA-HSA-Z(HER2:342) validated these results. At 24 h post injection, the biodistribution data revealed high tumor (16.26% ID/g) and liver (14.11% ID/g) uptake but relatively low kidney uptake (6.06% ID/g). Blocking studies with coinjected, nonlabeled Ac-Cys-Z(HER2:342) confirmed the in vivo specificity of HER2. Radiolabeled DOTA-HSA-Z(HER2:342) Affibody conjugates are promising SPECT and PET-type probes for the imaging of HER2 positive cancer. More importantly, DOTA-HSA-Z(HER2:342) is suitable for labeling with therapeutic radionuclides (e.g., ??Y or 1??Lu) for treatment studies. The approach of using HSA to optimize the pharmacokinetics and biodistribution profile of Affibodies may be extended to the design of many other targeting molecules.     

Development and Evaluation of a Novel 99mTc-Labeled Annexin A5 for Early Detection of Response to Chemotherapy

^99mTc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, ^99mTc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel ^99mTc-labeled annexin A5 using a bis(hydroxamamide) derivative [C₃(BHam)₂] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C₃(BHam)₂ was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. ^99mTc labeling was performed by a ligand exchange reaction with ^99mTc-glucoheptonate. Biodistribution experiments for both ^99mTc-C₃(BHam)₂-annexin A5 and ^99mTc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of ^99mTc-C₃(BHam)₂-annexin A5 just after the first treatment of 5-FU was evaluated. ^99mTc-C₃(BHam)₂-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, ^99mTc-C₃(BHam)₂-annexin A5 had a much lower kidney accumulation of radioactivity than ^99mTc-HYNIC-annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of ^99mTc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of ^99mTc-C₃(BHam)₂-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of ^99mTc-C₃(BHam)₂-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that ^99mTc-C₃(BHam)₂-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy.     

99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

^99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, ^99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a ^99mTc-labeled HYNIC-DAPI compound with that of ^99mTc pertechnetate (^99mTcO₄ ⁻). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by ^99mTc-HYNIC-DAPI (1.03) was twice that caused by ^99mTcO₄ ⁻ (0.51), and the number of DSBs increased fivefold in the ^99mTc-HYNIC-DAPI-treated sample compared with the ^99mTcO₄ ⁻ treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the ^99mTcO₄ ^– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the ^99mTc-HYNIC-DAPI-treated samples. These results indicated that ^99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the ^99mTc-labeled compound with DNA. In contrast to these results, ^99mTcO₄ ⁻ induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of ^99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.     

99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice

CD13 receptor plays a critical role in tumor angiogenesis and metastasis. We therefore aimed to develop ^99mTc-labeled monomeric and dimeric NGR-containing peptides, namely, NGR1 and NGR2, for SPECT imaging of CD13 expression in HepG2 hepatoma xenografts. Both NGR-containing monomer and dimer were synthesized and labeled with ^99mTc. In vivo receptor specificity was demonstrated by successful blocking of tumor uptake of ^99mTc-NGR dimer in the presence of 20 mg/kg NGR2 peptide. Western blot and immunofluorescence staining confirmed the CD13 expression in HepG2 cells. The NGR dimer showed higher binding affinity and cell uptake in vitro than the NGR-containing monomer, presumably due to a multivalency effect. ^99mTc-Labeled monomeric and dimeric NGR-containing peptides were subjected to SPECT imaging and biodistribution studies. SPECT scans were performed in HepG2 tumor-bearing mice at 1, 4, 12, and 24 h post-injection of ~7.4 MBq tracers. The metabolism of tracers was determined in major organs at different time points after injection which demonstrated rapid, significant tumor uptake and slow tumor washout for both traces. Predominant clearance from renal and hepatic system was also observed in ^99mTc-NGR1 and ^99mTc-NGR2. In conclusion, monomeric and dimeric NGR peptide were developed and labeled with ^99mTc successfully, while the high integrin avidity and long retention in tumor make ^99mTc-NGR dimer a promising agent for tumor angiogenesis imaging.     

99mTc标记丝状噬菌体肽库及其在正常小鼠体内的分布

利用噬菌体展示技术已选出了多条与靶结合的肽. 然而,即使是体内直接筛选得到的,肽与肿瘤或靶器官的体内结合并不理想. 为了更好地理解噬菌体在体内的循环, 通过MAG3 ^99mTc标记噬菌体肽库,研究了肽库在体内分布. 体内分布实验结果显示,^99mTc-噬菌体主要分布在肝和脾中,而心脏、肌肉、脑和胰腺这些器官或组织中的分布非常低. ^99mTc-噬菌体在胃、肠和骨中的累积,随着时间延长在不断升高,其他器官中的吸收则在不断降低. 从5 min到30 min,^99mTc-噬菌体在血中清除迅速. 当噬菌体在体内循环足够长的时间后,一些噬菌体颗粒可以穿透血管进入并内化在器官或组织中. 总之,为了筛选具有高特异性和亲和性的肽,应该根据靶器官和筛选部位的不同,在筛选前确定合适的噬菌体在体内的循环时间.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).     

Synthesis and evaluation of 99mTc–2-[(3-carboxy-1-oxopropyl)amino]-2-deoxy-d-glucose as a potential tumor imaging agent

The 2-[(3-carboxy-1-oxopropyl)amino]-2-deoxy-d-glucose (CPADG) was synthesized and radiolabeled with ^99mTcO₄⁻ to obtain the ^99mTc–CPADG complex in high yield. It was stable over 6 h in saline at room temperature and in serum at 37 °C. The partition coefficient and electrophoresis results indicated that the complex was hydrophilic and cationic. In vitro cell studies showed there was an increase in the uptake of ^99mTc–CPADG as a function of incubation time and ^99mTc–CPADG was possibly transported via the glucose transporters. The biodistribution of ^99mTc–CPADG in mice bearing S 180 tumor showed that the complex accumulated in the tumor with high uptake and good retention. The tumor/blood and tumor/muscle ratios increased with time and reached 1.91 and 5.05 at 4 h post-injection. Single photon emission computed tomography (SPECT) image studies showed there was an obvious accumulation in tumor sites, suggesting ^99mTc–CPADG would be a promising candidate for tumor imaging.     

Tetraamine‐modified octreotide and octreotate: labeling with 99mTc and preclinical comparison in AR4‐2J cells and AR4‐2J tumor‐bearing mice

Two somatostatin analogues, [^99mTc]Demotide and [^99mTc]Demotate 4, were compared with [^99mTc]Demotate 1, a previously reported somatostatin receptor subtype 2 (sst₂) targeting tracer. Conjugates were prepared by coupling an open‐chain tetraamine chelator to D ‐Phe¹ of [Tyr³]‐octreotide or [Tyr³]‐octreotate, respectively, via a p‐benzylaminodiglycolic acid spacer adopting solid‐phase peptide synthesis techniques. Peptide conjugates were collected in a highly pure form after chromatographic purification. Eventually, [^99mTc]Demotide and [^99mTc]Demotate 4 were obtained in ～1 Ci/µmol specific activity and >96% purity after labeling under alkaline conditions. Demotide and Demotate 4 exhibited similar high binding affinities for the sst₂ expressed in AR4‐2J cells with IC₅₀ values 0.16 and 0.10 nM, respectively. The (radio)metallated analogues [^99mTc]Demotide and [^99mTc]Demotate 4 showed equally high affinities to the sst₂ during saturation binding assays in AR4‐2J cell membranes (K_ds 0.08 and 0.07 nM, respectively). During incubation at 37 °C with AR4‐2J cells, the radiopeptides internalized effectively via a receptor‐mediated process, with [^99mTc]Demotate 4 exhibiting a faster internalization rate than [^99mTc]Demotide. After injection in athymic mice bearing sst₂‐expressing AR4‐2J tumors, the radiotracers showed high and specific uptake in the tumor (>25%ID/g at 1 h) and in the sst₂–positive organs. However, both [^99mTc]Demotide and [^99mTc]Demotate 4 showed unfavorably higher background activity, especially in the abdomen, in comparison to [^99mTc]Demotate 1 and are, therefore, less suited than [^99mTc]Demotate 1 for sst₂‐targeted tumor imaging in man. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.