Coligand effects on the solution stability, biodistribution and metabolism of the (99m)Tc-labeled cyclic RGDfK tetramer

In this study, we present the evaluation of two new ternary ligand (99m)Tc complexes [(99m)Tc(HYNIC tetramer)(tricine)(L)] [L=isonicotinic acid (ISONIC) and 2,5-pyridinedicarboxylic acid (PDA)] as potential radiotracers for tumor imaging. Athymic nude mice bearing MDA-MB-435 human breast cancer xenografts were used to evaluate their biodistribution and metabolic properties. Solution stability data showed that [(99m)Tc(HYNIC tetramer)(tricine)(L)] (L=ISONIC and PDA) had significant decomposition (14% and 35%, respectively) at 6 h in the absence of excess ISONIC or PDA coligand. Biodistribution data clearly showed that [(99m)Tc(HYNIC tetramer)(tricine)(PDA)] had a much lower uptake in most organs of interest than [(99m)Tc(HYNIC tetramer)(tricine)(ISONIC)] during the 2-h study period. Results from metabolism studies revealed that approximately 50% of [(99m)Tc(HYNIC tetramer)(tricine)(ISONIC)] remained intact in fecal samples at 120 min postinjection, whereas only 10% of [(99m)Tc(HYNIC tetramer)(tricine)(PDA)] remained intact in fecal samples. The extent of metabolism correlated well with radiotracer solution stability. The results from this and our previous studies clearly demonstrated that coligands [trisodium triphenylphosphine-3,3',3'-trisulfonate (TPPTS), ISONIC and PDA] have a significant impact on the tumor uptake, excretion kinetics and metabolism of the (99m)Tc-labeled cyclic RGDfK tetramer. Among the three radiotracers evaluated in this tumor-bearing animal model, [(99m)Tc(HYNIC tetramer)(tricine)(TPPTS)] remained the best with respect to blood clearance, tumor uptake and target/background ratios.     

Plasma protein binding of (99m)Tc-labeled hydrazino nicotinamide derivatized polypeptides and peptides

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that exhibit slow blood clearance. In addition, the molecular sizes of parental peptides played an important role in the progression of the exchange reaction of one of the tricine coligands with plasma proteins.     

Preclinical evaluation of [99mTc/EDDA/tricine/HYNIC0, 1-Nal3, Thr8]-octreotide as a new analogue in the detection of somatostatin-receptor-positive tumors

PURPOSE: Radiolabeled somatostatin analogues are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin-receptor-positive tumors. The aim of this study was to evaluate a new somatostatin analogue designed for the labeling with (99m)Tc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC(0)), 1-Nal(3), Thr(8)]-octreotide ([HYNIC]-NATE), using ethylenediamine-N,N'-diacetic acid (EDDA) and tricine as coligands. METHODS: Synthesis was preformed on a solid phase using a standard Fmoc strategy. Labeling with (99m)Tc was performed at 100 degrees C for 10 min using SnCl(2) as a reductant. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate affinity was determined in AR4-2J cell membranes. The internalization and externalization rates were studied in sstr(2)-expressing AR4-2J cells. Biodistribution of radiopeptide was studied in rats bearing the AR4-2J tumor. RESULTS: Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. Peptide conjugate showed high affinity binding for sstr(2). The radioligand showed a moderate and specific internalization into AR4-2J cells (14.13+/-0.61% at 4 h). In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in somatostatin-receptor-positive organs. After 4 h, uptake in the AR4-2J tumor was 1.33+/-0.23%ID/g (percentage of injected dose per gram of tissue). CONCLUSION: These data show that [(99m)Tc/EDDA/tricine/HYNIC]-NATE is a specific radioligand for the somatostatin-receptor-positive tumors and is a suitable candidate for clinical studies.     

(99m)Tc-HYNIC-derivatized ternary ligand complexes for (99m)Tc-labeled polypeptides with low in vivo protein binding

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ((99m)Tc)-labeled polypeptides with tricine as a coligand. However, (99m)Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [(99m)Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare (99m)Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [(99m)Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [(99m)Tc](HYNIC-Fab)(tricine)(2.) When injected into mice, [(99m)Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [(99m)Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [(99m)Tc](HYNIC-NGA)(tricine)(2.) In addition, [(99m)Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [(99m)Tc](HYNIC-NGA)(tricine)(2.) Similar results were observed with (99m)Tc-labeled Fab fragments where [(99m)Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [(99m)Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [(99m)Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [(99m)Tc](HYNIC)(tricine)(BP) complex attractive as a radiolabeling reagent for targeted imaging.     

Nitriles form mixed-coligand complexes with (99m)Tc-HYNIC-peptide.

Using a 12-amino acid peptide conjugated with HYNIC as a model, we investigated nitriles as possible coligands for labeling with (99m)Tc. After the preparation of the (99m)Tc labeled HYNIC-peptide using tricine as coligand, the addition of acetonitile was found by reverse phase HPLC to block further coligand exchange with ethylenediamine diacetic acid (EDDA) at room temperature. The addition of this nitrile changed the pharmacokinetics of the (99m)Tc labeled peptide in normal mice towards faster clearance and significant differences in accumulation in most tissues sampled. By replacing acetonitrile with cyanoacetate, a nitrile not present in the HPLC eluant, it was possible to show the existence of a new, more hydrophilic, species by reverse phase HPLC. We conclude that nitriles can act as coligands for HYNIC-conjugated peptides labeled with (99m)Tc and tricine. Furthermore, the presence of acetonitrile during Sep-Pak or HPLC purification may inadvertently generate a mixed tricine/acetonitile coligand (99m)Tc-HYNIC-peptide complex.     

In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide,a comparison of tricine and EDDA as co-ligands

The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. OBJECTIVE: The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. METHODS: The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. RESULTS: The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. CONCLUSIONS: Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of alpha(V)beta(3) integrin proteins per cell might be limited.     

Synthesis and biological evaluation of potent alphavbeta3-integrin receptor antagonists

INTRODUCTION: alpha(v)beta(3) Integrin is expressed in sprouting endothelial cells in growing tumors, whereas it is absent in quiescent blood vessels. In addition, various tumor cell types express alpha(v)beta(3) integrin. alpha(v)beta(3) Integrin, a transmembrane heterodimeric protein, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin and plays a pivotal role in invasion, proliferation and metastasis. Due to the selective expression of alpha(v)beta(3) integrin in tumors, radiolabeled RGD peptides and peptidomimetics are attractive candidates for tumor targeting. METHODS: A cyclic RGD peptide, a peptoid-peptide hybrid, an all-peptoid and a peptidomimetic compound were synthesized, conjugated with 1,4,7,10-tetraazadodecane-N,N',N',N'-tetraacetic acid (DOTA) and radiolabeled with (111)In. Their in vitro and in vivo alpha(v)beta(3)-binding characteristics were determined. RESULTS: IC(50) values were 236 nM for DOTA-E-c(RGDfK), 219 nM for DOTA-peptidomimetic, >10 mM for DOTA-all-peptoid and 9.25 mM for the peptoid-peptide hybrid DOTA-E-c(nRGDfK). (111)In-labeled compounds, except for [(111)In]DOTA-all-peptoid, showed specific uptake in human alpha(v)beta(3)-expressing tumors xenografted in athymic mice. Tumor uptake for [(111)In]DOTA-E-c(RGDfK) was 1.73+/-0.4% ID/g (2 h postinjection) and that of [(111)In]DOTA-peptidomimetic was 2.04+/-0.3% ID/g. Tumor uptake for the peptoid-peptide hybrid [(111)In]DOTA-E-c(nRGDfK) was markedly lower (0.45+/-0.07% ID/g). The all-peptoid [(111)In]DOTA-E-c(nRGnDnFnK) did not show specific uptake in tumors (0.11+/-0.04% ID/g). CONCLUSIONS: The peptidomimetic compound and the cyclic RGD peptide have a high affinity for alpha(v)beta(3) integrin, and these compounds have better tumor-targeting characteristics than the peptoid-peptide hybrid and the all-peptoid.     

Lys and Arg in UBI: a specific site for a stable Tc-99m complex?

The aim of this study was to help establish if ubiquicidin peptide 29-41 fragment (UBI) contains a specific site for 99mTc labeling by a new direct method under alkaline conditions. Since this peptide does not have cysteine residues, it is possible that neighboring arginine and lysine in the peptide amino acid sequence (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) could be a specific coordination site to form a stable 99mTc-UBI complex. Following direct labeling, the in vitro stability of 99mTc-UBI was compared to UBI radiolabeled by one indirect method using HYNIC/tricine and HYNIC/tricine/EDDA. Radiochemical purity of 99mTc-UBI averaged 97% compared to 88% for 99mTc-HYNIC-UBI/tricine and 98% for 99mTc-HYNIC-UBI/tricine/EDDA. Both 99mTc-HYNIC-UBI (tricine or EDDA) and 99mTc-UBI showed stability in human serum and solutions of cysteine. 99mTc-UBI radiochemical purity 24 h after dilution in 0.9% NaCl was greater than 90% at pH 9 and greater than 95% at pH 6.5. Under one set of experimental conditions, in vitro binding to bacteria of 99mTc-UBI was 35% and identical to that of 99mTc-HYNIC-UBI/tricine and 99mTc-HYNIC-UBI/tricine/EDDA at 32% and 31% respectively. The biodistribution of 99mTc-UBI in mice showed a rapid renal clearance. To help identify the site(s) of 99mTc binding following direct labeling, molecular mechanics and quantum-mechanical calculations were performed which showed that the amine groups of Arg(7) and Lys are the most probable site. The calculations show that these groups can form a square pyramid with two water molecules for the Tc cation (dxysp(3)). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results.     

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 N(3)S system is described and a comparison made with [Tyr(3)]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG(3) and an N(3)S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands for HYNIC conjugates. All conjugates and (99m)Tc-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 (99m)Tc-RC160 derivatives compared with (99m)Tc-EDDA/HYNIC-[Tyr(3)]-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 (99m)Tc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with (99m)Tc-EDDA/HYNIC-[Tyr(3)]-octreotide, (99m)Tc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives.     

An improved method of 18F peptide labeling: hydrazone formation with HYNIC-conjugated c(RGDyK)

Radiolabeled alpha(v)beta(3)-integrin antagonists are increasingly investigated as a means of imaging angiogenesis. Several methods of labeling alpha(v)beta(3)-integrin binding peptide with (18)F have been reported recently. In the present study, we devised a straightforward means for labeling Arg-Gly-Asp (RGD) peptide with (18)F via hydrazone formation between c(RGDyK)-hydrazinonicotinic acid (HYNIC) (3) and 4-[(18)F]-fluorobenzaldehyde ([(18)F]4). The resulting reaction mixture was purified by HPLC to give 4'-[(18)F]-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) ([(18)F]5). The conjugation efficiency of 3 and 4 to form [(18)F]5 was 95.2%, and the radiochemical purity of [(18)F]5 after purification was >99%. The specific activity of [(18)F]5 estimated by radio-HPLC was 20.5 GBq/mumol (end of synthesis). Competitive binding assay of c(RGDyK) (1) and 5 was performed using [(125)I]iodo-c(RGDyK) as a radioligand, and K(i) values were found to be 2.8 and 21.7 nM, respectively. For the biodistribution study, the angiogenic mouse model was established by inducing unilateral ischemia on the left hindlimbs of ICR mice after femoral artery ablation. Seven days after inducing ischemia, [(18)F]5 was administered to the mice through the tail vein. Ischemic muscle uptake of [(18)F]5 was significantly higher than that of normal muscle (P<.01). Specific uptake was confirmed by coinjection of 1 with [(18)F]5. Here, we successfully labeled RGD peptide with (18)F via hydrazone formation between 3 and 4, resulting to [(18)F]5. [(18)F]5 was found to have high affinity for alpha(v)beta(3)-integrin and to accumulate specifically in ischemic hindlimb muscle of mice. We suggest that (18)F labeling via formation of hydrazone between HYNIC peptide and [(18)F]4 is a useful method for labeling c(RGDyK), which can be applied for imaging angiogenesis.     

Preparation and characterization of 99mTc(CO)3-BPy-RGD complex as alphav beta3 integrin receptor-targeted imaging agent.

The aim of this study is to develop a novel arginine-glycine-aspartic acid (RGD) peptide-containing ligand for (99m)Tc labeling as alpha(v)beta(3) integrin receptor-targeted imaging agent. BPy-RGD conjugate was successfully synthesized by coupling of 5-carboxylate-2,2'-bipyridine and c(RGDyK) peptide through EDC/SNHS in aqueous solution and was characterized by MADLI-TOF-MS (m/z=802.72, C(38)H(48)N(11)O(9)). (99m)Tc(CO)(3)-BPy-RGD was prepared by exchange reaction between [(99m)Tc(H(2)O)(3)(CO)(3)](+) and BPy-RGD. Final product was purified by HPLC and tested for octanol/water partition coefficient. Cell-binding assays of BPy-RGD and unmodified c(RGDyK) were tested in MDA-MB-435 cells ((125)I-echistatin as radioligand). Preliminary biodistribution of the (99m)Tc(I)-labeled radiotracer in orthotopic MDA-MB-435 breast tumor xenograft model was also evaluated. The BPy-RGD conjugate had good integrin-binding affinity (50% inhibitory concentration (IC(50))=92.51+/-22.69 nM), slightly lower than unmodified c(RGDyK) (IC(50)=59.07+/-11.03 nM). The hydrophilic radiotracer also had receptor-mediated activity accumulation in MDA-MB-435 tumor (1.45+/-0.25 percentage of injected dose per gram (%ID/g) at 1.5h postinjection (p.i.)), which is known to be integrin positive. After blocking with c(RGDyK), the tumor uptake was reduced from 0.71+/-0.01%ID/g to 0.33+/-0.18%ID/g at 4h p.i. (99m)Tc(I) tricarbonyl complex of cyclic RGD peptide is a promising strategy for integrin targeting. Further modification of the bipyridine-conjugated RGD peptide by using more potent RGD peptides and fine tuning of the tether group between the RGD moiety and (99m)Tc(CO)(3)(+) core to improve the tumor targeting efficacy and in vivo kinetic profiles is currently in progress.     

Radiolabeling of annexin A5 with (99m)Tc: comparison of HYNIC-Tc vs. iminothiolane-Tc-tricarbonyl conjugates

In the perspective of expanding the use of annexin A5 (anx A5) as radioactive tracer of cell death in vivo, we recently described its radiolabeling with (99m)Tc-tricarbonyl [(99m)Tc(H(2)O)(3)(CO)(3)](+) via the mercaptobutyrimidyl group (anx A5-SH). The aim of the present article was to compare this new method with the HYNIC strategy (anx A5-HYNIC), recognized at present as the reference for the radiolabeling of proteins with (99m)Tc. Similar radiolabeling yields and better chemical stability were obtained with the [anx A5-SH-(99m)Tc-tricarbonyl] complex. Since the [anx A5-HYNIC-(99m)Tc(tricine)(2)] conjugate shows isomeric forms which can affect the biological properties whereas [anx A5-SH-(99m)Tc-tricarbonyl] is less or not prone to such drawback, the latter seems superior to the former. Furthermore, (anx A5-SH) is readily obtained via commercial sources of Traut's reagent whereas (anx A5-HYNIC) is not. The results provide encouraging evidence in the development of anx A5-labeled reagent for apoptose imaging.     

Evaluation of [99mTc/EDDA/HYNIC0]octreotide derivatives compared with [111In-DOTA0,Tyr3, Thr8]octreotide and [111In-DTPA0]octreotide: does tumor or pancreas uptake correlate with the rate of internalization?

Radiolabeled somatostatin analogs are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin receptor-positive tumors. The aim of this study was to compare 3 somatostatin analogs designed for the labeling with (99m)Tc (where HYNIC is 6-hydrazinopyridine-3-carboxylic acid): 6-hydrazinopyridine-3-carboxylic acid(0)-octreotide (HYNIC-OC/(99m)Tc-(1)), [HYNIC(0),Tyr(3)]octreotide (HYNIC-TOC/(99m)Tc-(2)), and [HYNIC(0),Tyr(3),Thr(8)]octreotide (HYNIC-TATE/(99m)Tc-(3)), using ethylenediamine-N,N'-diacetic acid (EDDA) as a coligand. In addition, we compared the (99m)Tc-labeled peptides [(111)In-diethylenetriaminepentaacetic acid(0)]octreotide ([(111)In-DTPA]-OC) and [(111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(0),Tyr(3),Thr(8)]octreotide ([(111)In-DOTA]-TATE) with regard to the rate of internalization and the biodistribution in AR4-2J (expressing the somatostatin receptor subtype 2) tumor-bearing rats. The main attention was directed toward a potential correlation between the rate of internalization and the tumor or pancreas uptake. METHODS: Synthesis was performed on solid phase using a standard Fmoc strategy. Internalization was studied in cell culture (AR4-2J) and biodistribution was studied using a Lewis rat tumor model (AR4-2J). RESULTS: The 5 radiopeptides showed a specific internalization into AR4-2J cells in culture (as shown by blocking experiments). The rate of internalization of the 5 radiopeptides differed significantly according to the following order: (99m)Tc-(1) approximately = [(111)In-DTPA]-OC < (99m)Tc-(2) < (99m)Tc-(3) approximately = [(111)In-DOTA]-TATE. All radiopeptides displayed a rapid blood clearance and a fast clearance from all somatostatin receptor-negative tissues predominantly via the kidneys. A receptor-specific uptake of radioactivity was observed for all compounds in somatostatin receptor-positive organs such as the pancreas, the adrenals, and the stomach. After 4 h, the uptake in the AR4-2J tumor was comparable for (99m)Tc-(2) (3.85 +/- 1.0 injected dose per gram tissue (%ID/g)), (99m)Tc-(3) (3.99 +/- 0.58%ID/g), and [(111)In-DOTA]-TATE (4.12 +/- 0.74%ID/g) but much lower for [(111)In-DTPA]-OC (0.99 +/- 0.08%ID/g) and (99m)Tc-(1) (0.70 +/- 0.13%ID/g). The specificity was determined by blocking experiments using a large excess of [Tyr(3)]octreotide. (99m)Tc-(3) displayed the highest tumor-to-kidney ratio (2.5:1), followed by (99m)Tc(2) (1.9:1) and [(111)In-DOTA]-TATE (1.7:1). CONCLUSION: These data show that the 5 radiopeptides are specific radioligands for the somatostatin receptor subtype 2. The rate of internalization correlates with the uptake in the tumor (R(2) = 0.75; P = 0.026) and pancreas (R(2) = 0.98; P = 7.4.10(-5)). [Tyr(3),Thr(8)]octreotide derivatives show superiority over the corresponding octreotide and [Tyr(3)]octreotide derivatives, indicating that [(111)In-DOTA]-TATE and [(99m)Tc/EDDA/HYNIC]-TATE are suitable candidates for clinical studies.     

Imaging of HER2/neu expression in BT-474 human breast cancer xenografts in athymic mice using [(99m)Tc]-HYNIC-trastuzumab (Herceptin) Fab fragments

OBJECTIVE: To evaluate the ability of trastuzumab (Herceptin) Fab, labelled with (99m)Tc through introduced hydrazinenicotinamide (HYNIC) functionalities, to image HER2/neu-overexpressing human breast cancer xenografts in athymic mice. METHODS: Fab fragments were produced by immobilized papain digestion of trastuzumab immunoglobulin G (IgG), followed by purification by ultrafiltration. The immunoreactivity of trastuzumab Fab was evaluated by receptor-binding assays against HER2/neu-positive SK-BR-3 human breast cancer cells. Trastuzumab Fab fragments were labelled with (99m)Tc following modification with HYNIC N-hydroxysuccinimide ester. Biodistribution and tumour imaging studies were performed in athymic mice bearing subcutaneous HER2/neu-overexpressing BT-474 human breast cancer xenografts following intravenous injection of 1.1 or 25 MBq of [(99m)Tc]-trastuzumab Fab (30 microg), respectively. The specificity of tumour uptake was assessed by comparison with that of [(99m)Tc]-labelled irrelevant anti-CD33 HuM195 Fab. RESULTS: Trastuzumab Fab was pure and exhibited preserved immunoreactivity towards SK-BR-3 cells (K(d) = 1.6 x 10(-8) M). Modification with HYNIC diminished its receptor-binding affinity fourfold. [(99m)Tc]-trastuzumab Fab localized avidly and specifically in BT-474 xenografts, achieving a tumour uptake of 10.7% of the injected dose (ID) per gram and a tumour to blood (T/B) ratio of 3 : 1 at 24 h. The tumour uptake and T/B ratio for [(99m)Tc]-trastuzumab Fab were significantly higher than those for control [(99m)Tc]-HuM195 Fab (2.6% ID x g(-1) and 0.9 : 1, respectively; P<0.05). Tumours were imaged as early as 2 h post-injection of [(99m)Tc]-trastuzumab Fab, but were more clearly visualized at 6 and 24 h post-injection. CONCLUSIONS: [(99m)Tc]-HYNIC-trastuzumab Fab localized specifically in HER2/neu-overexpressing human breast cancer xenografts in athymic mice, allowing imaging of the tumours within the useful lifetime of the radionuclide.     

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     

99Tcm-（HYNIC-[Lys3]-BBS）（tricine）（tricine）的制备及对胰腺癌荷瘤鼠显像研究

目的制备99Tcm-（联肼尼克酰胺-蛙皮素类似肽）（N-三羟甲基甘氨酸）（三苯基膦三间磺酸钠盐）[（HYNIC-[Lys3]-BBS）（tricine）（TPPTS）]三重配位化合物,评价其在正常小鼠及胰腺癌荷瘤裸小鼠的生物分布。方法双功能螯合剂HYNIC与[Lys3]-BBS偶联（pH值9．0）,以SnCl2为还原剂,tricine和TPPTS为协同配体,进行99Tcm-标记,合成三重配位化合物99Tcm-（HYNIC-[Lys。]-BBS）（tricine）（TPPTS）。用Sep-PakC18cartridge和HPLC对其纯化和分析,测定其标记率和放化纯,研究其在人血清中的稳定性,并进行正常小鼠体内的生物分布研究以及胰腺癌荷瘤裸小鼠活体显像。结果99Tcm-（HYNIC_[Lys3]-BBS）（tricine）（TPPTS）标记率为（90±2）％,放化纯〉95％,在人血清中放置4h其放化纯仍大于85％。正常小鼠体内分布结果表明,99Tcm-（HYNIC-[Lys3]-BBS）（tricine）（TPPTS）血液清除迅速,2h血液中放射性为（0．07±0．01）％ID／g,主要经。肾排泄,肝、胃肠道摄取较少,2h时肝放射性为（0．27±0．03）％ID／g,胃为（0．06±0．03）％ID／g,肠为（0．04±0．00）％ID／g。胰腺癌荷瘤裸小鼠吖显像可见肿瘤部位有放射性浓聚影,2h后肿瘤与对侧正常肌肉的T／NT比值最高达3．71±0．57。结论99Tcm-（HYNIC-[Lys3]-BBS）（tricine）（TPPTS）三重配位化合物制备成功,所用标记方法可行,标记物稳定性较好,标记率和放化纯较高,生物分布特性良好,有望用于胰腺癌的显像研究。     

99m-Technetium-labelled peptide-HYNIC conjugates: effects of lipophilicity and stability on biodistribution.

The aim of this study was to explore the effects of lipophilicity and stability on the biodistribution of 99mTc labelled peptides through the use of different co-ligands. 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was coupled to the somatostatin analogue RC160 and radiolabelled using a range of ethylendiaminediacetic acid (EDDA) and ethylenediaminetetraacetic acid (EDTA) derivatives as well as tricine and pyridine/tricine as co-ligands. After labelling with technetium-99m, chromatographic, stability, protein-binding, and rat biodistribution studies were performed. For most co-ligands, biodistribution correlated well with in vitro properties. Lipophilic substitution on EDDA resulted in higher protein binding, increased liver uptake, and intestinal excretion. Stabilisation of tricine with pyridines reduced blood levels and lowered liver uptake. EDTA derivatives showed high instability in vitro and in vivo.     

Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies

INTRODUCTION: Radionuclide imaging can be a useful tool for the diagnosis of prostate cancer. Bombesin (BBN) is a molecule with high affinity for gastrin releasing peptide (GRP) receptors which are over-expressed in that tumor. This report compares (99m)Tc-HYNIC-betaAla-BBN(7-14)NH2 [(99m)Tc-HYNIC-BBN] and (99m)Tc identical withN(PNP6)-Cys-betaAla-BBN(7-14)NH2 [(99m)TcN(PNP6)-Cys-BBN] with regard to labeling procedures as well as in vitro and in vivo evaluation (biodistribution and scintigraphic imaging). METHODS: Peptide synthesis was performed in an automated peptide synthesizer. HYNIC-BBN was radiolabeled with pertechnetate using tricine and ethylenediamine diacetic acid (EDDA) as coligands. Cys- BBN was radiolabeled in a two-step procedure with the preparation of the precursor (99m)Tc-Nitrido first and then introducing diphosphine (PNP6). Radiochemical evaluation of conjugates, as well as studies of stability, transchelation toward cysteine, and partition coefficient were done. Biological studies included internalization, biodistribution in healthy animals and in animals bearing PC3 cancer cells with acquisition of images from the tumor-bearing animals. RESULTS: Both complexes showed a high radiochemical yield along with good stability. Biodistribution studies pointed out strong renal excretion for the former complex due to its hydrophilic profile and marked hepatobiliary excretion for the latter, corresponding to observed lipophilicity. Tumor uptake was higher for (99m)Tc-HYNIC-BBN and the same occurred with internalization findings, which exceeded those of (99m)TcN(PNP6)-BBN. Blocking studies in mice bearing PC-3 tumor cells revealed significantly reduced pancreas and tumor uptake, demonstrating receptor specificity of the conjugates. CONCLUSION: The best radiotracer was (99m)Tc-HYNIC-BBN on the basis of high radiochemical yield, fast radiolabeling procedure without need for a purification step, and more consistent tumor uptake.     

^99Tc^m标记RGD环肽在荷肺腺癌裸鼠中的显像研究

目的制备^99Tc^m标记的含RGD序列的^99Tc^m-联肼尼克酰胺（HYNIC）-c（RGDfK）环肽单体,评价其在整合素表达阳性的肺腺癌严重联合免疫缺陷（SCID）小鼠肿瘤模型中的生物学分布,并进行显像研究。方法（1）以HYNIC为双功能螯合剂,以三羟甲基甘氨酸（tricine）和乙二胺二乙酸为协同配体,采用二步法制备^99Tc^m标记HYNIC—c（RGDfK）,进行细胞结合实验,测定标记物生物学活性;（2）将荷A549肺腺癌模型小鼠分为7组[第7组作为竞争性抑制组,注射显像剂前0．5h先注射HYNIC-c（CRDGfk）100μg],每组5只,经尾静脉注射7．4MBq的^99Tc^m-HYNIC-c（RGDfK）,于注射后0．5,1,2,4,8,12h处死,计算荷A549肺腺癌小鼠模型各脏器％ID／g,同时采用ROI技术研究^99Tc^m-HYNIC—c（RGDfK）在小鼠体内的生物学分布,计算不同时间点的T／NT比值（NT选取肌肉）;（3）取6只荷瘤裸鼠,其中3只为竞争性抑制组,经尾静脉注射7．4MBq的^99Tc^m-HYNIC—c（RGDfK）,于注射后0．5,1,2,4,8,12h进行静态1显像。结果^99Tc^m-HYNIC—c（RGDfK）的标记率〉90％,放化纯〉95％。^99Tc^m-HYNIC—c（RGDfK）与A549肺腺癌细胞特异性结合率最高为36．14％,体内分布实验显示^99Tc^m-HYNIC—c（RGDfK）在肾的摄取率始终高于20％ID／g,注射后0．5h肿瘤％ID／g为10．52±1．48,8h为17．26±2．81,12h为8．93±0．90,竞争性抑制组注射后0．5h为2．29±0．85。通过ROI技术测得T／NT在8h达6．87。注射后1h肿瘤可显影,4～8h显影更清晰。结论^99Tc^m标记HYNIC—c（RGDfK）易于制备,具有良好的靶向性。     

99Tcm标记RGD环肽四聚体在神经胶质瘤裸鼠模型中的显像研究

目的 制备99Tcm标记的含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)序列的环肽四聚体99Tcm-联肼尼克酰胺(HYNIC)-E{E[c(RGDfK)]2}2,评价其在整合素αvβ3表达阳性的荷人神经胶质瘤裸鼠模型的生物分布和显像.方法 以HYNIC为双功能螫合剂,以三羟甲基甘氨酸(tricine)和三苯基膦三磺酸钠(TPfffS)为协同配体,采用两步法制备99Tcm-HYNIC-E{E[c(RGDfK)2}2.通过体外受体竞争结合实验比较e(RGDyK)单体、HYNIC-E[c(RGDfK)2二聚体和HYNIC-E{E[c(RGDfK)]2}2四聚体与整合素αvβ3亲和力.生物分布实验数据显示,99Tcm-HYNIC-E{E[c(RGDtK)]2}2主要经肾排泄;注射后1h,肿瘤对99Tcm-HYNIC-E{E[c(RGDfK)]2}2的摄取为99Tcm-HYNIC-E[c(RG-DfK)]2的2倍,分别为(10.32±0．07)％ID／g和(5.15±O.52)％ID／g,与体外受体竞争结合实验数据相一致;注射后4h,肿瘤对99Tcm-HYNIC-E{E[c(RGDfK)]2}2的摄取仍达(9.35.4±1.35)％ID／g,表明标记物在肿瘤中的滞留时间足够长.r显像结果显示,注射后1h肿瘤清晰可见.注射后4h显像效果更佳.结论 99Tcm-HYNIC-E{E[c(RGDfK)]2}2具有较高的肿瘤摄取和较长的肿瘤滞留时间,可以用于整合素αvβ3表达阳性肿瘤的显像;放射性核素(如90Y)标记的RGD环肽四聚体可用于整合素(αvβ3表达阳性肿瘤的治疗.