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Introduction Sigma-1 receptor is a target for tumor imaging. In a previous study, we synthesized a vesamicol analog, (+)-2-[4-(4-bromophenyl)piperidino]cyclohexanol [(+)-pBrV], with a high affinity for sigma-1 receptor, and synthesized radiobrominated (+)-pBrV. This radiobrominated (+)-pBrV showed high tumor uptake in tumor-bearing mice; however, radioactivity accumulation in normal tissues, such as the liver, was high. We assumed that the accumulation of (+)-pBrV in the non-target tissues was partially derived from its high lipophilicity; therefore, we synthesized and evaluated (+)-4-[1-(2-hydroxycyclohexyl)piperidine-4-yl]-2-bromophenol [(+)-BrV-OH], which is a more hydrophilic compound. Although we aimed to develop a PET tracer using 76Br, in these initial studies, we used 77Br because of its longer half-life. Methods (+)-[77Br]BrV-OH was synthesized using the chloramine-T method with a radiochemical purity of 95%. Lipophilicity and affinity for sigma-1 receptor of (+)-[77Br]BrV-OH were determined, and biodistribution experiments were performed. We also performed an in vivo blocking study by co-injecting excess amounts of the sigma-1 receptor ligand, SA4503, into mice. Results The lipophilicity and affinity for sigma-1 receptor of (+)-[77Br]BrV-OH were lower than those of (+)-[77Br]pBrV. (+)-[77Br]BrV-OH also showed high tumor uptake in biodistribution experiments in DU-145 tumor-bearing mice,. Although (+)-[77Br]pBrV was retained in most tissues, (+)-[77Br]BrV-OH was cleared from these tissues. In blocking studies, the co-injection of SA4503 significantly decreased the tumor uptake of (+)-[77Br]BrV-OH. Conclusion These results indicate that (+)-[76Br]BrV-OH has potential as a PET probe for sigma-1 receptor imaging.

Introduction Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated 131 I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[ 131 I] p IV], which has a high affinity for sigma receptors. Therefore, (+)-[ 131 I] p IV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[ 211 At] p AtV, an 211 At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy. Methods The radiolabeled sigma receptor ligand (+)-[ 211 At] p AtV was prepared using a standard halogenation reaction generating a 91% radiochemical yield with 98% purity after HPLC purification. The partition coefficient of (+)-[ 211 At] p AtV was measured. Cellular uptake experiments and in vivo biodistribution experiments were performed using a mixed solution of (+)-[ 211 At] p AtV and (+)-[ 125 I] p IV; the human prostate cancer cell line DU-145, which expresses high levels of the sigma receptors, and DU-145 tumor-bearing mice. Results The lipophilicity of (+)-[ 211 At] p AtV was similar to that of (+)-[ 125 I] p IV. DU-145 cellular uptake and the biodistribution patterns in DU-145 tumor-bearing mice at 1h post-injection were also similar between (+)-[ 211 At] p AtV and (+)-[ 125 I] p IV. Namely, (+)-[ 211 At] p AtV demonstrated high uptake and retention in tumor via binding to sigma receptors. Conclusion These results indicate that (+)-[ 211 At] p AtV could function as an new agent for alpha-radionuclide receptor therapy.

Introduction Sigma receptors are appropriate targets for tumor imaging because they are highly expressed in a variety of human tumors. Previously, we synthesized a vesamicol analog, (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol ((+)- p IV), with high affinity for sigma receptors, and prepared radioiodinated (+)- p IV. In this study, to develop a radiobromine-labeled vesamicol analog as a sigma receptor imaging agent for PET, nonradioactive and radiobromine-labeled (+)-2-[4-(4-bromophenyl)piperidino]cyclohexanol ((+)- p BrV) was prepared and evaluated in vitro and in vivo . In these initial studies, 77 Br was used because of its longer half-life. Methods (+)-[ 77 Br] p BrV was prepared by a bromodestannylation reaction with radiochemical purity of 98.8% after HPLC purification. The partition coefficient of (+)-[ 77 Br] p BrV was measured. In vitro binding characteristics of (+)- p BrV to sigma receptors were assayed. Biodistribution experiments were performed by intravenous administration of a mixed solution of (+)-[ 77 Br] p BrV and (+)-[ 125 I] p IV into DU-145 tumor-bearing mice. Results The lipophilicity of (+)-[ 77 Br] p BrV was lower than that of (+)-[ 125 I] p IV. As a result of in vitro binding assay to sigma receptors, the affinities of (+)- p BrV to sigma receptors were competitive to those of (+)- p IV. In biodistribution experiments, (+)-[ 77 Br] p BrV and (+)-[ 125 I] p IV showed high uptake in tumor via sigma receptors. The biodistributions of both radiotracers showed similar patterns. However, the accumulation of radioactivity in liver after injection of (+)-[ 77 Br] p BrV was significantly lower compared to that of (+)-[ 125 I] p IV. Conclusion These results indicate that radiobromine-labeled p BrV possesses great potential as a sigma receptor imaging agent for PET.

The radioiodinated (+)-p-iodovesamicol [(+)-pIV], which shows a high binding affinity for sigma-1 (sigma-1) receptors, is prepared by an exchange reaction. The specific activity (SA) is fairly low and therefore is insufficient for clinical use. In this study, we prepared (+)-[(125)I]pIV with a high SA from tributylstannyl precursor and compared the in vivo characteristics between high and low SA by imaging sigma-1 receptors in the central nervous system. In the biodistribution study, a difference in brain accumulation was observed between the two methods. At 30 min postinjection, the brain accumulation (1.58%ID/g) of low SA [0.6-1.1 TBq/mmol (16-30 Ci/mmol)] (+)-[(125)I]pIV was higher than that (1.34%ID/g) of high SA [88.8 TBq/mmol (2400 Ci/mmol)] (+)-[(125)I]pIV. In the blocking study, the brain uptake of high SA (+)-[(125)I]pIV was reduced more significantly by the coadministration of sigma ligands such as pentazocine, haloperidol or SA4503 than that of low SA (+)-[(125)I]pIV. These results showed that nonspecific binding of high SA (+)-[(125)I]pIV in the brain was lower than that of low SA (+)-[(125)I]pIV, and high SA (+)-[(125)I]pIV bound more specifically to sigma-1 receptors in the brain than low SA (+)-[(125)I]pIV. In contrast, in the blood-binding study, high SA (+)-[(125)I]pIV (58.4%) bound to blood cells with higher affinity than low SA (+)-[(125)I]pIV (46.0%). In metabolite studies, blood metabolites of high SA (+)-[(125)I]pIV (57.3+/-3.5%) were higher than those of low SA (+)-[(125)I]pIV (45.5+/-4.1%) at 30 min postinjection. Higher SA may be apt to bind to blood cells with higher affinity and to be metabolized faster.

In this study, the (+)-enantiomer of radioiodinated 2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[(125)I]-p-iodovesamicol] [(+)-[(125)I]pIV], which is reported to bind with high affinity to sigma-1 receptors in vitro, was tested for its usefulness in imaging sigma-1 receptors in the central nervous system (CNS) in vivo. In biodistribution studies, significant amounts (approximately 3% of the injected dose) of (+)-[(125)I]pIV accumulated in rat brain, and its retention was prolonged. In blocking studies, the accumulation of (+)-[(125)I]pIV in the rat brain was significantly reduced by the coadministration of sigma-ligands such as pentazocine (5.0 micromol), haloperidol (0.5 micromol) or SA4503 (0.5 micromol). The blocking effect of pentazocine (selective sigma-1 ligand) was similar to the blocking effects of SA4503 and haloperidol [nonselective sigma (sigma-1 and sigma-2) ligands]. Ex vivo autoradiography of the rat brain at 45 min following intravenous injection of (+)-[(125)I]pIV showed high localization in brain areas rich in sigma-1 receptors. Thus, the distribution of (+)-[(125)I]pIV was thought to bind to sigma-1 receptors in the CNS in vivo. These results indicate that radioiodinated (+)-pIV may have the potential to image sigma-1 receptors in vivo.

Since elevated levels of gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are associated with a poor prognosis in cancer patients, these enzymes are potential targets for tumor imaging. In the present study, a cyclic decapeptide, cCTTHWGFTLC (CTT), was selected as a mother compound because of its selective inhibitory activity toward gelatinases. For imaging gelatinase activity in tumors, we designed a CTT-based radiopharmaceutical taking into consideration that (1) the HWGF motif of the peptide is important for the activity, (2) hydrophilic radiolabeled peptides show low-level accumulation in the liver and (3) an increase in the negative charge of radiolabeled peptides is effective in reducing renal accumulation. Thus, a highly hydrophilic and negatively charged radiolabel, indiun-111-diethylenetriaminepentaacetic acid ( 111 In-DTPA), was attached to an N-terminal residue distant from the HWGF motif ( 111 In-DTPA-CTT). In MMP-2 inhibition assays, In-DTPA-CTT significantly inhibited the proteolytic activity in a concentration-dependent fashion. When injected into normal mice, 111 In-DTPA-CTT showed low levels of radioactivity in the liver and kidney. A comparison of the pharmacokinetic characteristics of 111 In-DTPA-CTT with those of other CTT derivatives having different physicochemical properties revealed that the increase in hydrophilicity and negative charge caused by the conjugation of 111 In-DTPA reduced levels of radioactivity in the liver and kidney. In tumor-bearing mice, a significant correlation was observed between the accumulation in the tumor as well as tumor-to-blood ratio of 111 In-DTPA-CTT and gelatinase activity. These findings support the validity of the chemical design of 111 In-DTPA-CTT for reducing accumulation in nontarget tissues and maintaining the inhibitory activity of the mother compound. Furthermore, 111 In-DTPA-CTT derivatives would be potential radiopharmaceuticals for the imaging of gelatinase activity in metastatic tumors in vivo.

The chemokine receptor CXCR4 is highly expressed in tumor cells and plays an important role in tumor metastasis. The aim of this study was to develop a radiopharmaceutical for the imaging of CXCR4-expressing tumors in vivo. Based on structure-activity relationships, we designed a 14-residue peptidic CXCR4 inhibitor, Ac-TZ14011, as a precursor for radiolabeled peptides. For 111In-labeling, diethylenetriaminepentaacetic acid (DTPA) was attached to the side chain of d-Lys(8) which is distant from the residues indispensable for the antagonistic activity. In-DTPA-Ac-TZ14011 inhibited the binding of a natural ligand, stromal cell-derived factor-1alpha, to CXCR4 in a concentration-dependent manner with an IC50 of 7.9 nM (Ac-TZ14011: 1.2 nM). In biodistribution experiments, more 111In-DTPA-Ac-TZ14011 accumulated in the CXCR4-expressing tumor than in blood or muscle. Furthermore, the tumor-to-blood and tumor-to-muscle ratios were significantly reduced by coinjection of Ac-TZ14011, indicating a CXCR4-mediated accumulation in tumor. These findings suggested that 111In-DTPA-Ac-TZ14011 would be a potential agent for the imaging of CXCR4 expression in metastatic tumors in vivo.

Metabolic studies of 111 In-DTPA-labeled polypeptides and peptides showed that the radiolabeled (poly)peptides generated 111 In-DTPA-adducts of amino acid that possess long residence times in the lysosomal compartment of the tissues where (poly)peptides accumulated. However, a recent study suggested that metal-chelate-methionine (Met) might possess in vivo behaviors different from metal-chelate adducts of other amino acids. In this study, to elucidate whether some biological characteristics of Met may accelerate the renal elimination rate of 111 In-DTPA-adduct of Met into urine, 111 In-DTPA-Met 1 -octreotide was synthesized and the renal handling of 111 In-DTPA-Met was investigated using 111 In-DTPA-l-Phe 1 -octreotide (Phe represents phenylalanine), which was reported previously, as a reference. Both 111 In-DTPA-conjugated octreotide analogs were stable against 3-h incubation in murine serum at 37°C. Both 111 In-DTPA-octreotide analogs also showed rapid clearance of the radioactivity from the blood and similar accumulation of the radioactivity in the kidney. No significant differences were observed in the renal radioactivity levels from 10 min to 24 h postinjection between the two. Metabolic studies indicated that 111 In-DTPA-Met 1 -octreotide and 111 In-DTPA-l-Phe 1 -octreotide generated 111 In-DTPA-adducts of Met and Phe, respectively, as the final radiometabolites at similar rates. These findings suggested that the long residence times of the radioactivity in tissues after administration of 111 In-DTPA-labeled peptides and polypeptides would be attributed to inherent characteristics of 111 In-DTPA chelate.

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.

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.

Recent development of a variety of thiol-free chelating agents has facilitated the design of 99mTc-labeled somatostatin analogs suitable for receptor imaging of somatostatin-positive tumors. However, it remains ambiguous whether the disulfide bonds in cyclic peptides are stable during 99mTc complexation reactions, and contradictory results have been reported regarding the integrity of disulfide bonds in cyclic somatostatin analogs. To estimate the stability of the disulfide bond in a synthetic somatostatin analog at low peptide concentrations, [125I]I-RC-160, in which radioiodine was incorporated into the 3-Tyr residue, was synthesized and the integrity of the disulfide bond of the peptide was investigated in the presence of reducing agents such as ascorbic acid, dithionite, and stannous ions. The disulfide bond in [125I]I-RC-160 remained stable in the presence of ascorbic acid in boiling water. The disulfide bond was also stable when treated with stannous ions at concentrations sufficient to reduce 99mTc for complexation with a thiol-free chelating agent, bis(hydroxamamide) analog when the 99mTc complexation reaction was performed at room temperature. However, the disulfide bond of [125I]I-RC-160 was slightly cleaved in the presence of a small amount of stannous ions when the reaction was performed in boiling water. Treatment of [125I]I-RC-160 with dithionite in boiling water markedly reduced the disulfide bond of the parental peptide. These findings indicated that synthetic somatostatin analogs may be labeled with 99mTc with stannous ions as the reducing agent without impairing their structure after conjugation of thiol-free chelating agents that provide 99mTc chelates under mild reaction conditions.

To elucidate the cause for the different levels of hepatic radioactivity among mammals after injection of protein radiopharmaceuticals, the metabolism of radiolabeled proteins and the fate of their radiometabolites in the parenchymal cells of rat liver were investigated and compared with those of mice. We used galactosyl-neoglycoalbumin (NGA) as a carrier protein, and NGA was labeled with 111In via 1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) or 1-[p-(5-maleimidopentyl)aminobenzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) and with 125I via direct iodination. All radiolabeled NGAs exhibited rapid accumulation in liver parenchymal cells after intravenous injection into rats. Radioactivity was eliminated following NGA-125I injection at similar rates from rat and mouse liver. In contrast, both 111In-labeled NGAs demonstrated much slower elimination of radioactivity in rat when compared with mouse liver. Analyses of radioactivity in bile and liver indicated that both SCN-Bz-EDTA and EMCS-Bz-EDTA rendered mono-amino acid adducts as the final radiometabolites, which were generated in rat liver within 1 h postinjection. Subcellular distribution studies suggested that these radiometabolites were copurified with lysosome in rat liver. Because similar results were observed in mice previously, the difference between rats and mice in radioactivity elimination from liver parenchymal cells would be predominantly attributable to the different efflux rate of the 111In-labeled metabolites from the lysosome between these species. Such differences in the efflux rates of radiometabolites from the lysosome among mammals may also account for the different hepatic radioactivity levels of radiolabeled proteins between animal and clinical studies.

金沢大学新学術創成研究機構 / 金沢大学医薬保健研究域薬学系, Introduction: 68Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing 68Ga-labeled bone imaging agents for PET, in these initial studies 67Ga was used because of its longer half-life. Methods: DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). 67Ga-DOTA-Bn-SCN-HBP was prepared by coordination with 67Ga, and its in vitro and in vivo evaluations were performed. Results: 67Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. 67Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, 67Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of 67Ga-DOTA-Bn-SCN-HBP. Conclusions: 67Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with 68Ga. © 2010 Elsevier Inc. All rights reserved.

Introduction Based on the concept of bifunctional radiopharmaceuticals, we have previously developed 186 Re-complex-conjugated bisphosphonate analogs for palliation of painful bone metastases and have demonstrated the utility of these compounds. By applying a similar concept, we hypothesized that a bone-specific directed 90 Y-labeled radiopharmaceutical could be developed. Methods In this study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as the chelating site, and DOTA was conjugated with 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. [ 90 Y]DOTA-complex-conjugated bisphosphonate ([ 90 Y]DOTA-HBP) was prepared by coordination with 90 Y, and its biodistribution was studied in comparison to [ 90 Y]citrate. Results In biodistribution experiments, [ 90 Y]DOTA-HBP and [ 90 Y]citrate rapidly accumulated and resided in the bone. Although [ 90 Y]citrate showed a higher level of accumulation in the bone than [ 90 Y]DOTA-HBP, the clearances of [ 90 Y]DOTA-HBP from the blood and from almost all soft tissues were much faster than those of [ 90 Y]citrate. As a result, the estimated absorbed dose ratios of soft tissues to osteogenic cells (target organ) of [ 90 Y]DOTA-HBP were lower than those of [ 90 Y]citrate. Conclusions [ 90 Y]DOTA-HBP showed superior biodistribution characteristics as a bone-seeking agent and led to a decrease in the level of unnecessary radiation compared to [ 90 Y]citrate. Since the DOTA ligand forms a stable complex not only with 90 Y but also with lutetium ( 177 Lu), indium ( 111 In), gallium ( 67/68 Ga), gadolinium (Gd) and so on, complexes of DOTA-conjugated bisphosphonate with various metals could be useful as agents for palliation of metastatic bone pain, bone scintigraphy and magnetic resonance imaging.

Introduction The preferable pharmacokinetics of rhenium-186 ( 186 Re)-monoaminemonoamidedithiol-conjugated or 186 Re-mercaptoacetyltriglycine-conjugated bisphosphonates (BPs) suggested that the molecular design would be applicable to other radionuclides such as 68 Ga, 99m Tc, 153 Sm and 177 Lu. In this study, a key factor affecting the pharmacokinetics of a chelate-conjugated BP was investigated to estimate the validity and the applicability of molecular design. Methods Chemically inert and well-characterized tricarbonyl[ 186 Re][(cyclopentadienylcarbonyl amino)-acetic acid]rhenium ([ 186 Re]CpTR-Gly) was conjugated with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate and purified by high-performance liquid chromatography (HPLC) to prepare [ 186 Re](1-{3-[tricarbonyl(cyclopentadienylcarbonyl amino)-acetylamido]-1-hydroxy-1-phosphono-propyl}-phosphonic acid)rhenium ([ 186 Re]CpTR-Gly-APD). Plasma stability, plasma protein binding, hydroxyapatite (HA) binding and the pharmacokinetics of [ 186 Re]CpTR-Gly-APD were compared with those of 186 Re 1-hydroxyethylidene-1,1-diphosphonate (HEDP). The effect of HEDP coadministration and preadministration on the pharmacokinetics of [ 186 Re]CpTR-Gly-APD was also determined. Results The HPLC-purified [ 186 Re]CpTR-Gly-APD showed higher plasma stability, higher HA binding, higher bone accumulation and lower plasma protein binding than did 186 Re-HEDP. However, HA binding of [ 186 Re]CpTR-Gly-APD decreased to levels slightly higher than that of 186 Re-HEDP at similar HEDP concentrations. Bone accumulation of [ 186 Re]CpTR-Gly-APD also decreased to levels similar to that of 186 Re-HEDP when [ 186 Re]CpTR-Gly-APD was coinjected with HEDP equivalent to that in 186 Re-HEDP. In contrast, HEDP pretreatment did not impair bone accumulation of the two 186 Re-labeled compounds. However, a delay in blood clearance and an increase in renal radioactivity levels were observed particularly with 186 Re-HEDP. Conclusions Although 186 Re-HEDP possessed HA binding and bone accumulation similar to those of [ 186 Re]CpTR-Gly-APD, the specific activity of 186 Re-labeled BPs was found to play a crucial role in bone accumulation and blood clearance. Thus, the molecular design of chelate-conjugated BP would be useful for the development of bone-seeking radiopharmaceuticals with a variety of radionuclides by selecting chelating molecules that provide high specific activities.

To develop a radiopharmaceutical for the palliation of painful bone metastases based on the concept of bifunctional radiopharmaceuticals, we synthesized a bisphosphonate derivative labeled with rhenium-186 (186Re) that contains a hydroxyl group at the central carbon of its bisphosphonate structure, we attached a stable 186Re-MAMA chelate to the amino group of a 4-amino butylidene-bisphosphonate derivative [N-[2-[[4-[(4-hydroxy-4,4-diphosphonobutyl)amino]-4-oxobutyl]-2-thioethylamino]acetyl]-2-aminoethanethiolate] oxorhenium (V) (186Re-MAMA-HBP) and we investigated the effect of a hydroxyl group at the central carbon of its bisphosphonate structure on affinity for hydroxyapatite and on biodistribution by conducting a comparative study with [N-[2-[[3-(3,3-diphosphonopropylcarbamoyl)propyl]-2-thioethylamino]acetyl]-2-aminoethanethiolate] oxorhenium (V) (186Re-MAMA-BP). The precursor of 186Re-MAMA-HBP, trityl (Tr)-MAMA-HBP, was obtained by coupling a Tr-MAMA derivative to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. 186Re-MAMA-HBP was prepared by a reaction with 186ReO(4-) and SnCl2 in citrate buffer after the deprotection of the Tr groups of Tr-MAMA-HBP. After reversed-phase high-performance liquid chromatography, 186Re-MAMA-HBP had a radiochemical purity of over 95%. Compared with 186Re-MAMA-BP, 186Re-MAMA-HBP showed a greater affinity for hydroxyapatite beads in vitro and accumulated a significantly higher level in the femur in vivo. Thus, the introduction of a hydroxyl group into 186Re complex-conjugated bisphosphonates would be effective in enhancing accumulation in bones. These findings provide useful information on the design of bone-seeking therapeutic radiopharmaceuticals.

The effect of molecular charges on renal accumulation of 111In-DTPA-labeled low molecular weight (LMW) peptides was investigated using 111In-DTPA-octreotide derivatives as models to design radiolabeled peptides that are taken up less by renal cells. The N-terminal D-phenylalanine (Phe) of 111In-DTPA-D-Phe(1)-octreotide was replaced with L-aspartic acid (Asp), L-lysine (Lys), L-methionine (Met) or L-Phe. Cellulose acetate electrophoresis indicated that both 111In-DTPA-L-Phe(1)-octreotide and 111In-DTPA-L-Met(1)-octreotide showed similar net charges, whereas 111In-DTPA-L-alphaLys(1)-octreotide and 111In-DTPA-L-Asp(1)-octreotide had more positive and negative charges, respectively, at pH values similar to those in blood and glomerular filtrate. When injected into mice, significant differences were observed in the renal radioactivity levels. 111In-DTPA-L-alphaLys(1)-octreotide showed the highest radioactivity levels from 10 min to 6 h postinjection, whereas the lowest radioactivity levels were observed with 111In-DTPA-L-Asp(1)-octreotide at all the postinjection intervals. These findings indicated that the replacement of only one amino acid in 111In-DTPA-D-Phe(1)-octreotide significantly altered net molecular charges of the resulting peptides and that the net charges of the 111In-DTPA-octreotide derivatives significantly affected their renal uptake. Thus, an increase of negative charges in peptide molecules may constitute a strategy for designing 111In-DTPA-conjugated LMW peptides with low renal radioactivity levels.