OtherBASIC SCIENCE INVESTIGATIONS

Development of a Novel 99mTc-Chelate–Conjugated Bisphosphonate with High Affinity for Bone as a Bone Scintigraphic Agent

Kazuma Ogawa, Takahiro Mukai, Yasuyuki Inoue, Masahiro Ono and Hideo Saji
Journal of Nuclear Medicine December 2006, 47 (12) 2042-2047;

Abstract

In bone scintigraphy using 99mTc with methylenediphosphonate (99mTc-MDP) and hydroxymethylenediphosphonate (99mTc-HMDP), it takes 2−6 h after an injection before imaging can start. This interval could be shortened with a new radiopharmaceutical with higher affinity for bone. Here, based on the concept of bifunctional radiopharmaceuticals, we designed a 99mTc-mercaptoacetylglycylglycylglycine (MAG3)-conjugated hydroxy-bisphosphonate (HBP) (99mTc-MAG3-HBP) and a 99mTc-6-hydrazinopyridine-3-carboxylic acid (HYNIC)-conjugated hydroxy-bisphosphonate (99mTc-HYNIC-HBP). Methods: 99mTc-MAG3-HBP was prepared by complexation of MAG3-HBP with 99mTc using SnCl2 as a reductant. The precursor of 99mTc-HYNIC-HBP, HYNIC-HBP, was obtained by deprotection of the Boc group after the coupling of Boc-HYNIC to a bisphosphonate derivative. 99mTc-HYNIC-HBP was prepared by a 1-pot reaction of HYNIC-HBP with 99mTcO4−, tricine, and 3-acetylpyridine in the presence of SnCl2. Affinity for bone was evaluated in vitro by hydroxyapatite-binding assays for 99mTc-HMDP, 99mTc-MAG3-HBP, and 99mTc-HYNIC-HBP. Biodistribution experiments for the 3 99mTc-labeled compounds were performed on normal rats. Results: 99mTc-MAG3-HBP and 99mTc-HYNIC-HBP were each prepared with a radiochemical purity of >95%. In the in vitro binding assay, 99mTc-MAG3-HBP and 99mTc-HYNIC-HBP had greater affinity for hydroxyapatite than 99mTc-HMDP. In the biodistribution experiments, 99mTc-MAG3-HBP and 99mTc-HYNIC-HBP had higher levels of radioactivity in bone than 99mTc-HMDP. 99mTc-MAG3-HBP was cleared from the blood slower than 99mTc-HMDP, whereas there was no significant difference in clearance between 99mTc-HYNIC-HBP and 99mTc-HMDP. Consequently, 99mTc-HYNIC-HBP showed a higher bone-to-blood ratio than 99mTc-HMDP. Conclusion: We developed a novel 99mTc-chelate−conjugated bisphosphonate with high affinity for bone and rapid clearance from blood, based on the concept of bifunctional radiopharmaceuticals. The present findings indicate that 99mTc-HYNIC-HBP holds great potential for bone scintigraphy.

Over the last quarter of a century, complexes of 99mTc with methylenediphosphonate (99mTc-MDP) and hydroxymethylenediphosphonate (99mTc-HMDP) have been widely used as radiopharmaceuticals for bone scintigraphy in cases of metastatic bone disease, Paget's disease, fractures in osteoporosis, and so forth (1–4). With these 99mTc-labeled bisphosphonates, however, an interval of 2–6 h is needed between injection and bone imaging (3). Shorting this interval would lessen the burden on patients in terms of the total length of the examination and the dose of radiation absorbed. To enable imaging at an earlier time after injection, a radiopharmaceutical with higher affinity for bone is required.

Bisphosphonate analogs accumulate in bone because their phosphonate groups bind to the Ca2+ of hydroxyapatite crystals (5). In the case of 99mTc-MDP and 99mTc-HMDP, the phosphonate groups coordinate with technetium (6), which might decrease the inherent accumulation of MDP and HMDP in bone. Thus, we hypothesized that bone affinity of 99mTc-labeled bisphosphonate would be increased by the design of a bisphosphonate in which the phosphonate groups do not coordinate with technetium, and we attempted to design a 99mTc-chelate–conjugated bisphosphonate based on the concept of bifunctional radiopharmaceuticals (Fig. 1). In this study, mercaptoacetylglycylglycylglycine (MAG3) and 6-hydrazinopyridine-3-carboxylic acid (HYNIC) were chosen as chelating sites because they have been widely used for 99mTc labeling of proteins and peptides (7–11) and conjugated to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. Then, 99mTc-[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate (99mTc-MAG3-HBP; Fig. 1A) and [99mTc][[4-[(6-hydrazinopyridine-3-carbonyl)amino]-1-hydroxy-1-phosphonobutyl]phosphonic acid](tricine)(3-acetylpyridine) (99mTc-HYNIC-HBP; Fig. 1B) were prepared by coordination with 99mTc, and their properties in vitro and in vivo were compared with these of 99mTc-HMDP.

FIGURE 1. 
FIGURE 1. 

Chemical structures of 99mTc-MAG3-HBP (A) and 99mTc-HYNIC-HBP (B).

MATERIALS AND METHODS

Materials

Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AC-200 spectrometer (JEOL Ltd.), and the chemical shifts were reported in parts per million downfield from an internal 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt standard. Electrospray ionization mass spectra (ESI-MS) were obtained with a LCMS-QP8000α (Shimadzu). Thin-layer chromatographic analyses were performed with silica plates (Silica gel 60; Merck KGaA) with acetone as a developing solvent. 99mTc-Pertechnetate (99mTcO4−) was eluted in a saline solution on a daily basis from generators (Daiichi Radioisotope Laboratories, Ltd.). 99mTc-HMDP was prepared by reconstitution with a conventional HMDP labeling kit (Nihon Medi-Physics Co., Ltd.) with a 99mTcO4− solution. Other reagents were of reagent grade and were used as received.

Preparation of 99mTc-MAG3-HBP

The precursor of 99mTc-MAG3-HBP, [1-hydroxy-1-phosphono-4-[2-[2-[2-(2-tritylmercaptoacetylamino)-acetylamino]acetylamino]acetylamino]butyl]phosphonic acid (Tr-MAG3-HBP) was synthesized according to procedures described previously (12). The trityl group of Tr-MAG3-HBP (0.1 mg) was dissolved in 190 μL of trifluoroacetic acid (TFA) and 10 μL of triethylsilane. After removal of the solvent under a stream of N2, 80 μL of 0.1 mol/L borate buffer (pH 9.5) were added to the residue. Next, 3 μL of SnCl2·2H2O solution in 0.1 mol/L citrate buffer (pH 5.0) (1 mg/mL) and 200 μL of 99mTcO4− solution were added, and the reaction mixture was vigorously stirred and allowed to react at 95°C for 1 h. 99mTc-MAG3-HBP was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) performed with a Cosmosil 5C18-AR-300 column (4.6 × 150 mm; Nacalai Tesque) at a flow rate of 1 mL/min with a mixture of 0.2 mol/L phosphate buffer (pH 6.0) and ethanol (90:10) containing 10 mmol/L tetrabutylammonium hydroxide.

Preparation of [[[[(4-Hydroxy-4,4-Diphosphonobutyl)Carbamoylmethyl]Carbamoylmethyl]Carbamoylmethyl]Carbamoylmethanethiolate] Oxorhenium(V) (Re-MAG3-HBP)

Re-MAG3-HBP was synthesized according to procedures described previously (12). ESI-MS calculated for C12H20N4O12P2187ReS (M- H)−: m/z 692. Found: 692 C12H20N4O12P2185ReS (M- H)−: m/z 690. Found: 690.

Preparation of 99mTc-HYNIC-HBP

2,3,5,6-Tetrafluorophenyl 6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carboxylate (Boc-HYNIC-TFP) and 4-amino-1-hydroxybutylidene-1,1-bisphosphonate were synthesized according to procedures described preciously (12,13). 4-Amino-1-hydroxybutylidene-1,1-bisphosphonate (10.3 mg, 41.3 μmol) was suspended in 1.33 mL of distilled water, and triethylamine (25.1 mg, 248 μmol) was added to the suspension. After a few seconds of stirring at room temperature, Boc-HYNIC-TFP (17.8 mg, 44.4 μmol) dissolved in 1.33 mL of acetonitrile was added. The reaction mixture was stirred for 3 h at room temperature. RP-HPLC was performed with a Hydrosphere 5C18 column (20 × 150 mm; YMC Co., Ltd) at a flow rate of 16 mL/min with a mixture of water, acetonitrile, and formic acid (90:10:1). Chromatograms were obtained by monitoring the ultraviolet (UV) adsorption at a wavelength of 254 nm. The fraction containing [4-[[6-(tert-butoxycarbonyl)-hydrazinopyridine-3-carbonyl]amino]-1-hydroxy-1-phosphonobutyl]phosphonic acid (Boc-HYNIC-HBP) was identified by mass spectrometry and collected. The solvent was removed by lyophilization to provide Boc-HYNIC-HBP (178 mg, 36.7%) as white crystals. 1H NMR (D2O): δ 8.42 (1H, s), 8.33 (1H, d), 7.21 (1H, d), 3.43 (2H, t), 1.92−2.04 (4H, m), 1.47 (9H, s). ESI-MS calculated for C15H26N4O10P2 (M- H)−: m/z 483. Found: 483.

Boc-HYNIC-HBP (10.4 mg, 21.5 μmol) was stirred in a mixed solution of TFA (720 μL) and anisole (80 μL) for 10 min at room temperature. After removal of the solvent under a stream of N2, the residue was washed with dry ether to produce [4-[(6-hydrazinopyridine-3-carbonyl)amino]-1-hydroxy-1-phosphonobutyl]phosphonic acid (HYNIC-HBP) quantitatively as white crystals. 1H NMR (D2O): δ 8.33 (1H, s), 8.13 (1H, d), 7.01 (1H, d), 3.42 (2H, t), 1.94−2.04 (4H, m). ESI-MS calculated for C10H18N4O8P2 (M- H)−: m/z 383. Found: 383.

Forty microliters of HYNIC-HBP solution (3.75 mg/mL in 0.1 mol/L borate buffer, pH 9.5) were mixed successively with 200 μL of tricine solution (30 mg/mL in 10 mmol/L citrate buffer, pH 5.2), 200 μL of 3-acetylpyridine solution (10 μL/mL in 10 mmol/L citrate buffer, pH 5.2), 200 μL of 99mTcO4− solution, and 25 μL of SnCl2 solution (1.0 mg/mL in 0.1N HCl). The reaction mixture was vigorously stirred and allowed to react at 95°C for 35 min. 99mTc-HYNIC-HBP was purified by RP-HPLC under the same conditions as for the preparation of 99mTc-MAG3-HBP.

Hydroxyapatite-Binding Assay

The hydroxyapatite-binding assay was performed according to procedures described preciously with a slight modification (14–16). In brief, hydroxyapatite beads (Bio-Gel; Bio-Rad) were suspended in Tris/HCl-buffered saline (50 mmol/L, pH 7.4) at 1, 2.5, and 10 mg/mL. For the solutions of 99mTc-labeled compounds (99mTc-MAG3-HBP, 99mTc-HYNIC-HBP, and 99mTc-HMDP), the bisphosphonate concentrations were adjusted to 0.60 μmol/L. One hundred microliters of each solution of 99mTc-labeled compound were added to 100 μL of the hydroxyapatite suspension, and samples were gently shaken for 1 h at room temperature because it has been reported that 1 h was enough to attain binding equilibrium (14). After centrifugation at 10,000g for 5 min, the radioactivity of the supernatant was measured with an auto well γ-counter (ARC-2000; Aloka). Control experiments were performed with a similar procedure in the absence of hydroxyapatite beads. The rate of binding was determined as follows:Math

Biodistribution Experiments

Animal experiments were conducted in accordance with our institutional guidelines, and the experimental procedures were approved by the Kyoto University Animal Care Committee. Biodistribution experiments were performed with an intravenous administration of 250 μL of each diluted tracer solution (370–740 kBq) to male Wistar rats (200–230 g). Groups of at least 4 rats each were sacrificed by decapitation at 5, 10, 30, and 60 min after injection. Organs of interest were removed and weighed, and radioactivity counts were determined with an auto well γ-counter and corrected for background radiation and physical decay during counting.

Serum Protein-Binding Assay

The binding of 99mTc-labeled compounds to serum protein was evaluated by ultrafiltration. Male Wistar rats (200–230 g) received a bolus of 99mTc-labeled compound by intravenous injection. At 3 min after the injection, the rats were anesthetized with ether and blood was collected by heart puncture. Each serum sample was prepared and applied to Centrifree units (Millipore). The units were centrifuged at 1,000g at room temperature. The radioactivity counts of the initial samples and filtrates were determined with an auto well γ-counter.

Statistical Analysis

Data are expressed as means ± SD where appropriate. Results of biodistribution experiments were statistically analyzed using a 1-way ANOVA followed by the Dunnett post hoc test. Differences were considered statistically significant when P values were less than 0.05.

RESULTS

Preparation of 99mTc-Labeled Compounds

99mTc-MAG3-HBP was prepared by complexation with 99mTc using SnCl2 as a reductant. The radiochemical yield of 99mTc-MAG3-HBP was 73%. After purification by RP-HPLC, 99mTc-MAG3-HBP had a radiochemical purity of >95%.

HYNIC-HBP, the precursor of 99mTc-HYNIC-HBP, was synthesized by the coupling of the carboxyl group of Boc-HYNIC with the amino group of the bisphosphonate derivative and the subsequent deprotection of the Boc group. 99mTc-HYNIC-HBP was prepared by a 1-pot reaction of HYNIC-HBP with 99mTcO4−, tricine, and 3-acetylpyridine in the presence of SnCl2. The radiochemical yield of 99mTc-HYNIC-HBP was 39%. After purification by RP-HPLC, 99mTc-HYNIC-HBP had a radiochemical purity of >95%.

Hydroxyapatite-Binding Assay

Figure 2 shows the percentage of each 99mTc-labeled compound bound to hydroxyapatite beads. With an increase in the amount of hydroxyapatite, the rate of binding rose. Both 99mTc-chelate−comjugated bisphosphonates (99mTc-MAG3-HBP and 99mTc-HYNIC-HBP) showed a significantly higher rate of binding than 99mTc-HMDP.

FIGURE 2. 
FIGURE 2. 

Binding of hydroxyapatite to 99mTc-MAG3-HBP, 99mTc-HYNIC-HBP, and 99mTc-HMDP. Data are expressed as mean ± SD for 3 or 4 experiments. Asterisks indicate statistically significant differences compared with 99mTc-HMDP with the Dunnett test (P < 0.05).

Biodistribution Experiments

The biodistributions of 99mTc-MAG3-HBP, 99mTc-HYNIC-HBP, and 99mTc-HMDP in normal rats are presented in Tables 1, 2, and 3. All 99mTc-labeled compounds accumulated rapidly and resided a long time in the femur. 99mTc-Chelate−conjugated bisphosphonates, 99mTc-MAG3-HBP and 99mTc-HYNIC-HBP, accumulated in bone in significantly larger amounts than 99mTc-HMDP. However, the bone-to-blood ratio of 99mTc-MAG3-HBP was decreased because its clearance from blood was delayed compared with that of 99mTc-HMDP. 99mTc-HYNIC-HBP had a significantly higher bone-to-blood ratio than 99mTc-HMDP because its clearance was equivalent to that of 99mTc-HMDP.

View this table:
TABLE 1

Biodistribution of Radioactivity After Intravenous Administration of 99mTc-MAG3-HBP in Rats

View this table:
TABLE 2

Biodistribution of Radioactivity After Intravenous Administration of 99mTc-HYNIC-HBP in Rats

View this table:
TABLE 3

Biodistribution of Radioactivity After Intravenous Administration of 99mTc-HMDP in Rats

Serum Protein-Binding Assay

The proportions of 99mTc-MAG3-HBP and 99mTc-HYNIC-HBP bound to serum protein were 97.7% ± 0.2% and 88.7% ± 2.7%, respectively.

DISCUSSION

Radiopharmaceuticals with greater affinity for bone are expected to be effective in shortening the time interval between injection and bone imaging. Thus, based on the concept of bifunctional radiopharmaceuticals, 99mTc-chelate–conjugated bisphosphonates were designed in this study. First, MAG3 was chosen as a 99mTc-chelating group because this N3S ligand forms a relatively compact, hydrophilic, and stable complex with 99mTc in high yields (17). As expected, 99mTc-MAG3-HBP bound to the hydroxyapatite beads in vitro and accumulated in the rat femur in vivo to a greater extent than 99mTc-HMDP. However, the level of 99mTc-MAG3-HBP in blood was higher than that of 99mTc-HMDP, which resulted in a decreased bone-to-blood ratio. We attribute this high radioactivity of 99mTc-MAG3-HBP in blood to the binding of serum proteins because the 99mTc-MAG3 complex shows strong binding to serum proteins (18,19). In fact, the level of binding of 99mTc-MAG3-HBP to protein was very high.

HYNIC is a representative bifunctional chelating agent used to prepare 99mTc-labeled proteins and peptides with tricine as a coligand (10,20–22). However, it has been reported that the complex [99mTc](HYNIC)(tricine)2 is not stable and exists in multiple forms, and the pharmacokinetics could be affected by the exchange reaction between tricine and protein in the plasma and tissues (22–24). To solve these problems, Liu et al. used several pyridine derivatives as coligands to form ternary ligand complexes, [99mTc](HYNIC)(tricine)(pyridine derivative), with greater stability and fewer isomers (25). Furthermore, in a previous study, we showed that little binding to plasma protein was displayed by 99mTc-HYNIC-labeled polypeptides derivatized with this ternary ligand complex (13). Then, 99mTc-HYNIC-HBP in which [99mTc](HYNIC)(tricine)(3-acetylpyridine) was conjugated with a bisphosphonate derivative, was designed for high bone affinity and rapid washout from the blood.

The rate of binding of 99mTc-HYNIC-HBP to serum protein was significantly lower than that of 99mTc-MAG3-HBP. When these values were expressed as a nonprotein−binding rate, the value of 99mTc-HYNIC-HBP (11.3%) is about 5 times the value of 99mTc-MAG3-HBP (2.3%). The difference in the binding of the compound to the serum protein affects its blood clearance (26–28). In fact, the blood clearance of 99mTc-HYNIC-HBP was significantly faster than that of 99mTc-MAG3-HBP and was equivalent to that of 99mTc-HMDP, although factors other than protein binding may also be responsible for rapid blood clearance. Furthermore, 99mTc-HYNIC-HBP accumulated in larger amounts in femur than did 99mTc-HMDP, which could be attributable to its design based on the bifunctional radiopharmaceutical concept. Consequently, the bone-to-blood ratio of 99mTc-HYNIC-HBP was significantly higher than that of 99mTc-HMDP.

We assume that 99mTc-chelate−conjugated bisphosphonates in bone remain intact because their 99mTc complexes are stable. On the other hand, previous studies suggested that there was a separation of the 99mTc-bisphosphonate complex into its 99mTc and bisphosphonate components before incorporation into the mineral phase (29,30). However, the separation of 99mTc-bisphosphonate must occur at the bone site because reduced 99mTc alone was not taken up by bone (30). Namely, there is little difference from the point of view that bisphosphonate is used as a carrier to bone in both cases. Thus, we hypothesized that the affinity of bisphosphonate for hydroxyapatite is important for bone accumulation and it would be increased by the design of a bisphosphonate in which the phosphonate groups do not coordinate with technetium. In fact, 99mTc-chelate–conjugated bisphosphonates had better affinity for hydroxyapatite and accumulated in the rat femur in vivo to greater extent than 99mTc-HMDP. These results support our hypothesis. However, other factors such as the blood circulation and delivery of the agents may also be important. Further study may be required to elucidate the mechanism for bone localization.

Because MAG3-HBP and HYNIC-HBP contain a bisphosphonate site, there is the possibility that 99mTc coordinates not with the MAG3 moiety or the HYNIC moiety but rather with the bisphosphonate moiety. To ascertain that 99mTc is chelated with only the MAG3 moiety, Re-MAG3-HBP was prepared by the coupling of nonradioactive Re-MAG3 complexed previously with the bisphosphonate analog. By RP-HPLC analysis, identical retention times between 99mTc-MAG3-HBP and Re-MAG3-HBP were exhibited (Fig. 3), revealing their structural analogy. In the case of the HYNIC complex, because it is difficult to synthesize the corresponding stable Re-HYNIC complex, 99mTc-HYNIC-HBP was prepared by the coupling of [99mTc](HYNIC-TFP)(tricine)(3-acetylpyridine) with the bisphosphonate derivative. RP-HPLC analysis revealed this 99mTc-labeled product to be identical to that obtained from the reaction of HYNIC-HBP with 99mTcO4−, tricine, and 3-acetylpyridine in the presence of SnCl2 (Fig. 4). These findings exclude the possibility of complexation between technetium and the bisphosphonate structure and indicate the chelation of 99mTc with only the MAG3 moiety in MAG3-HBP and the HYNIC moiety in HYNIC-HBP.

FIGURE 3. 
FIGURE 3. 

RP-HPLC chromatograms of nonradioactive Re-MAG3-HBP (A) and 99mTc-MAG3-HBP (B). Conditions: flow rate of 1 mL/min with 10% ethanol in 200 mmol/L phosphate buffer (pH 6.0) containing 10 mmol/L tetrabutylammoniumhydroxide.

FIGURE 4. 
FIGURE 4. 

RP-HPLC chromatograms of 99mTc-HYNIC-HBP prepared by coupling of [99mTc](HYNIC-TFP)(tricine)(3-acetylpyridine) with bisphosphonate derivative (A) and labeling of HYNIC-HBP with 99mTcO4−, tricine, and 3-acetylpyridine (B). Conditions: flow rate of 1 mL/min with 10% ethanol in 200 mmol/L phosphate buffer (pH 6.0) containing 10 mmol/L tetrabutylammoniumhydroxide.

CONCLUSION

As a radiopharmaceutical that accumulates at high level in bone and is rapidly cleared from blood, we designed a novel 99mTc-chelate−conjugated bisphosphonate, 99mTc-HYNIC-HBP. 99mTc-HYNIC-HBP had good affinity for hydroxyapatite crystals and showed lower binding to serum proteins. In rat biodistribution experiments, 99mTc-HYNIC-HBP had a higher bone-to-blood accumulation ratio of radioactivity at early times after injection than 99mTc-HMDP. These results indicate that 99mTc-HYNIC-HBP holds great potential for bone scintigraphy.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a research grant from the Sagawa Foundation for Promotion of Cancer Research.

Footnotes

  • COPYRIGHT © 2006 by the Society of Nuclear Medicine, Inc.

References

  1. 1.↵
    Subramanian G, McAfee JG, Blair RJ, Kallfelz FA, Thomas FD. Technetium-99m-methylene diphosphonate: a superior agent for skeletal imaging—comparison with other technetium complexes. J Nucl Med. 1975;16:744–755.
    OpenUrlAbstract/FREE Full Text
  2. 2.
    Domstad PA, Coupal JJ, Kim EE, Blake JS, DeLand FH. 99mTc-Hydroxymethane diphosphonate: a new bone imaging agent with a low tin content. Radiology. 1980;136:209–211.
    OpenUrlPubMed
  3. 3.↵
    Love C, Din AS, Tomas MB, Kalapparambath TP, Palestro CJ. Radionuclide bone imaging: an illustrative review. Radiographics. 2003;23:341–358.
    OpenUrlCrossRefPubMed
  4. 4.↵
    Mari C, Catafau A, Carrio I. Bone scintigraphy and metabolic disorders. Q J Nucl Med. 1999;43:259–267.
    OpenUrlPubMed
  5. 5.↵
    Meyer JL, Nancollas GH. The influence of multidentate organic phosphonates on the crystal growth of hydroxyapatite. Calcif Tissue Res. 1973;13:295–303.
    OpenUrlCrossRefPubMed
  6. 6.↵
    Libson K, Deutsch E, Barnett BL. Structural characterization of a technetium-99-diphosphonate complex: implications for the chemistry of technetium-99m skeletal imaging agents. J Am Chem Soc. 1980;102:2476–2478.
    OpenUrl
  7. 7.↵
    Hnatowich DJ, Qu T, Chang F, Ley AC, Ladner RC, Rusckowski M. Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine. J Nucl Med. 1998;39:56–64.
    OpenUrlAbstract/FREE Full Text
  8. 8.
    Liu S, Edwards DS, Barrett JA. 99mTc labeling of highly potent small peptides. Bioconjug Chem. 1997;8:621–636.
    OpenUrlCrossRefPubMed
  9. 9.
    Abrams MJ, Juweid M, tenKate CI, et al. Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats. J Nucl Med. 1990;31:2022–2028.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    Ohtsuki K, Akashi K, Aoka Y, et al. Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med. 1999;26:1251–1258.
    OpenUrlCrossRefPubMed
  11. 11.↵
    Steffens MG, Oosterwijk E, Kranenborg MH, et al. In vivo and in vitro characterizations of three 99mTc-labeled monoclonal antibody G250 preparations. J Nucl Med. 1999;40:829–836.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    Ogawa K, Mukai T, Arano Y, et al. Development of a rhenium-186-labeled MAG3-conjugated bisphosphonate for the palliation of metastatic bone pain based on the concept of bifunctional radiopharmaceuticals. Bioconjug Chem. 2005;16:751–757.
    OpenUrlPubMed
  13. 13.↵
    Ono M, Arano Y, Mukai T, et al. 99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding. Nucl Med Biol. 2001;28:215–224.
    OpenUrlPubMed
  14. 14.↵
    Fujisawa R, Kuboki Y. Preferential adsorption of dentin and bone acidic proteins on the (100) face of hydroxyapatite crystals. Biochim Biophys Acta. 1991;1075:56–60.
    OpenUrlPubMed
  15. 15.
    Kasugai S, Fujisawa R, Waki Y, Miyamoto K, Ohya K. Selective drug delivery system to bone: small peptide (Asp)6 conjugation. J Bone Miner Res. 2000;15:936–943.
    OpenUrlCrossRefPubMed
  16. 16.↵
    Ogawa K, Mukai T, Arano Y, et al. Rhemium-186-monoaminemonoamidedithiol-conjugated bisphosphonate derivatives for bone pain palliation. Nucl Med Biol. 2006;33:513–520.
    OpenUrlPubMed
  17. 17.↵
    Fritzberg AR, Kasina S, Eshima D, Johnson DL. Synthesis and biological evaluation of technetium-99m MAG3 as a hippuran replacement. J Nucl Med. 1986;27:111–116.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    Verbruggen AM, Nosco DL, Van Nerom CG, Bormans GM, Adriaens PJ, De Roo MJ. Technetium-99m-L,L-ethylenedicysteine: a renal imaging agent. I. Labeling and evaluation in animals. J Nucl Med. 1992;33:551–557.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Taylor A Jr, Eshima D, Christian PE, Wooten WW, Hansen L, McElvany K. Technetium-99m MAG3 kit formulation: preliminary results in normal volunteers and patients with renal failure. J Nucl Med. 1988;29:616–622.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Verbeke K, Hjelstuen O, Debrock E, Cleynhens B, De Roo M, Verbruggen A. Comparative evaluation of 99Tcm-Hynic-HSA and 99Tcm-MAG3-HSA as possible blood pool agents. Nucl Med Commun. 1995;16:942–957.
    OpenUrlPubMed
  21. 21.
    Van der Laken CJ, Boerman OC, Oyen WJG, et al. Technetium-99m-labeled chemotactic peptides in acute infection and sterile inflammation. J Nucl Med. 1997;38:1310–1315.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Liu S, Edwards DS, Looby RJ, et al. Labeling a hydrazino nicotinamide-modified cyclic IIb/IIIa receptor antagonist with 99mTc using aminocarboxylates as coligands. Bioconjug Chem. 1996;7:63–71.
    OpenUrlCrossRefPubMed
  23. 23.
    Ono M, Arano Y, Uehara T, et al. Intracellular metabolic fate of radioactivity after injection of technetium-99m-labeled hydrazino nicotinamide derivatized proteins. Bioconjug Chem. 1999;10:386–394.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Ono M, Arano Y, Mukai T, et al. Plasma protein binding of 99mTc-labeled hydrazino nicotinamide derivatized polypeptides and peptides. Nucl Med Biol. 2001;28:155–164.
    OpenUrlCrossRefPubMed
  25. 25.↵
    Liu S, Edwards DS, Harris AR. A novel ternary ligand system for 99mTc-labeling of hydrazino nicotinamide-modified biologically active molecules using imine-N-containing heterocycles as coligands. Bioconjug Chem. 1998;9:583–595.
    OpenUrlCrossRefPubMed
  26. 26.↵
    Vallner JJ. Binding of drugs by albumin and plasma protein. J Pharm Sci. 1977;66:447–465.
    OpenUrlCrossRefPubMed
  27. 27.
    Daneshmend TK, Warnock DW. Clinical pharmacokinetics of systemic antifungal drugs. Clin Pharmacokinet. 1983;8:17–42.
    OpenUrlCrossRefPubMed
  28. 28.↵
    Ogiso T, Iwaki M, Konishi Y. Effect of furosemide on plasma clearance, anticoagulant effect and protein binding of warfarin in rats. J Pharmacobiodyn. 1982;5:829–840.
    OpenUrlPubMed
  29. 29.↵
    van Langevelde A, Driessen OMJ, Pauwels EKJ, Thesingh CW. Aspects of 99mtechnetium binding from an ethane-1-hydroxy-1,1-diphosphonate 99mTc complex to bone. Eur J Nucl Med. 1977;2:47–51.
    OpenUrlPubMed
  30. 30.↵
    Schwartz Z, Shani J, Soskolne WA, Touma H, Amir D, Sela J. Uptake and biodistribution of technetium-99m-MD32P during rat tibial bone repair. J Nucl Med. 1993;34:104–108.
    OpenUrlAbstract/FREE Full Text
  • Received for publication May 9, 2006.
  • Accepted for publication September 6, 2006.
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Bookmark this article

Jump to section