Nhat Le

Multimodal Nanoprobes for Radionuclide and Five-Color Near-Infrared Optical Lymphatic Imaging

Current contrast agents generally have one function and can only be imaged in monochrome; therefore, the majority of imaging methods can only impart uniparametric information. A single nanoparticle has the potential to be loaded with multiple payloads. Such multimodality probes have the ability to be imaged by more than one imaging technique, which could compensate for the weakness or even combine the advantages of each individual modality. Furthermore, optical imaging using different optical probes enables us to achieve multicolor in vivo imaging, wherein multiple parameters can be read from a single image. To allow differentiation of multiple optical signals in vivo, each probe should have a close but different near-infrared emission. To this end, we synthesized nanoprobes with multimodal and multicolor potential, which employed a polyamidoamine dendrimer platform linked to both radionuclides and optical probes, permitting dual-modality scintigraphic and five-color near-infrared optical lymphatic imaging using a multiple-excitation spectrally resolved fluorescence imaging technique.

Pulsed High-Intensity Focused Ultrasound Enhances Uptake of Radiolabeled Monoclonal Antibody to Human Epidermoid Tumor in Nude Mice

The aim of this study was to determine if pulsed high-intensity focused ultrasound (HIFU) exposures could enhance tumor uptake of 111In-MX-B3, a murine IgG1κ monoclonal antibody directed against the Ley antigen. Methods: MX-B3 was labeled with 111In, purified, and confirmed for its binding to the antigen-positive A431 cell line. Groups of nude mice were inoculated subcutaneously with A431 tumor cells on both hind flanks. A tumor on one flank was treated with pulsed-HIFU; the other tumor was used as an untreated control. Within 10 min after the HIFU exposure, the mice received intravenous 111In-MX-B3 for imaging and biodistribution studies. Mice were euthanized at 1, 24, 48, and 120 h after injection for biodistribution studies. Results: The HIFU exposure shortened the peak tumor uptake time (24 vs. 48 h for the control) and increased the peak tumor uptake value (38 vs. 25 %ID/g [percentage injected dose per gram] for the control). The HIFU effect on enhancing tumor uptake was greater at earlier times up to 24 h, but the effect was gradually diminished thereafter. The HIFU effect on enhancing tumor uptake was substantiated by nuclear imaging studies. HIFU also increased the uptake of the antibody in surrounding tissues, but the net increase was marginal compared with the increase in tumor uptake. Conclusion: This study demonstrates that pulsed-HIFU significantly enhances the delivery of 111In-MX-B3 in human epidermoid tumors xenografted in nude mice. The results of this pilot study warrant further evaluation of other treatment regimens, such as repeated HIFU exposures for greater delivery enhancement of antibodies labeled with cytotoxic radioisotopes or pulsed-HIFU exposure in addition to a combined therapy of 90Y-B3 and taxol to enhance the synergistic effect.

Synergistic Antitumor Activity of Taxol and Immunotoxin SS1P in Tumor-Bearing Mice

Purpose: To investigate the combined antitumor activity in mice of immunotoxin SS1P and Taxol. Methods: Immunodeficient mice were implanted with A431/K5 tumors expressing mesothelin. Established tumors were treated i.v. with immunotoxin SS1P alone, i.p. with Taxol alone, or with the two agents together. SS1P was radiolabeled with 111In and used to study the effect of Taxol on its uptake by A431/K5 tumors. Results: Using doses at which either agent alone caused stabilization of tumor growth, the combination was synergistic causing long-lasting complete remissions in many animals. In contrast, synergy was not observed when the same cells were treated with these agents in vitro. Tumor uptake of 111In-SS1P was not affected by treatment with Taxol. Conclusion: The combination of Taxol and SS1P exerts a synergistic antitumor effect in animals but not in cell culture. This effect is not secondary to increased tumor uptake of the immunotoxin. Synergy could be due to improved immunotoxin distribution within the tumor or could involve factors released by other cell types in the tumors.

Lowering of pI by acylation improves the renal uptake of 99mTc-labeled anti-Tac dsFv: effect of different acylating reagents

Anti-Tac disulfide-stabilized variable region fragment (dsFv) was labeled with 99mTc by a preformed chelate approach using 99mTc-MAG3-trifluorophenyl (TFP) ester. Simultaneously it was acylated with TFP-lactate or succinic anhydride to decrease the isoelectric point of dsFv (pI 10). Acylation of dsFv (0.04 mM) with the lactate at a 73 times molar excess reduced the pI to 5.0-6.7, whereas acylation with succinic anhydride at a 30 times molar excess reduced the pI to 4.9-8.7. Comparative biodistribution studies performed in mice (n = 5) showed the reduced renal accumulation of the 99mTc proportional to the pI reduction. The effect of the pI on the reduced renal uptake was especially pronounced at 15 min postinjection. The reduced renal uptake was also reflected in the reduced whole-body retention, indicating that lowering the pI inhibited the tubular reabsorption of the labeled dsFv.

Similarities in the Biodistribution of Iodine-Labeled Anti-Tac Single-Chain Disulfide-Stabilized Fv Fragment and Anti-Tac Disulfide-Stabilized Fv Fragment

We evaluated the biodistribution and pharmacokinetics of two different iodine-labeled Fv fragments of anti-Tac monoclonal antibody (MAb) in normal and tumor-bearing nude mice. One was a disulfide-stabilized Fv fragment (dsFv), and the other was a single-chain disulfide-stabilized Fv fragment (scdsFv). The scdsFv is a newly developed type of Fv fragment superior to the dsFv in which the VH and VL are linked by covalent bonds through a spacer arm and by an internal disulfide bond. These modifications increase the yield of scdsFv. Both reagents recognize the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The biodistribution of the Fv fragments was evaluated in normal mice co-injected with 50 mg of L-lysine and in a no-lysine control group. Biodistribution was also evaluated in nude mice bearing subcutaneous tumor xenografts derived from IL-2Ralpha-positive ATAC4 cells and receptor-negative A431 cells. These mice were co-injected with 125I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 131I-labeled anti-Tac dsFv (2 microCi/0.7 microg) or with 131I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 125I-labeled anti-Tac dsFv (4 microCi/0.7 microg). The biodistribution of 125I-labeled anti-Tac scdsFv and 131I-labeled anti-Tac dsFv was very similar in all organs and the tumors. The renal uptake of both reagents was blocked effectively (<93%) and similarly by lysine. The scdsFv cleared slightly faster from the circulation than did the dsFv because there were more aggregates of dsFv than of scdsFv (3% vs. 1%, respectively). The scdsFv-to-dsFv ratio ranged from 0.79 to 1.20 in all organs at all time points we examined. In conclusion, the first biodistribution study of an scdsFv molecule shows that the scdsFv had a biodistribution very similar to that of the dsFv and seems to be a good alternative to the dsFv because of its higher production yield.

An improved synthesis of [125I]N-(diethylaminoethyl)-4-iodobenzamide: a potential ligand for imaging malignant melanoma.

To improve the radiolabeling yield and the specific activity of [125I]N-(2-diethylaminoethyl)-4-iodobenzamide (DAB), the aryltributyltin precursor was synthesized from the N-(2-diethylaminoethyl)-4-bromobenzamide derivative by palladium catalyzed stannylation using bis(tributyltin). The radiolabeled product, [125I]DAB, was obtained by an iododestannylation reaction in high radiochemical yields (85-94%, radiochemical purity, > 98%) using chloramine-T as an oxidizing agent. The specific activity was greater than 1600 Ci/mmol. The biodistribution studies in nude mice implanted with human malignant melanoma xenograft showed a good tumor uptake (6.14% ID/g at 1 h, 2.81% ID/g at 6 h and 0.42% ID/g at 24 h) of [125I]DAB. Unfortunately, a high uptake in the non-target organs, such as liver and lung, was found. At 1 h post-injection the activity level in liver and lung was 11.76 and 7.58% ID/g, respectively. A slow clearance of activity from liver and lung was observed at 6 h (3.43 and 0.49% ID/g). These results demonstrate that iodinated IDAB is a potential radiopharmaceutical for the management of patients with malignant melanoma.

Evaluation of 99mTc-Mercaptoacetyltriglycine-Biocytin as a new hepatobiliary imaging agent in mice coinjected with bilirubin

We evaluated 99mTc-labeled mercaptoacetyltriglycine (99mTc-MAG3)-biocytin as a hepatobiliary imaging agent in the absence and presence of bilirubin in mice. We then compared its pharmacokinetic parameters; peak liver/heart activity ratio (rmax) and half clearance time (HCT) with those of 99mTc-labeled diisopropyl-iminodiacetic acid (99mTc-disofenin). Balb/c mice were injected intravenously with hepatobiliary agent (99mTc-MAG3-biocytin or 99mTc-disofenin) alone or in combination with bilirubin at two doses (7 and 14 mg/kg) dissolved in 5% human serum albumin. Images were acquired every 15 s for 30 min with a gamma-camera equipped with a pinhole collimator. Dynamic images showed rapid hepatic uptake of 99mTc-MAG3-biocytin, with rapid clearance from the blood and rapid excretion via the biliary system. Its hepatic uptake was not affected by bilirubin coinjection, whereas 99mTc-disofenin coinjected with bilirubin showed a higher blood background than 99mTc-disofenin alone. These qualitative findings were reflected in pharmacokinetic parameters, rmax and HCT. The rmax was obtained from plots of time versus liver/heart activity ratios obtained in equal-area regions of interest over the heart and liver. The HCT was calculated from the hepatic clearance curve from plots of time versus liver activity. 99mTc-MAG3-biocytin without bilirubin coinjection showed an rmax of 8.9+/-1.3 and an HCT of 399+/-36 s. These values did not change even when 14 mg/kg of bilirubin were coinjected. By contrast, the parameters for 99mTc-disofenin with bilirubin were significantly (p < 0.01) affected by 14 mg/kg of bilirubin coinjection: rmax was decreased from 7.9+/-2.5 to 1.4+/-0.2 and HCT was increased from 292+/-32 s to 782+/-133 s. 99mTc-MAG3-biocytin hepatobiliary scintigraphy in mice is not affected by bilirubin coinjection, and this hepatobiliary agent appears to offer promise for estimating hepatic function in patients with high bilirubin levels.

Epitope Blocking: Positive and Negative Effects on the Biodistribution of 125I‐Labeled Anti‐Tac Disulfide‐stabilized Fv Fragment of Two Antibodies against Different Epitopes of the Circulating Antigen

Prior in vivo studies using the 125I‐labeled anti‐Tac disulfide‐stabilized variable region fragment (125I‐anti‐Tac dsFv) of monoclonal antibody in the presence of the circulating soluble alpha subunit of the interleukin‐2 receptor (sIL‐2Rα) have shown formation of complexes which interfere with biodistribution. In this study we evaluated the effects of preinjecting HuTac and 7G7/B6, two immunoglobulin Gs (IgGs) that recognize different epitopes of sIL‐2Rα, on the biodistribution of 125I‐anti‐Tac dsFv in mice bearing SP2/Tac tumor xenografts, which produce sIL‐2Rα, or on nude mice injected with 500 ng of sIL‐2Rα. We also evaluated the biodistribution in mice of 125I‐labeled sIL‐2Rα injected alone or with HuTac and 7G7/B6. Injection of either HuTac or 7G7/B6 resulted in complexes with the sIL‐2Rα in serum. Injection of HuTac before 125I‐anti‐Tac dsFv, in SP2/Tac tumor‐bearing mice, resulted in faster clearance of the dsFv from the blood (7.6%ID/g at 30 min), compared to 23.2%ID/g for the no‐antibody control; preinjection of 7G7/B6 prolonged the retention of 125I‐anti‐Tac dsFv to 35.3%ID/g, with more complexes in serum. In mice pre‐injected with 7G7/B6 the concentration of 125I‐anti‐Tac dsFv in tumor was lower (5.2±0.3%ID/g) than in mice preinjected with HuTac (7.9±1.2%ID/g) or in the control group (5.6±0.7%ID/g). In conclusion, while both IgGs formed complexes with sIL‐2Rα and prolonged its retention, preinjection of 7G7/ B6 was detrimental, because the increased circulating sIL‐2Rα still had the epitope recognized by the dsFv available for binding and neutralized the anti‐Tac dsFv upon injection, whereas preinjection of HuTac blocked the epitope.