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PURPOSE:

This report presents the synthesis of a cyclic RGD dimer conjugate, MAG(2)-G(3)-E[G(3)-c(RGDfK)](2) (MAG(2)-3G(3)-dimer, G(3) = Gly-Gly-Gly, MAG(2) = S-benzoyl mercaptoacetylglycylglycyl), and evaluation of its (99m)Tc complex, (99m)TcO(MAG(2)-3G(3)-dimer), as a new radiotracer for imaging the tumor integrin alpha(v)beta(3) expression.

METHODS:

An in vitro displacement assay was used to determine the integrin alpha(v)beta(3) binding affinity of MAG(2)-3G(3)-dimer against (125)I-c(RGDyK) bound to U87MG human glioma cells. The athymic nude mice bearing U87MG glioma xenografts were used for biodistribution and planar imaging studies.

RESULTS:

We found that (1) MAG(2) is such a highly effective bifunctional chelator that (99m)TcO(MAG(2)-3G(3)-dimer) can be prepared in high yield (radiochemical purity >95%) and with high specific activity ( approximately 5 Ci/micromol) using a kit formulation; (2) (99m)TcO(MAG(2)-3G(3)-dimer) has very high solution stability in the kit matrix; and (3) (99m)TcO(MAG(2)-3G(3)-dimer) has very fast clearance kinetics from the intestine, liver, and kidneys. Among the (99m)Tc-labeled cyclic RGD peptides evaluated in the xenografted U87MG glioma models, (99m)TcO(MAG(2)-3G(3)-dimer) has the best pharmacokinetics and tumor to background ratios (tumor/liver = 4.29 +/- 1.00 at 30 min postinjection and 8.29 +/- 1.50 at 120 min postinjection; tumor/kidney = 1.16 +/- 0.19 at 30 min postinjection and 2.49 +/- 0.25 at 120 min postinjection). Planar imaging studies showed that tumors in the glioma-bearing mice administered with (99m)TcO(MAG(2)-3G(3)-dimer) can be visualized with excellent contrast as early as 15 min postinjection. (99m)TcO(MAG(2)-3G(3)-dimer) was able to maintain its chemical integrity in kidneys (>80% intact) and liver (>95% intact) over the 2-h period. However, there was significant metabolism (>50% of the injected radioactivity) detected in both urine and feces samples.

CONCLUSION:

(99m)TcO(MAG(2)-3G(3)-dimer) is a very attractive radiotracer for early detection of integrin alpha(v)beta(3)-positive tumors and has significant advantages over the (18)F-labeled RGD peptide radiotracers with respect to the cost, availability, and easiness for routine clinical preparation.

Radiolabeled RGD (Arg-Gly-Asp) and bombesin (BBN) radiotracers that specifically target integrin alpha(v)beta(3) and gastrin releasing peptide receptor (GRPR) are both promising radiopharmaceuticals for tumor imaging. We recently designed and synthesized a RGD-BBN heterodimeric peptide with both RGD and BBN motifs in one single molecule. The (18)F-labeled RGD-BBN heterodimer exhibited dual integrin alpha(v)beta(3) and GRPR targeting in a PC-3 prostate cancer model. In this study we investigated whether radiolabeled RGD-BBN tracers can be used to detect breast cancer by using microPET. Cell binding assay demonstrated that the high GRPR expressing breast cancer cells typically express low to moderate level of integrin alpha(v)beta(3), while high integrin alpha(v)beta(3) expressing breast cancer cells have negligible level of GRPR. We labeled RGD-BBN heterodimer with three positron emitting radionuclides (18)F, (64)Cu, and (68)Ga and investigated the corresponding PET radiotracers in both orthotopic T47D (GRPR(+)/low integrin alpha(v)beta(3)) and MDA-MB-435 (GRPR(-)/integrin alpha(v)beta(3)(+)) breast cancer models. The three radiotracers all possessed in vitro dual integrin alpha(v)beta(3) and GRPR binding affinity. The advantages of the RGD-BBN radiotracers over the corresponding BBN analogues are obvious for imaging MDA-MB-435 (GRPR(-)/integrin alpha(v)beta(3)(+)) tumor. (18)F-FB-PEG(3)-RGD-BBN showed lower tumor uptake than (64)Cu-NOTA-RGD-BBN and (68)Ga-NOTA-RGD-BBN but was able to visualize breast cancer tumors with high contrast. Synthesis of (64)Cu-NOTA-RGD-BBN and (68)Ga-NOTA-RGD-BBN is much faster and easier than (18)F-FB-PEG(3)-RGD-BBN. (64)Cu-NOTA-RGD-BBN showed prolonged tumor uptake but also higher liver retention and kidney uptake than (68)Ga-NOTA-RGD-BBN and (18)F-FB-PEG(3)-RGD-BBN. (68)Ga-NOTA-RGD-BBN possessed high tumor signals but also relatively high background uptake compared with the other two radiotracers. In summary, the prosthetic labeling groups, chelators, and isotopes all have a profound effect on the tumor targeting efficacy and in vivo kinetics of the RGD-BBN tracers for dual integrin and GRPR recognition. Further development of suitably labeled RGD-BBN tracers for PET imaging of cancer is warranted.

PURPOSE:

Various radiolabeled Arg-Gly-Asp (RGD) peptides have been previously investigated for tumor integrin alpha(v)beta(3) imaging. To further develop RGD radiotracers with enhanced tumor-targeting efficacy and improved in vivo pharmacokinetics, we designed a new RGD homodimeric peptide with two PEG(4) spacers (PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) between the two monomeric RGD motifs and one PEG(4) linker on the glutamate alpha-amino group ((18)F-labeled PEG(4)-E[PEG(4)-c(RGDfK)](2), P-PRGD2), as a promising agent for noninvasive imaging of integrin expression in mouse models.

METHODS:

P-PRGD2 was labeled with (18)F via 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-FP) prosthetic group. In vitro and in vivo characteristics of the new dimeric RGD peptide tracer (18)F-FP-P-PRGD2 were investigated and compared with those of (18)F-FP-P-RGD2 ((18)F-labeled RGD dimer without two PEG(4) spacers between the two RGD motifs). The ability of (18)F-FP-P-PRGD2 to image tumor vascular integrin expression was evaluated in a 4T1 murine breast tumor model.

RESULTS:

With the insertion of two PEG(4) spacers between the two RGD motifs, (18)F-FP-P-PRGD2 showed enhanced integrin alpha(v)beta(3)-binding affinity, increased tumor uptake and tumor-to-nontumor background ratios compared with (18)F-FP-P-RGD2 in U87MG tumors. MicroPET imaging with (18)F-FP-P-PRGD2 revealed high tumor contrast and low background in tumor-bearing nude mice. Biodistribution studies confirmed the in vivo integrin alpha(v)beta(3)-binding specificity of (18)F-FP-P-RGD2. (18)F-FP-P-PRGD2 can specifically image integrin alpha(v)beta(3) on the activated endothelial cells of tumor neovasculature.

CONCLUSION:

(18)F-FP-P-PRGD2 can provide important information on integrin expression on the tumor vasculature. The high integrin binding affinity and specificity, excellent pharmacokinetic properties and metabolic stability make the new RGD dimeric tracer (18)F-FP-P-PRGD2 a promising agent for PET imaging of tumor angiogenesis and for monitoring the efficacy of antiangiogenic treatment.

Radiolabeled RGD and bombesin peptides have been extensively investigated for tumor integrin alpha(v)beta(3) and GRPR imaging, respectively. Due to the fact that many tumors are both integrin and GRPR positive, we designed and synthesized a heterodimeric peptide Glu-RGD-BBN, which is expected to be advantageous over the monomeric peptides for dual-receptor targeting. A PEG(3) spacer was attached to the glutamate alpha-amino group of Glu-RGD-BBN to enhance the (18)F labeling yield and to improve the in vivo kinetics. PEG(3)-Glu-RGD-BBN possesses the comparable GRPR and integrin alpha(v)beta(3) receptor-binding affinities as the corresponding monomers, respectively. The dual-receptor targeting properties of (18)F-FB-PEG(3)-Glu-RGD-BBN were observed in PC-3 tumor model. (18)F-FB-PEG(3)-Glu-RGD-BBN with high tumor contrast and favorable pharmacokinetics is a promising PET tracer for dual integrin and GRPR positive tumor imaging. This heterodimer strategy may also be an applicable method to develop other molecules with improved in vitro and in vivo characterizations for tumor diagnosis and therapy.

PURPOSE:

Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides have great potential for the early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response. (18)F-labeled RGD analogs ([(18)F]-AH111585 and [(18)F]Galacto-RGD) have been investigated in clinical trials for positron emission tomography (PET) imaging of integrin expression in cancer patients. To develop new RGD radiotracers with higher tumor accumulation, improved in vivo kinetics, easy availability and low cost, we developed two new RGD peptides and labeled them with generator-eluted (68)Ga (t(1/2) = 68 min) for PET imaging of integrin alpha(v)beta(3) expression in tumor xenograft models.

MATERIALS AND METHODS:

The two new cyclic RGD dimers, E[PEG(4)-c(RGDfK)](2) (P(4)-RGD2, PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and E[Gly(3)-c(RGDfK)](2) (G(3)-RGD2, G(3) = Gly-Gly-Gly) were designed, synthesized and conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) for (68)Ga labeling. The microPET imaging and biodistribution of the (68)Ga labeled RGD tracers were investigated in integrin alpha(v)beta(3)-positive tumor xenografts.

RESULTS:

The new RGD dimers with the Gly(3) and PEG(4) linkers showed higher integrin alpha(v)beta(3) binding affinity than no-linker RGD dimer (RGD2). NOTA-G(3)-RGD2 and NOTA-P(4)-RGD2 could be labeled with (68)Ga within 30 min with higher purity (>98%) and specific activity (8.88-11.84 MBq/nmol). Both (68)Ga-NOTA-P(4)-RGD2 and (68)Ga-NOTA-G(3)-RGD2 exhibited significantly higher tumor uptake and tumor-to-normal tissue ratios than (68)Ga-NOTA-RGD2.

CONCLUSION:

Because of their high affinity, high specificity and excellent pharmacokinetic properties, further investigation of the two novel RGD dimers for clinical PET imaging of integrin alpha(v)beta(3) expression in cancer patients is warranted.

http://dx.doi.org/10.1007/s00259-008-1045-1

A novel dual-modality molecular probe composed of biocompatible Fe(3)O(4) nanocrystal, monoclonal antibody and radionuclide was designed and prepared. All functional components in the dual-modality molecular probe, i.e., Fe(3)O(4), PEG, mAb 3H11 and (125)I, were chemically bonded together for forming a stable molecular probe. Systematic in vitro experiments were carried out for evaluating the biological activity of the antibody in the targeting probe. A series of in vivo experiments were performed based on the dual-modality imaging probe for detecting xenografted tumors in nude mice by MRI and gamma-imaging techniques. The pharmacokinetics of the dual-modality molecular probe in tumor-bearing nude mice was studied.

PURPOSE:

Internalization is one of the key steps for anticancer immunoconjugates to deliver the drugs inside of cancer cells. Herein, the internalization property of antiepidermal growth factor receptor (EGFR) monoclonal antibody (mAb) LA22 was evaluated.

MATERIALS AND METHODS:

The binding and internalization properties of LA22 on A549 cells were investigated by using 125I-LA22. In vitro internalization was also confirmed by indirect fluorescent staining. In vivo tumor targeting and internalization of 125I-LA22 were evaluated in the A549 nude mice model.

RESULTS:

The mAb LA22 showed a high affinity to EGFRs expressed on A549 cells (Kd = 0.69 +/- 0.13 nM). The in vitro internalization of LA22 was time- and temperature dependent. The cell-surface-bound LA22 was rapidly internalized at 37 degrees C. The experimental results of LA22 internalization obtained from radioassay and fluorescent staining were consistent with a good linear correlation. At 72 hours postinjection, a clear gamma-image of tumor was obtain in A549 tumor xenografts, and the tumor uptake of 125I-LA22 was 8.00 +/- 0.61 percent injected dose per gram (%ID/g) (2.19 +/- 0.37 %ID/g for 125I-mIgG). Similar to the in vitro observation, 64.06% of the cell-bound mAb LA22 was internalized into the tumor cells in vivo.

CONCLUSIONS:

The mAb, LA22, is a rapid, high-internalizing antibody, and this property makes it a promising vehicle for tumor-targeted drug delivery.