Jiyun Shi

Improving Tumor Uptake and Pharmacokinetics of 64Cu-Labeled Cyclic RGD Peptide Dimers with Gly3 and PEG4 Linkers

Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides represent a new class of radiotracers with potential for early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response in cancer patients. This article describes the synthesis of two cyclic RGD peptide dimer conjugates, DOTA-PEG(4)-E[PEG(4)-c(RGDfK)](2) (DOTA-3PEG(4)-dimer: DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and DOTA-G(3)-E[G(3)-c(RGDfK)](2) (DOTA-3G(3)-dimer: G(3) = Gly-Gly-Gly). Integrin alpha(v)beta(3) binding affinities of cyclic RGD peptides were determined by competitive displacement of (125)I-echistatin bound to U87MG human glioma cells and follow the order of DOTA-E{E[c(RGDfK)](2)}(2) (DOTA-tetramer: IC(50) = 10 +/- 2 nM) > DOTA-3G(3)-dimer (IC(50) = 62 +/- 6 nM) approximately DOTA-3PEG(4)-dimer (IC(50) = 74 +/- 3 nM) > DOTA-E[c(RGDfK)](2) (DOTA-dimer: IC(50) = 102 +/- 5 nM). The addition of PEG(4) and G(3) linkers between two cyclic RGD motifs in DOTA-3G(3)-dimer and DOTA-3PEG(4)-dimer makes it possible for them to achieve the simultaneous integrin alpha(v)beta(3) binding in a bivalent fashion. Both (64)Cu(DOTA-3PEG(4)-dimer) and (64)Cu(DOTA-3G(3)-dimer) were prepared in high yield with specific activity being >50 Ci/mmol. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG human glioma xenografts. The results from those studies show that PEG(4) and G(3) linkers are particularly useful for improving tumor uptake and clearance kinetics of (64)Cu radiotracers from the nontumor organs, such as kidneys, liver, and lungs. There is a linear relationship between the tumor size and %ID tumor uptake, suggesting that (64)Cu(DOTA-3PEG(4)-dimer) and (64)Cu(DOTA-3PEG(4)-dimer) might be useful for noninvasive monitoring of tumor growth or shrinkage during antiangiogenic therapy. MicroPET imaging data clearly demonstrate the utility of (64)Cu(DOTA-3G(3)-dimer) as a new PET radiotracer for imaging integrin alpha(v)beta(3)-positive tumors.

Intrinsically Copper‐64‐Labeled Organic Nanoparticles as Radiotracers

PET friendly: labels for PET imaging are incorporated into completely organic porphysomes by using a fast (30 min), one-pot, high-yielding (>95 %) procedure to produce highly stable (>48 h) radiolabeled nanoparticles that show the highest specific activity ever reported for a (64) Cu-labeled nanoparticle. These (64) Cu-porphysomes can be accurately and noninvasively tracked in vivo.

99mTc-3PRGD2 for Integrin Receptor Imaging of Lung Cancer: A Multicenter Study

99mTc-3PRGD2 is a new SPECT tracer targeting integrin αVβ3 receptor for detecting tumors, imaging angiogenesis, and evaluating tumor response to therapy. A multicenter study was designed to investigate the efficacy of 99mTc-3PRGD2 for the evaluation of patients with lung cancer. Methods: Seventy patients (51 men, 19 women; mean age ± SD, 63 ± 9 y) with a suspected lung lesion and for whom definite pathologic diagnosis was finally obtained (malignant, n = 58; benign, n = 12) were recruited from 6 centers. Whole-body planar scanning and chest SPECT were performed at 1 and 4 h, respectively, after intravenous injection of 11.1 MBq/kg (0.3 mCi/kg) of 99mTc-3PRGD2. The images were read in consensus by 6 experienced nuclear medicine physicians masked to the source, history, and pathologic diagnosis. The tumor-to-background (T/B) ratios were calculated for semiquantitative analysis. A Student t test was used for statistical analysis, and a P value less than 0.05 was considered significant. Results: With low 99mTc-3PRGD2 background in the lungs and mediastinum, most lung malignancies were prominent on the 1-h images (T/B ratio, 1.65 ± 0.47 for the planar imaging and 2.78 ± 1.52 for SPECT). The T/B ratios were significantly lower in the benign lesions (P < 0.05). The sensitivity was 88% for semiquantitative analysis and could reach 93%–97% in visual analysis when considering the volume effect, necrosis, and metastasis. However, the specificity was only 58%–67%. Most lymph node and bone metastases could also be detected. Conclusion: 99mTc-3PRGD2 imaging at 1 h is sensitive for the detection of lung cancer, meriting further investigation of 99mTc-3PRGD2 as a novel clinical tracer for integrin receptor imaging.

A Novel Type of Dual-Modality Molecular Probe for MR and Nuclear Imaging of Tumor: Preparation, Characterization and in Vivo Application

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.

99mTc-Labeled Cyclic RGD Peptides for Noninvasive Monitoring of Tumor Integrin αvβ3 Expression

This report describes the biologic evaluations of [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (99mTc-3P-RGD2: 6-hydrazinonicotinyl; 3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid; and TPPTS = trisodium triphenylpho-sphine-3,3′,3“-trisulfonate), [99mTc(HYNIC-3G-RGD2)(tricine)(TPPTS)] (99mTc-3G-RGD2: 3G-RGD2 = G3-E[G3-c(RGDfK)]2 and G3 = Gly-Gly-Gly), and 99mTcO(MAG2−3G-RGD2) (MAG2 = mercaptoacetylglycylglycyl) as radiotracers for noninvasive imaging of tumor integrin αvβ3 expression in five xenografted tumor-bearing models. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG, MDA-MB-435, A549, HT29, or PC-3 tumor xenografts. Immunochemistry was performed using the cultured primary tumor cells and xenografted tumor tissues. It was found that the radiotracer tumor uptake followed the trend U87MG > MDA-MB-435 ≈ HT29 ≈ A549 > PC-3. The total integrin β3 expression levels followed the general trend: U87MG > MDA-MB-435 ≈ A549~HT29 > PC-3. There is a linear relationship between the radiotracer injected dose per gram tumor uptake and the total integrin β3 expression levels. On the basis of these, it was concluded that radiotracer tumor uptake is contributed by integrin αVβ3 expressed on tumor cells and activated endothelial cells of the tumor neovasculature. 99mTc-3P-RGD2 has the capability to monitor integrin αvβ3 expression in a noninvasive fashion.

Evaluation of 111In-Labeled Cyclic RGD Peptides: Tetrameric Not Tetravalent

This report presents the synthesis and evaluation of (111)In(DOTA-6G-RGD(4)) (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid; 6G-RGD(4) = E{G(3)-E[G(3)-c(RGDfK)](2)}(2) and G(3) = Gly-Gly-Gly), (111)In(DOTA-RGD(4)) (RGD(4) = E{E[c(RGDfK)](2)}(2)) and (111)In(DOTA-3G-RGD(2)) (3G-RGD(2) = G(3)-E[G(3)-c(RGDfK)](2)) as new radiotracers for imaging integrin alpha(v)beta(3)-positive tumors. The IC(50) values of DOTA-6G-RGD(4), DOTA-RGD(4), and DOTA-3G-RGD(2) were determined to be 0.4 +/- 0.1, 1.4 +/- 0.1 and 1.1 +/- 0.1 nM against (125)I-c(RGDyK) bound to integrin alpha(v)beta(3)-positive U87MG human glioma cells. (111)In(DOTA-6G-RGD(4)), (111)In(DOTA-RGD(4)), and (111)In(DOTA-3G-RGD(2)) were prepared by reacting (111)InCl(3) with the respective DOTA conjugate in NH(4)OAc buffer (100 mM, pH = 5.5). Radiolabeling could be completed by heating the reaction mixture at 100 degrees C for 15-20 min. The specific activity was approximately 1850 MBq/micromol for (111)In(DOTA-3G-RGD(2)) and approximately 1480 MBq/micromol for (111)In(DOTA-6G-RGD(4)). The athymic nude mice bearing U87MG human glioma xenografts were used to evaluate tumor uptake and excretion kinetics of (111)In(DOTA-6G-RGD(4)), (111)In(DOTA-RGD(4)), and (111)In(DOTA-3G-RGD(2)). The results from both the integrin alpha(v)beta(3) binding assay and biodistribution studies suggest that the tetrameric cyclic RGD peptides, such as RGD(4) and 6G-RGD(4), are most likely bivalent in binding to the integrin alpha(v)beta(3). Both (111)In(DOTA-6G-RGD(4)) and (111)In(DOTA-RGD(4)) had significantly higher tumor uptake than (111)In(DOTA-3G-RGD(2)) at 24-72 h postinjection due to the extra RGD motifs in RGD(4) and 6G-RGD(4). (111)In(DOTA-3G-RGD(2)) had very little metabolism, while (111)In(DOTA-6G-RGD(4)) had significant metabolism during its excretion via both renal and hepatobiliary routes over the 2 h period, probably due to its much larger size. The combination of high tumor uptake with long tumor retention suggests that their corresponding (90)Y and (177)Lu analogues M(DOTA-6G-RGD(4)) (M = (90)Y and (177)Lu) might be useful as therapeutic radiotracers for treatment of integrin alpha(v)beta(3)-positive solid tumors.

Two (90)Y-Labeled Multimeric RGD Peptides RGD4 and 3PRGD2 for Integrin Targeted Radionuclide Therapy

We have recently developed a series of new Arg-Gly-Asp (RGD) dimeric peptides for specific targeting of integrin α(v)β₃ with enhanced tumor uptake and improved pharmacokinetics. In this study, we investigated ⁹⁰Y-labeled RGD tetramer (RGD4) and the new type of RGD dimer (3PRGD2), for the radionuclide therapy of integrin α(v)β₃-positive tumors. Biodistribution and gamma imaging studies of ¹¹¹In labeled RGD4 and 3PRGD2 were performed. Groups of nude mice were used to determine maximum tolerated dose (MTD) of ⁹⁰Y-DOTA-RGD4 and ⁹⁰Y-DOTA-3PRGD2. The radionuclide therapeutic efficacy of ⁹⁰Y-DOTA-RGD4 and ⁹⁰Y-DOTA-3PRGD2 was evaluated in U87MG tumor-bearing nude mice. The U87MG tumor uptake of ¹¹¹In-DOTA-3PRGD2 was slightly lower than that of the ¹¹¹In-DOTA-RGD4 (e.g., 6.13 ± 0.82%ID/g vs 6.43 ± 1.6%ID/g at 4 h postinjection), but the uptake of ¹¹¹In-DOTA-3PRGD2 in normal organs, such as liver and kidneys, was much lower than that of ¹¹¹In-DOTA-RGD4, which resulted in much higher tumor-to-nontumor ratios and lower toxicity. The MTD of ⁹⁰Y-DOTA-RGD4 in nude mice is less than 44.4 MBq, while the MTD of ⁹⁰Y-DOTA-3PRGD2 in mice is more than 55.5 MBq. ⁹⁰Y-DOTA-3PRGD2 administration exhibited a similar tumor inhibition effect as compared with ⁹⁰Y-DOTA-RGD4 at the same dose. The tumor vasculature in the ⁹⁰Y-DOTA-3PRGD2 treatment group was much less than the control groups. Radionuclide therapy studies exhibited that both ⁹⁰Y-DOTA-RGD4 and ⁹⁰Y-DOTA-3PRGD2 caused significant tumor growth delay in the U87MG tumor model. Compared to ⁹⁰Y-DOTA-RGD4, the low accumulation of ⁹⁰Y-DOTA-3PRGD2 in normal organs led to lower toxicity and higher MTD in nude mice, which would make it more suitable for high dose or multiple-dose regimens, in order to achieve maximum therapeutic efficacy.

64Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium cations as highly selective tumor imaging agents: effects of linkers and chelates on radiotracer biodistribution characteristics.

Radiolabeled organic cations, such as triphenylphosphonium (TPP), represents a new class of radiotracers for imaging cancers and the transport function of multidrug resistance P-glycoproteins (particularly MDR1 Pgp) by single photon emission computed tomography (SPECT) or positron emission tomography (PET). This report presents the synthesis and biological evaluation of (64)Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium (TPEP) cations as novel PET radiotracers for tumor imaging. Biodistribution studies were performed using the athymic nude mice bearing subcutaneous U87MG human glioma xenografts to explore the impact of linkers, bifunctional chelators (BFCs), and chelates on biodistribution characteristics of the (64)Cu-labeled TPEP cations. Metabolism studies were carried out using normal athymic nude mice to determine the metabolic stability of four (64)Cu radiotracers. It was found that most (64)Cu radiotracers described in this study have significant advantages over (99m)Tc-Sestamibi for their high tumor/heart and tumor/muscle ratios. Both BFCs and linkers have significant impact on biological properties of (64)Cu-labeled TPEP cations. For example, (64)Cu(DO3A-xy-TPEP) has much lower liver uptake and better tumor/liver ratios than (64)Cu(DO3A-xy-TPP), suggesting that TPEP is a better mitochondrion-targeting molecule than TPP. Replacing DO3A with DO2A results in (64)Cu(DO2A-xy-TPEP) (+), which has a lower tumor uptake than (64)Cu(DO3A-xy-TPEP). Substitution of DO3A with NOTA-Bn leads to a significant decrease in tumor uptake for (64)Cu(NOTA-Bn-xy-TPEP). The use of DOTA-Bn to replace DO3A has little impact on the tumor uptake, but the tumor/liver ratio of (64)Cu(DOTA-Bn-xy-TPEP) (-) is not as good as that of (64)Cu(DO3A-xy-TPEP), probably due to the aromatic benzene ring in DOTA-Bn. Addition of an extra acetamido group in (64)Cu(DOTA-xy-TPEP) results in a lower liver uptake, but tumor/liver ratios of (64)Cu(DOTA-xy-TPEP) and (64)Cu(DO3A-xy-TPEP) are comparable due to a faster tumor washout of (64)Cu(DOTA-xy-TPEP). Substitution of xylene with the PEG 2 linker also leads to a significant reduction in both tumor and liver uptake. MicroPET imaging studies on (64)Cu(DO3A-xy-TPEP) in athymic nude mice bearing U87MG glioma xenografts showed that the tumor was clearly visualized as early as 1 h postinjection with very high T/B contrast. There was very little metabolite (<2%) detectable in the urine and feces samples for (64)Cu(DO3A-xy-TPEP), (64)Cu(DOTA-Bn-xy-TPEP)(-), and (64)Cu(NOTA-Bn-xy-TPEP). Considering both tumor uptake and T/B ratios (particularly tumor/heart, tumor/liver, and tumor/muscle), it was concluded that (64)Cu(DO3A-xy-TPEP) is a promising PET radiotracer for imaging the MDR-negative tumors.

(68)Ga-PRGD2 PET/CT in the evaluation of Glioma: a prospective study.

Integrin αvβ3 is overexpressed in both neovasculature and glioma cells. We aimed to evaluate 68gallium-BNOTA-PRGD2 (68Ga-PRGD2) as a new reagent for noninvasive integrin αvβ3 imaging in glioma patients. With informed consent, 12 patients with suspicious brain glioma, as diagnosed by enhanced magnetic resonance imaging (MRI) scanning, were enrolled to undergo 68Ga-PRGD2 PET/CT and 18F-FDG PET/CT scans before surgery. The preoperative images were compared and correlated with the pathologically determined WHO grade. Next, the expression of integrin αvβ3, CD34, and Ki-67 were determined by immunohistochemical staining of the resected brain tumor tissue. Our findings demonstrated that 68Ga-PRGD2 specifically accumulated in the brain tumors that were rich of integrin αvβ3 and other neovasculature markers, but not in the brain parenchyma other than the choroid plexus. Therefore, 68Ga-PRGD2 PET/CT was able to evaluate the glioma demarcation more specifically than 18F-FDG PET/CT. The maximum standardized uptake values (SUVmax) of 68Ga-PRGD2, rather than those of 18F-FDG, were significantly correlated with the glioma grading. The maximum tumor-to-brain ratios (TBRmax) of both tracers were significantly correlated with glioma grading, whereas 68Ga-PRGD2 seemed to be more superior to 18F-FDG in differentiating high-grade glioma (HGG) from low-grade glioma (LGG). Moreover, 68Ga-PRGD2 PET/CT showed different accumulation patterns for HGG of WHO grades III and IV. This is the first noninvasive integrin imaging study, to the best of our knowledge, conducted in preoperative patients with different grades of glioma, and it preliminarily indicated the effectiveness of this novel method for evaluating glioma grading and demarcation.

Integrin αvβ6–Targeted SPECT Imaging for Pancreatic Cancer Detection

Integrin αvβ6, a member of the integrin family, is specifically expressed in many malignancies but not in normal organs. Overexpression of integrin αvβ6 is usually correlated with malignant potential and poor prognosis. In this study, we describe the synthesis and evaluation of a 99mTc-labeled integrin αvβ6–targeting peptide as a SPECT radiotracer for the in vivo imaging of integrin αvβ6 expression. Methods: An integrin αvβ6–targeting peptide (denoted as the HK peptide) was conjugated with 6-hydrazinonicotinyl (HYNIC) and radiolabeled with 99mTc using tricine and TPPTS (trisodium triphenylphosphine-3,3′,3″-trisulfonate) as coligands. The in vitro and in vivo characteristics of 99mTc-HYNIC(tricine)(TPPTS)-HK (99mTc-HHK) were investigated in BxPC-3 (integrin αvβ6–positive) and HEK293 (integrin αvβ6–negative) models. The ability of 99mTc-HHK to detect liver metastasis of pancreatic cancer was evaluated using small-animal SPECT/CT. Results: 99mTc-HHK showed high integrin αvβ6–binding specificity both in vitro and in vivo. 99mTc-HHK was cleared rapidly from the blood and normal organs except for the kidneys. The highest uptake (0.88 ± 0.12 percentage injected dose per gram) of 99mTc-HHK in BxPC-3 tumors was observed at 0.5 h after injection. High-contrast images of integrin αvβ6–positive tumors were obtained using 99mTc-HHK. The minimum nonspecific activity accumulation in normal liver tissues rendered high-quality SPECT/CT images of metastatic lesions. Conclusion: 99mTc-HHK is a promising SPECT radiotracer for the noninvasive imaging of integrin αvβ6 expression in vivo. SPECT/CT with 99mTc-HHK could provide an effective approach for the noninvasive detection of primary and metastatic lesions of integrin αvβ6–positive tumors.