Technetium-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptides for human melanoma imaging

IntroductionThe purpose of this study was to examine whether ^99mTc-labeled Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide targeting both melanocortin-1 (MC1) and α_vβ₃ integrin receptors was superior in melanoma targeting to ^99mTc-labeled α-MSH or RGD peptide targeting only the MC1 or α_vβ₃ integrin receptor.MethodsRGD-Lys-(Arg¹¹)CCMSH, RAD-Lys-(Arg¹¹)CCMSH and RGD-Lys-(Arg¹¹)CCMSHscramble were designed to target both MC1 and α_vβ₃ integrin receptors, MC1 receptor only and α_vβ₃ integrin receptor only, respectively. The MC1 or α_vβ₃ integrin receptor binding affinities of three peptides were determined in M21 human melanoma cells. The melanoma targeting properties of ^99mTc-labeled RGD-Lys-(Arg¹¹)CCMSH, RAD-Lys-(Arg¹¹)CCMSH and RGD-Lys-(Arg¹¹)CCMSHscramble were determined in M21 human melanoma-xenografted nude mice. Meanwhile, the melanoma uptake of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH was blocked with various non-radiolabeled peptides in M21 melanoma xenografts.ResultsRGD-Lys-(Arg¹¹)CCMSH displayed 2.0 and 403 nM binding affinities to both MC1 and α_vβ₃ integrin receptors, whereas RAD-Lys-(Arg¹¹)CCMSH or RGD-Lys-(Arg¹¹)CCMSHscramble lost their α_vβ₃ integrin receptor binding affinity by greater than 248-fold or MC1 receptor binding affinity by more than 100-fold, respectively. The melanoma uptake of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH was 2.49 and 2.24 times (P < .05) the melanoma uptakes of ^99mTc-RAD-Lys-(Arg¹¹)CCMSH and ^99mTc-RGD-Lys-(Arg¹¹)CCMSHscramble at 2 h post-injection, respectively. Either RGD or (Arg¹¹)CCMSH peptide co-injection could block 42% and 57% of the tumor uptake of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH, whereas the coinjection of RGD+(Arg¹¹)CCMSH peptide mixture could block 66% of the tumor uptake of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH.ConclusionsTargeting both MC1 and α_vβ₃ integrin receptors enhanced the melanoma uptake of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH in M21 human melanoma xenografts. Flank M21 human melanoma tumors were clearly visualized by single photon emission computed tomography/computed tomographic imaging using ^99mTc-RGD-Lys-(Arg¹¹)CCMSH as an imaging probe, highlighting its potential use as a dual-receptor-targeting imaging probe for human melanoma detection.     

[68Ga]NS3-RGD and [68Ga] Oxo-DO3A-RGD for imaging αvβ3 integrin expression: synthesis,evaluation, and comparison

Introduction⁶⁸Ga-labeled RGD peptides in combination with PET allow non-invasive determination of α_vβ₃ integrin expression which is highly increased during tumor-induced angiogenesis. The aim of this study was to synthesize and evaluate two RGD peptides containing alternative chelating systems, namely [⁶⁸Ga]NS₃-RGD and [⁶⁸Ga]Oxo-DO3A-RGD and to compare their in vitro and in vivo properties with [⁶⁸Ga]DOTA- and [⁶⁸Ga]NODAGA-RGD.MethodsSyntheses of both radiotracers followed standard SPPS protocols. For in vitro characterization distribution coefficients, protein binding abilities, serum stabilities, and α_vβ₃ integrin binding affinities were determined. For in vitro tests as well as for the biodistribution assay α_vβ₃ positive human melanoma M21 and α_vβ₃ negative M21-L cells were used.Results⁶⁸Ga-labeling of NS₃-RGD resulted in good radiochemical purity, whereas HPLC analysis showed two peaks with a ratio of 1:6 for [⁶⁸Ga]Oxo-DO3A-RGD. Distribution coefficients were ? 3.4 for [⁶⁸Ga]Oxo-DO3A-RGD and ? 2.9 for [⁶⁸Ga]NS₃-RGD. Both radiotracers were stable in PBS solution at 37 °C for 2 h but lack stability in human serum. Protein binding was approximately 40% of the total activity for [⁶⁸Ga]NS₃-RGD and 70% for [⁶⁸Ga]Oxo-DO3A-RGD, respectively, resulting in high blood pool activities. Biodistribution assays confirmed these findings and showed an additional high uptake in liver and kidneys, especially for [⁶⁸Ga]NS₃-RGD. Furthermore, [⁶⁸Ga]Oxo-DO3A-RGD showed nearly the same activity concentrations in α_vβ₃ positive and α_vβ₃ negative tumors.Conclusions[⁶⁸Ga]Oxo-DO3A-RGD and [⁶⁸Ga]NS₃-RGD have inferior characteristics compared to already existing ⁶⁸Ga-labeled RGD peptides and thus, both are not suited to image α_vβ₃ integrin expression. Of all our tested RGD peptides, [⁶⁸Ga]NODAGA-RGD still possesses the most favorable imaging properties. Moreover this study shows that the use of appropriate chelators to achieve good targeting properties of ⁶⁸Ga-labeled biomolecules and careful in vitro and in vivo evaluation including comparative studies of different strategies are essential components in designing an effective imaging agent for PET.     

Specific uptake of 99mTc-NC100692, an αvβ3-targeted imaging probe,in subcutaneous and orthotopic tumors

IntroductionThe α_vβ₃ integrin, which is expressed by angiogenic epithelium and some tumor cells, is an attractive target for the development of both imaging agents and therapeutics. While optimal implementation of α_vβ₃-targeted therapeutics will require a priori identification of the presence of the target, the clinical evaluation of these compounds has typically not included parallel studies with α_vβ₃-targeted diagnostics. This is at least partly due to the relatively limited availability of PET radiopharmaceuticals in comparison to those labeled with ^99mTc. In an effort to begin to address this limitation, we evaluated the tumor uptake of ^99mTc-NC100692, a cyclic RGD peptide that binds to α_vβ₃ with ~ 1-nM affinity, in an α_vβ₃-positive tumor model as well as its in vivo specificity.MethodsMicroSPECT imaging was used to assess the ability of cilengitide, a therapeutic with high affinity for α_vβ₃, to block and displace ^99mTc-NC100692 in an orthotopic U87 glioma tumor. The specificity of ^99mTc-NC100692 was quantitatively evaluated in mice bearing subcutaneous U87MG tumors, by comparison of the biodistribution of ^99mTc-NC100692 with that of the non-specific structural analogue ^99mTc-AH-111744 and by blocking uptake of ^99mTc-NC100692 with excess unlabeled NC100692.ResultsMicroSPECT imaging studies demonstrated that uptake of ^99mTc-NC100692 in the intracranial tumor model was both blocked and displaced by the α_vβ₃-targeted therapeutic cilengitide. Biodistribution studies provided quantitative confirmation of these imaging results. Tumor uptake of ^99mTc-NC100692 at 1 h post-injection was 2.8 ± 0.7% ID/g compared to 0.38 ± 0.1% ID/g for ^99mTc-AH-111744 (p < 0.001). Blocking ^99mTc-NC100692 uptake by pre-injecting the mice with excess unlabeled NC100692 reduced tumor uptake by approximately five-fold, to 0.68 ± 0.3% ID/g (p = 0.01).ConclusionThese results confirm that ^99mTc-NC100692 does, in fact, target the α_vβ₃ integrin and may, therefore, be useful in identifying patients prior to anti-α_vβ₃ therapy as well as monitoring the response of these patients to therapy.     

SPECT/CT of lung nodules using 111In-DOTA-c(RGDfK) in a mouse lung carcinogenesis model

Objective

Lung cancer is one of the leading causes of cancer-related deaths worldwide, including Japan. Although computed tomography (CT) can detect small lung lesions such as those appearing as ground glass opacity, it cannot differentiate between malignant and non-malignant lesions. Previously, we have shown that single photon emission computed tomography (SPECT) imaging using ¹¹¹In-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid-cyclo-(Arg-Gly-Asp-d-Phe-Lys) (DOTA-c(RGDfK)), an imaging probe of α_vβ₃ integrin, is useful for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model. In this study, we aimed to assess the usefulness of SPECT/CT with ¹¹¹In-DOTA-c(RGDfK) for the evaluation of the malignancy of lung cancer.

Methods

Lung tumors were induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice. Twenty-six weeks after the urethane treatment, SPECT was performed an hour after injection of ¹¹¹In-DOTA-c(RGDfK). Following this, the radioactivity ratios of tumor to normal lung tissue were measured by autoradiography (ARG) in the excised lung samples. We also examined the expression of α_vβ₃ integrin in mouse and human lung samples.

Results

Urethane treatment induced 5 hyperplasias, 41 adenomas and 12 adenocarcinomas in the lungs of 8 A/J mice. SPECT with ¹¹¹In-DOTA-c(RGDfK) could clearly visualize lung nodules, though we failed to detect small lung nodules like adenoma and hyperplasias (adenocarcinoma: 66.7 %, adenoma: 33.6 %, hyperplasia: 0.0 %). ARG analysis revealed significant uptake of ¹¹¹In-DOTA-c(RGDfK) in all the lesions. Moreover, tumor to normal lung tissue ratios increased along with the progression of carcinogenesis. Histopathological examination using human lung tissue samples revealed clear up-regulation of α_vβ₃ integrin in well-differentiated adenocarcinoma (Noguchi type B and C) rather than atypical adenomatous hyperplasia.

Conclusion

Although there are some limitations in evaluating the malignancy of small lung tumors using ¹¹¹In-DOTA-c(RGDfK), SPECT with ¹¹¹In-DOTA-c(RGDfK) might be a useful non-invasive imaging approach for evaluating the characteristics of lung tumors in mice, thus showing potential for use in humans.     

Inter-heterogeneity and intra-heterogeneity of α<Subscript>v</Subscript>β<Subscript>3</Subscript> in non-small cell lung cancer and small cell lung cancer patients as revealed by <Superscript>68</Superscript>Ga-RGD<Subscript>2</Subscript> PET imaging

Purpose

Integrin α_vβ₃ is the therapeutic target of the anti-angiogenic drug cilengitide. The objective of this study was to compare α_vβ₃ levels in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients, by using the positron emission tomography (PET) tracer ⁶⁸Ga-labeled dimerized-RGD (⁶⁸Ga-RGD₂).

Methods

Thirty-one patients with pathologically confirmed lung cancer were enrolled (21 were NSCLC and 10 were SCLC). PET/CT images were acquired using ⁶⁸Ga-RGD₂.¹⁸F-FDG PET/CT images were also acquired on the consecutive day as reference. The standard uptake values (SUV) and the tumor/nontarget (T/NT) values were quantitatively compared. Expression of the angiogenesis marker α_vβ₃ in NSCLC and SCLC lesions was analyzed by immunohistochemistry.

Results

The ¹⁸F-FDG SUVmax and the SUVmean were not significantly different between NSCLC and SCLC patients. The ⁶⁸Ga-RGD₂ uptake of SCLC patients was at background levels in both SUV and T/NT measurements and was significantly lower than that of NSCLC patients. The range value of ⁶⁸Ga-RGD₂ SUVmean was 4.5 in the NSCLC group and 2.2 in the SCLC group, while the variation coefficient was 36.2% and 39.3% in NSCLC and SCLC primary lesions, respectively. Heterogeneity between primary lesions and putative distant metastases was also observed in some NSCLC cases. Immunostaining showed that α_vβ₃ integrin was expressed in the cells and neovasculature of NSCLC lesions, while SCLC samples had negative expression.

Conclusions

The uptake of ⁶⁸Ga-RGD₂ in SCLC patients is significantly lower than that in NSCLC patients, indicating a lower α_vβ₃ target level for cilengitide in SCLC. Apparent intra-tumor heterogeneities of α_vβ₃ also exist in both NSCLC and SCLC. Such inter- and intra-heterogeneity of α_vβ₃ may potentially improve current applications of α_vβ₃-targeted therapy and diagnostic imaging in lung cancer.

     

In vitro and in vivo evaluation of the effects of aluminum [18F]fluoride radiolabeling on an integrin αvβ6-specific peptide

IntroductionIncorporation of fluorine-18 (¹⁸F) into radiotracers by capturing ionic [¹⁸F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [¹⁸F]fluoride (Al[¹⁸F]^2 +) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[¹⁸F]^2 +-radiolabeling of an α_vβ₆ integrin-targeted peptide (NOTA-PEG₂₈-A20FMDV2) and its in vitro and in vivo evaluation.MethodsAluminum [¹⁸F]fluoride was prepared at r.t. from [¹⁸F]fluoride (40 MBq–11 GBq) and introduced into NOTA-PEG₂₈-A20FMDV2 (1) in sodium acetate (pH 4.1; 100°C, 15 min). The radiotracer Al[¹⁸F] NOTA-PEG₂₈-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in α_vβ₆(+) and α_vβ₆(−) cells) and in vivo (paired α_vβ₆(+) and α_vβ₆(−) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites).ResultsThe radiotracer 2 was prepared in 90 ± 6 min (incl. formulation; n = 3) in 19.3 ± 5.4% decay corrected radiochemical yield (radiochemical purity: > 99%; specific activity: 158 ± 36 GBq/μmol) and was stable in PBS and serum (2 h). During in vitro cell binding studies, 2 showed high, α_vβ₆-targeted binding (α_vβ₆(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(−) = 8.4/1) and internalization (α_vβ₆(+): 28.3 ± 0.5% of total radioactivity, (+)/(−) = 11.7/1). In vivo, 2 maintained α_vβ₆-targeted binding (biodistribution; 1 h: α_vβ₆(+): 1.74 ± 0.38% ID/g, (+)/(−) = 2.72/1; 4 h: α_vβ₆(+): 1.21 ± 0.56% ID/g, (+)/(−) = 4.0/1; 11% intact 2 in tumor at 1 h), with highest uptake around the tumor edge (autoradiography). Most of the radioactivity cleared rapidly in the urine within one hour, but a significant fraction remained trapped in the kidneys (4 h: 229 ± 44% ID/g).ConclusionThe Al[¹⁸F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good α_vβ₆-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [¹⁸F]-prosthetic groups (e.g., [¹⁸F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2.     

64Cu-Labeled CB-TE2A and diamsar-conjugated RGD peptide analogs for targeting angiogenesis: comparison of their biological activity

ObjectivesThe α_vβ₃ integrin is a cell adhesion molecule known to be involved in stages of angiogenesis and metastasis. In this study, the chelators CB-TE2A and diamsar were conjugated to cyclic RGDyK and RGDfD and the biological properties of ⁶⁴Cu-labeled peptides were compared.MethodsCB-TE2A-c(RGDyK) and diamsar-c(RGDfD) were labeled with ⁶⁴Cu in 0.1 M NH₄OAc (pH=8) at 95°C and 25°C, respectively. PET and biodistribution studies were carried out on M21 (α_vβ₃-positive) and M21L (α_v-negative) melanoma-bearing mice. Binding affinity of the Cu-chelator–RGD peptides to α_vβ₃ integrins was determined by a competitive binding affinity assay.ResultsBiological studies showed higher concentration of ⁶⁴Cu-CB-TE2A-c(RGDyK) in M21 tumor compared to M21L tumor at 1 and 4 h pi. Tumor concentration of ⁶⁴Cu-CB-TE2A-c(RGDyK) was higher than that of ⁶⁴Cu-diamsar-c(RGDfD). The difference is not due to differing binding affinities, since similar values were obtained for the agents. Compared to ⁶⁴Cu-diamsar-c(RGDfD), there is more rapid liver and blood clearance of ⁶⁴Cu-CB-TE2A-c(RGDyK), resulting in a lower liver and blood concentration at 24 h pi. Both ⁶⁴Cu-labeled RGD peptides show similar binding affinities to α_vβ₃. The differences in their biodistribution properties are likely related to different linkers, charges and lipophilicities. The M21 tumor is clearly visualized with ⁶⁴Cu-CB-TE2A-c(RGDyK) by microPET imaging. Administration of c(RGDyK) as a block significantly reduced the tumor concentration; however, the radioactivity background was also decreased by the blocking dose.ConclusionsBoth ⁶⁴Cu-CB-TE2A-c(RGDyK) and ⁶⁴Cu-diamsar-c(RGDfD) are potential candidates for imaging tumor angiogenesis. For diamsar-c(RGDfD), a linker may be needed between the Cu-chelator moiety and the RGD peptide to achieve optimal in vivo tumor concentration and clearance from nontarget organs.     

[18F]Fluciclatide in the in vivo evaluation of human melanoma and renal tumors expressing αvβ3 and αvβ5 integrins

Purpose

[¹⁸F]Fluciclatide is an integrin-targeted PET radiopharmaceutical. α_vβ₃ and α_vβ₅ are upregulated in tumor angiogenesis as well as on some tumor cell surfaces. Our aim was to use [¹⁸F]fluciclatide (formerly known as [¹⁸F]AH111585) for PET imaging of angiogenesis in melanoma and renal tumors and compare with tumor integrin expression.

Methods

Eighteen evaluable patients with solid tumors ≥2.0 cm underwent [¹⁸F]fluciclatide PET/CT. All patients underwent surgery and tumor tissue samples were obtained. Immunohistochemical (IHC) staining with mouse monoclonal antibodies and diaminobenzidine (DAB) was applied to snap-frozen tumor specimens, and additional IHC was done on formalin-fixed paraffin-embedded samples. DAB optical density (OD) data from digitized whole-tissue sections were compared with PET SUV_{80% max}, and Patlak influx rate constant (K _i) data, tumor by tumor.

Results

Tumors from all 18 patients demonstrated measurable [¹⁸F]fluciclatide uptake. At the final dynamic time-point (55 min after injection), renal malignancies (in 11 patients) demonstrated an average SUV_{80% max} of 6.4?±?2.0 (range 3.8 – 10.0), while the average SUV_{80% max} for metastatic melanoma lesions (in 6 patients) was 3.0?±?2.0 (range 0.7 – 6.5). There was a statistically significant difference in [¹⁸F]fluciclatide uptake between chromophobe and nonchromophobe renal cell carcinoma (RCCs, with SUV_{80% max} of 8.2?±?1.8 and 5.4?±?1.4 (P?=?0.020) and tumor-to-normal kidney (T/N) ratios of 1.5?±?0.4 and 0.9?±?0.2, respectively (P?=?0.029). The highest Pearson's correlation coefficients were obtained when comparing Patlak K _i and α_vβ₅ OD when segregating the patient population between melanoma and RCC (r?=?0.83 for K _i vs. melanoma and r?=?0.91 for K _i vs. RCC). SUV_{80% max} showed a moderate correlation with α_vβ₅ and α_vβ₃ OD.

Conclusion

[¹⁸F]Fluciclatide PET imaging was well tolerated and demonstrated favorable characteristics for imaging α_vβ₃ and α_vβ₅ expression in melanoma and RCC. Higher uptake was observed in chromophobe than in nonchromophobe RCC. [¹⁸F]Fluciclatide may be a useful radiotracer to improve knowledge of integrin expression.     

Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

ObjectiveThe goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the α_vβ₃ integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process.MethodsA hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing an RGD motif for binding to the α_vβ₃integrin, a metal chelator (DOTA) for radiolabeling with [⁶⁴Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [¹²³I] was synthesized, labeled with [⁶⁴Cu] and [¹²³I], and evaluated in vitro as a potential imaging agent.ResultsThe peptide was synthesized and labeled with [⁶⁴Cu] and [¹²³I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of > 98%. c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for α_vβ₃ integrins (Kd = 83.4 + 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2.ConclusionsThese results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity.     

Biological evaluation of an ornithine-modified 99mTc-labeled RGD peptide as an angiogenesis imaging agent

IntroductionRadiolabeled RGD peptides that specifically target integrin α_νβ₃ have great potential in early tumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous findings of our group on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfK derivative, c(RGDfK)-(Orn)₃-CGG. This new peptide availing the polar linker (Orn)₃ and the ^99mTc-chelating moiety CGG (Cys-Gly-Gly) is appropriately designed for ^99mTc-labeling, as well as consequent conjugation onto nanoparticles.MethodsA tumor imaging agent, c(RGDfK)-(Orn)₃-[CGG-^99mTc], is evaluated with regard to its radiochemical, radiobiological and imaging characteristics.ResultsThe complex c(RGDfK)-(Orn)₃-[CGG-^99mTc] was obtained in high radiochemical yield (> 98%) and was stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized ^185/187Re complex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the new radiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake (3.87 ± 0.48% ID/g at 60 min p.i.) was observed and was maintained relatively high even at 24 h p.i. (1.83 ± 0.05 % ID/g), thus providing well-defined scintigraphic imaging. Accumulation in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells.ConclusionDue to its relatively high tumor uptake, renal elimination and negligible abdominal localization, the new ^99mTc-RGD peptide is considered promising in the field of imaging α_νβ₃-positive tumors. However, the preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dual modality imaging of integrin expressing tumors should be further investigated.     

A heterodimeric [RGD-Glu-[64Cu-NO2A]-6-Ahx-RM2] αvβ3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors

IntroductionIn the present study, we describe a ⁶⁴Cu-radiolabeled heterodimeric peptide conjugate for dual α_vβ₃/GRPr (α_vβ₃ integrin/gastrin releasing peptide receptor) targeting of the form [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] (RGD: the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide used for α_vβ₃ integrin receptor targeting; Glu: glutamic acid; NO2A: 1,4,7-triazacyclononane-1,4-diacetic acid; 6-Ahx: 6-amino hexanoic acid; and RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂), an antagonist analogue of bombesin (BBN) peptide used for GRPr targeting).MethodsRGD-Glu-6Ahx-RM2] was conjugated to a NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) complexing agent to produce [RGD-Glu-[NO2A]-6-Ahx-RM2], which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by electrospray ionization–mass spectrometry (ESI-MS). Radiolabeling of the conjugate with ⁶⁴Cu produced [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2 in high radiochemical yield (≥ 95%). In vivo behavior of the radiolabeled peptide conjugate was investigated in normal CF-1 mice and in the PC-3 human prostate cancer experimental model.ResultsA competitive displacement receptor binding assay in human prostate PC-3 cells using ¹²⁵I-[Tyr⁴]BBN as the radioligand showed high binding affinity of [RGD-Glu-[^natCu-NO2A]-6-Ahx-RM2] conjugate for the GRPr (3.09 ± 0.34 nM). A similar assay in human, glioblastoma U87-MG cells using ¹²⁵I-Echistatin as the radioligand indicated a moderate receptor-binding affinity for the αvβ₃ integrin (518 ± 37.5 nM). In vivo studies of [RGD-Glu-[⁶⁴Cu-NO2A]-6-Ahx-RM2] showed high accumulation (4.86 ± 1.01 %ID/g, 1 h post-intravenous injection (p.i.)) and prolonged retention (4.26 ± 1.23 %ID/g, 24 h p.i.) of tracer in PC-3 tumor-bearing mice. Micro-positron emission tomography (microPET) molecular imaging studies produced high-quality, high contrast images in PC-3 tumor-bearing mice at 4 h p.i.ConclusionsThe favorable pharmacokinetics and enhanced tumor uptake of ⁶⁴Cu-NOTA-RGD-Glu-6Ahx-RM2 warrant further investigations for dual integrin and GRPr-positive tumor imaging and possible radiotherapy.     

Evaluation of an integrin αvβ6-specific peptide labeled with [18F]fluorine by copper-free,strain-promoted click chemistry

IntroductionClick chemistry, particularly the Huisgen 1,3-dipolar cycloaddition of an alkyne with an azide, has quickly become popular for site-specific radiolabeling. Recently, strain-promoted click chemistries have been developed, eliminating the need for potentially toxic copper catalysts. This study presents radiolabeling of an α_vβ₆ integrin targeting peptide (A20FMDV2) via strain-promoted click using a fluorine-18 prosthetic group, and in vitro and in vivo evaluation.MethodsThe radiotracer [¹⁸F]FBA-C₆-ADIBON₃-PEG₇-A20FMDV2 (1) was prepared from [¹⁸F]FBA-C₆-ADIBO (2) and N₃-PEG₇-A20FMDV2 (ethanol; 10 min; 35–45 °C). HPLC-purified and formulated radiotracer 1 was evaluated in vitro by cell binding (DX3puroβ6, α_vβ₆-positive; and DX3puro, α_vβ₆-negative control) and serum stability, and in vivo using PET/CT imaging and biodistribution studies in mice.ResultsThe radiotracer 1 was readily prepared and purified (from 2: 40 ± 4 min including HPLC, 11.9 ± 3.2% decay corrected isolated radiochemical yield, > 99% radiochemical purity, n = 4) and displayed good stability (1 h: > 99%, saline; 94.6%, serum). Strong α_vβ₆-targeted binding was observed in vitro (DX3puroβ6 cells, 15 min: 43.2% binding, > 6:1 for DX3puroβ6:DX3puro). In the mouse model DX3puroβ6-tumor binding was low (1 h: 0.47 ± 0.28% ID/g, 4 h: 0.14 ± 0.09% ID/g) and clearing from the bloodstream was via the renal and hepatobiliary routes (urine: 167 ± 84% ID/g at 1 h, 10.3 ± 4.8% ID/g at 4 h; gall bladder: 95 ± 33% ID/g at 1 h, 63 ± 11% ID/g at 4 h).ConclusionCopper-free, strain-promoted click chemistry is an attractive, straightforward approach to radiolabeling. Although the [¹⁸F]FBA-C₆-ADBIO-based prosthetic group did not interfere with α_vβ₆-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature.     

Longitudinal monitoring of tumor antiangiogenic therapy with near-infrared fluorophore-labeled agents targeted to integrin αvβ3 and vascular endothelial growth factor

Purpose

Optical imaging is emerging as a powerful tool for the noninvasive imaging of the biological processes in living subjects. This study aimed to investigate whether optical imaging of integrin α_vβ₃ and vascular endothelial growth factor (VEGF) expression can serve as sensitive biomarkers for tumor early response to antiangiogenic therapy.

Methods

We synthesized two near-infrared fluorescence (NIRF) imaging agents, CF680R-3PRGD2 and CF750-BevF(ab')₂, which were designed to specifically bind to integrin α_vβ₃ and VEGF, respectively. The ability of optical imaging using the two imaging agents for early monitoring the antiangiogenic effect of sunitinib was evaluated.

Results

CF680R-3PRGD2 and CF750-BevF(ab')₂ specifically bound to their respective targets in vitro and in HT-29 tumor-bearing nude mice. Sunitinib treatment led to significantly decreased tumor uptake of CF680R-3PRGD2 (e.g., 7.47?±?1.62 % vs. 4.24?±?0.16 % on day 4; P?<?0.05) and CF750-BevF(ab')₂ (e.g., 7.43?±?2.43 % vs. 4.04?±?1.39 % on day 2; P?<?0.05) in vivo. Immunofluorescence staining and an enzyme-linked immunosorbent assay confirmed that sunitinib-induced changes in tumor uptake of CF680R-3PRGD2 and CF750-BevF(ab')₂ were correlated with changes in the levels of integrin α_vβ₃ and VEGF. Radiobiodistribution of ^99mTc-3PRGD2 and ¹²⁵I-BevF(ab')₂, the radiocounterparts of CF680R-3PRGD2 and CF750-BevF(ab')₂, respectively, also validated optical imaging results.

Conclusion

Longitudinal monitoring of tumor integrin α_vβ₃ and VEGF expression could be used as early biomarkers for tumor response to antiangiogenic therapy. This strategy may facilitate the development of new antiangiogenic drugs, and be used for elucidation of the underlying mechanisms of therapies involving the integrin and the VEGF signaling pathway.     

Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

IntroductionAffibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL).MethodsThe HER2-targeting NCL-cyclized Affibody molecule Z_HER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. The binding affinity of DOTA-Z_HER2:342min was evaluated in vitro. The targeting properties of ¹¹¹In- and ⁶⁸ Ga-DOTA-Z_HER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of ¹¹¹In- and ⁶⁸ Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge.ResultsThe dissociation constant (K_D) for DOTA-Z_HER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K_D1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 ± 1.3 for ¹¹¹In- DOTA-Z_HER2:342min and 4.6 ± 0.7 for ⁶⁸ Ga-DOTA-Z_HER2:342min. However, the uptake of DOTA-Z_HER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.ConclusionsNative chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z_HER2:342min) with low nanomolar target affinity and specific tumor uptake.     

Design and biological evaluation of 99mTc-N2S2-Tat(49–57)-c(RGDyK): A hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins

The α(ν)β(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) (^99mTc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional ^99mTc-RGD peptide (^99mTc-EDDA/HYNIC-E-[c(RGDfK)]₂). Methods: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys¹² side chain of the Tat(49–57)-spacer-N₂S₂ peptide. ^99mTc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)β(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. Results: ^99mTc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for ^99mTc-Tat-RGD and 5.6% for ^99mTc-RGD. The IC₅₀ values were 6.7 nM (^99mTc-Tat-RGD) and 4.6 nM (^99mTc-RGD). Internalization in C6 cells was higher in ^99mTc-Tat-RGD (37.5%) than in ^99mTc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for ^99mTc-Tat-RGD (6.98% ± 1.34% ID/g at 3 h) than that of ^99mTc-RGD (3.72% ± 0.52% ID/g at 3 h) with specific recognition for α(v)β(3) integrins. Conclusions: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)β(3) integrins, the hybrid ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.     

��v��3 imaging can accurately distinguish between mature teratoma and necrosis in 18F-FDG-negative residual masses after treatment of non-seminomatous testicular cancer: a preclinical study

Purpose

We assessed whether imaging ??_v??₃ integrin could distinguish mature teratoma from necrosis in human non-seminomatous germ cell tumour (NSGCT) post-chemotherapy residual masses.

Methods

Human embryonal carcinoma xenografts (six/rat) were untreated (controls) or treated to form mature teratomas with low-dose cisplatin and all-trans retinoic acid (ATRA) over a period of 8?weeks. In another group, necrosis was induced in xenografts with high-dose cisplatin plus etoposide (two cycles).¹⁸F-Fluorodeoxyglucose (¹⁸F-FDG) small animal positron emission tomography (SA PET) imaging was performed in three rats (one control and two treated for 4 and 8?weeks with cisplatin+ATRA). Imaging of ??_v??₃ expression was performed in six rats bearing mature teratomas and two rats with necrotic lesions on a microSPECT/CT device after injection of the tracer [^99mTc]HYNIC-RGD [6-hydrazinonicotinic acid conjugated to cyclo(Arg-Gly-Asp-D-Phe-Lys)]. Correlative immunohistochemistry studies of human and mouse ??_v??₃ expression were performed.

Results

Cisplatin+ATRA induced differentiation of the xenografts. After 8?weeks, some glandular structures and mesenchymal cells were visible; in contrast, control tumours showed undifferentiated tissues. SA PET imaging showed that mature teratoma had very low avidity for ¹⁸F-FDG [mean standardised uptake value (SUV_mean)?=?0.48?±?0.05] compared to untreated embryonal carcinoma (SUV_mean?=?0.92?±?0.13) (p?=?0.005). ??_v??₃ imaging accurately distinguished mature teratoma (tumour to muscle ratio?=?4.29?±?1.57) from necrosis (tumour to muscle ratio?=?1.3?±?0.26) (p?=?0.0002). Immunohistochemistry studies showed that ??_v??₃ integrin expression was strong in the glandular structures of mature teratoma lesions and negative in host stroma.

Conclusion

Imaging ??_v??₃ integrin accurately distinguished mature teratoma from necrosis following cisplatin-based treatment in human NSGCT xenografts.     

Be spoilt for choice with radiolabelled RGD peptides: Preclinical evaluation of 68 Ga-TRAP(RGD)3

Gallium-68 is rapidly gaining importance, as this generator-produced PET isotope is available independent of on-site cyclotrons, enabling radiopharmaceutical production with comparably simple techniques at low cost. The recently introduced TRAP chelator combines the advantage of straightforward design of multimeric ⁶⁸ Ga-radiopharmaceuticals with very fast and efficient ⁶⁸ Ga-labeling. We synthesized a series of five cyclo(RGDfK) peptide trimers and determined their α_vβ₃ integrin affinities in competition assays on α_vβ₃-expressing M21 human melanoma cells against ¹²⁵I-echistatin. The compound with highest IC₅₀, Ga-TRAP(RGD)₃, showed more than 7-fold higher affinity compared to the monomers F-Galacto-RGD and Ga-NODAGA-c(RGDyK). TRAP(RGD)₃ was radiolabeled with ⁶⁸ Ga in a fully automated GMP compliant manner. CD-1 athymic nude mice bearing M21/M21L human melanoma xenografts were used for biodistribution studies, blockade experiments, metabolite studies and PET imaging. ⁶⁸ Ga-TRAP(RGD)₃ exhibited high M21 tumor uptake (6.08 ± 0.63% ID/g, 60 min p.i.), was found to be fully stable in vivo, and showed a fast renal clearance. Blockade studies showed that uptake in the tumor, as well as in all other tissues, is highly integrin specific. A comparison of biodistribution and PET data of ⁶⁸ Ga-TRAP(RGD)₃ with those of ⁶⁸ Ga-NODAGA-c(RGDyK) and ¹⁸ F-Galacto-RGD showed that the higher affinity of the trimer effects a larger dynamic response of tracer uptake to integrin expression, i.e., enhanced integrin-specific uptake in all tissues. We conclude that ⁶⁸ Ga-TRAP(RGD)₃ could allow for imaging of low-level integrin expression in tissues which are not visible with the two competitors. Overall, the study constitutes proof of concept for the favourable in vivo properties of TRAP-based ⁶⁸ Ga radiopharmaceuticals.     

Decay data of 123Tem

Measurements of coincidences between conversion electrons of the 88 keV transition in the ¹²³Te^m decay and subsequent 159 keV quanta have been made with a 4πβ-γ coincidence system equipped with a pressurized proportional counter (PPC) in the beta channel. With ¹²³Te^m sources prepared for activity determination, a detection probability for conversion electrons, ϵ, very close to ϵ = 1 was achieved. The following data have been derived from the slope of a polynomial fit to n_βn_γ/n_c vs (1 − ϵ)/ϵ by varying ϵ by discrimination and taking into account the γ sensitivity of the PPC (n_β, n_γ, n_c are the counting rates in the β, γ and coincidence channel): the total conversion coefficient of the 159 keV transition α_t = 0.1895(13), the γ-ray emission probability of the 159 keV photons p = 0.8407(9). These data agree with the theoretically calculated values, and with less accurate values determined experimentally by other authors.     

Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with 111In using a maleimido derivative of NODAGA

IntroductionAffibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule.MethodsThe maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z_HER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 were studied. Biodistribution of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 and [¹¹¹In-MMA-DOTA-Cys⁶¹]-Z_HER2:2395 was compared in mice.ResultsThe affinity of [MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 binding to HER2 was 67 pM. The ¹¹¹In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [¹¹¹In-MMA-DOTA-Cys⁶¹]-Z_HER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 due to higher clearance rate from normal tissues.ConclusionsMMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.     

PET imaging of α<Subscript>v</Subscript>β<Subscript>3</Subscript> integrin expression in tumours with <Superscript>68</Superscript>Ga-labelled mono-, di- and tetrameric RGD peptides

Purpose  

Due to the restricted expression of α_vβ₃ in tumours, α_vβ₃ is considered a suitable receptor for tumour targeting. In this study the α_vβ₃-binding characteristics of ⁶⁸Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their ¹¹¹In-labelled counterparts.