Dual‐isotope 111In/177Lu SPECT imaging as a tool in molecular imaging tracer design

The synthesis, design and subsequent pre‐clinical testing of new molecular imaging tracers are topic of extensive research in healthcare. Quantitative dual‐isotope SPECT imaging is proposed here as a tool in the design and validation of such tracers, as it can be used to quantify and compare the biodistribution of a specific ligand and its nonspecific control ligand, labeled with two different radionuclides, in the same animal. Since the biodistribution results are not blurred by experimental or physiological inter‐animal variations, this approach allows determination of the ligand's net targeting effect. However, dual‐isotope quantification is complicated by crosstalk between the two radionuclides used and the radionuclides should not influence the biodistribution of the tracer. Here, we developed a quantitative dual‐isotope SPECT protocol using combined ¹¹¹Indium and ¹⁷⁷Lutetium and tested this tool for a well‐known angiogenesis‐specific ligand (cRGD peptide) in comparison to a potential nonspecific control (cRAD peptide). Dual‐isotope SPECT imaging of the peptides showed a similar organ and tumor uptake to single‐isotope studies (cRGDfK–DOTA, 1.5 ± 0.8%ID cm^?3; cRADfK–DOTA, 0.2 ± 0.1%ID cm^?3), but with higher statistical relevance (p‐value 0.007, n = 8). This demonstrated that, for the same relevance, seven animals were required in case of a single‐isotope test design as compared with only three animals when a dual‐isotope test was used. Interchanging radionuclides did not influence the biodistribution of the peptides. Dual‐isotope SPECT after simultaneous injection of ¹¹¹In and ¹⁷⁷Lu‐labeled cRGD and cRAD was shown to be a valuable method for paired testing of the in vivo target specificity of ligands in molecular imaging tracer design. Copyright © 2012 John Wiley & Sons, Ltd.     

SPECT imaging of fibrin using fibrin‐binding peptides

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision‐making on possible therapeutic options in atherosclerosis, cancer and thrombus‐related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel ¹¹¹In‐labeled fibrin‐binding peptide (FibPep) to visualize thrombi in mice noninvasively using single‐photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin‐binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K_d) of 0.8 μ m , whereas NCFibPep displayed at least a 100‐fold lower affinity towards fibrin. A FeCl₃‐injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus‐containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g^?1), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g^?1, respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

Imaging integrin alpha‐v‐beta‐3 expression in tumors with an 18F‐labeled dimeric RGD peptide

Integrin α_vβ₃ receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine–glycine–aspartatic acid (RGD) peptide and integrin α_vβ₃, but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of ¹⁸F. In this study, the dimeric RGD peptide, E‐[c(RGDfK)]₂, was conjugated with NODAGA and radiolabeled with ¹⁸F in a simple one‐pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA‐E‐[c(RGDfK)]₂ labeled with ¹⁸F at a specific activity of 1.8 MBq nmol^?1 and a radiochemical purity of 100% could be achieved. The logP value of ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂ was ?4.26 ± 0.02. In biodistribution studies, ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g^?1) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ (3.44 ± 0.20 %ID g^?1, 2 h p.i.) was significantly lower than that of reference compounds ⁶⁸Ga‐labeled NODAGA‐E‐[c(RGDfK)]₂ (6.26 ± 0.76 %ID g^?1; p <0.001) and ¹¹¹In‐labeled NODAGA‐E‐[c(RGDfK)]₂ (4.99 ± 0.64 %ID g^?1; p < 0.01). Co‐injection of an excess of unlabeled NODAGA‐E‐[c(RGDfK)]₂ along with ¹⁸F‐NODAGA‐E‐[c(RGDfK)]₂ resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g^?1). The α_vβ₃ integrin‐expressing SK‐RC‐52 tumor could be successfully visualized by microPET with ¹⁸F‐labeled NODAGA‐E‐[c(RGDfK)]₂. In conclusion, NODAGA‐E‐[c(RGDfK)]₂ could be labeled rapidly with ¹⁸F using a direct aqueous, one‐pot method and it accumulated specifically in α_vβ₃ integrin‐expressing SK‐RC‐52 tumors, allowing for visualization by microPET. Copyright © 2013 John Wiley & Sons, Ltd.     

[68Ga]‐HP‐DO3A‐nitroimidazole: a promising agent for PET detection of tumor hypoxia

The goal of this study is to evaluate a new ⁶⁸Ga‐based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [⁶⁸Ga]‐HP‐DO3A‐nitroimidazole ([⁶⁸Ga]‐HP‐DO3A‐NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP‐DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [⁶⁸Ga]‐HP‐DO3A‐NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O₂. The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post‐injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [⁶⁸Ga]‐HP‐DO3A‐NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [⁶⁸Ga]‐HP‐DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor‐bearing mice breathing air versus 100% O₂. Given the commercial availability of cGMP ⁶⁸Ge/⁶⁸Ga generators and the ease of ⁶⁸Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy. Copyright © 2015 John Wiley & Sons, Ltd.     

Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

Tissue‐engineered nanoclay‐based 3D in vitro breast cancer model for studying breast cancer metastasis to bone

Breast cancer (BrCa) preferentially spreads to bone and colonises within the bone marrow to cause bone metastases. To improve the outcome of patients with BrCa bone metastasis, we need to understand better the mechanisms underlying bone metastasis. Researchers have relied heavily upon in vivo xenografts due to limited availability of human bone metastasis samples. A significant limitation of these is that they do not have a human bone microenvironment. To address this issue, we have developed a nanoclay‐based 3D in vitro model of BrCa bone metastasis using human mesenchymal stem cells (MSCs) and human BrCa cells mimicking late stage of BrCa pathogenesis at the metastatic site. This 3D model can provide a microenvironment suitable for cell–cell and cell–matrix interactions whilst retaining the behaviour of BrCa cells with different metastasis potential (i.e., highly metastatic MDA‐MB‐231 and low metastatic MCF‐7) as shown by the production of alkaline phosphatase and matrix metalloproteinase‐9. The sequential culture of MSCs with MCF‐7 exhibited 3D tumouroids formation and also occurrence of mesenchymal to epithelial transition of cancer metastasis as evidenced by gene expression and immunocytochemistry. The unique and distinct behaviour of highly metastatic MDA‐MB‐231 and the low metastatic MCF‐7 was observed at the bone metastasis site. The changes to migratory capabilities and invasiveness in MDA‐MB‐231 in comparison with tumour growth with MCF‐7 was observed. Together, a novel bone‐mimetic 3D in vitro BrCa model has been developed that could be used to study mechanisms governing the later stage of cancer pathogenesis in bone.     

PET of EGFR with 64Cu‐cetuximab‐F(ab′)2 in mice with head and neck squamous cell carcinoma xenografts

Overexpression of the epidermal growth factor receptor (EGFR) is linked to an adverse outcome in various solid tumors. Cetuximab is an EGFR inhibitor, which in combination with radiotherapy improves locoregional control and survival in a subgroup of patients with head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to develop and characterize an EGFR‐directed PET tracer, ⁶⁴Cu‐cetuximab‐F(ab′)₂, to determine the systemic accessibility of EGFR. Mice with HNSCC xenografts, UT‐SCC‐8 (n = 6) or UT‐SCC‐45 (n = 6), were imaged 24 h post injection with ⁶⁴Cu‐NODAGA‐cetuximab‐F(ab′)₂ using PET/CT. One mouse for each tumor model was co‐injected with excess unlabeled cetuximab 3 days before radiotracer injection to determine non‐EGFR‐mediated uptake. Ex vivo biodistribution of the tracer was determined and tumors were analyzed by autoradiography and immunohistochemistry. The SUV_max of UT‐SCC‐8 tumors was higher than that of UT‐SCC‐45: 1.5 ± 1.0 and 0.8 ± 0.2 (p < 0.05), respectively. SUV_max after in vivo blocking of EGFR with cetuximab was 0.4. Immunohistochemistry showed that UT‐SCC‐8 had a significantly higher EGFR expression than UT‐SCC‐45: 0.50 ± 0.19 versus 0.12 ± 0.08 (p < 0.005), respectively. Autoradiography indicated that ⁶⁴Cu‐cetuximab‐F(ab′)₂ uptake correlated with EGFR expression in both tumors: r = 0.86 ± 0.06 (UT‐SCC‐8) and 0.90 ± 0.06 (UT‐SCC‐45). ⁶⁴Cu‐cetuxmab‐F(ab′)₂ is a promising PET tracer to determine expression of EGFR in vivo. Clinically, this tracer has the potential to be used to determine cetuximab targeting of tumors and possibly to non‐invasively monitor the response to EGFR‐inhibitor treatment. Copyright © 2015 John Wiley & Sons, Ltd.     

A novel PET tracer for the imaging of αvβ3 and αvβ5 integrins in experimental breast cancer bone metastases

The aim of this study was the evaluation of ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ as a novel PET tracer to image αvβ3 and αvβ5 integrins. For this purpose, DOTA‐E‐[c(RGDfK)]₂ was labeled with ⁶⁸Ga, which was obtained from a ⁶⁸Ge/⁶⁸Ga generator, purified by solid‐phase extraction and the radiochemical purity analyzed by radio‐RP‐HPLC. ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ was obtained reproducibly in radiochemical yields of 60 ± 6% and with an excellent radiochemical purity of >99%. In nude rats bearing bone metastases after injection of MDA‐MB‐231 human breast cancer cells, biodistribution studies were performed to evaluate the accumulation of the radiotracer in selected organs, blood and bone metastases 0.5, 1, 2 and 3 h post injection. A rapid uptake into the bone metastases and rapid blood clearance was observed, resulting in tumor–blood ratios of up to 26.6 (3 h post injection) and tumor–muscle ratios of up to 7.9 (3 h post injection). A blocking experiment with coinjected αvβ3/αvβ5 antagonist showed the tumor uptake to be receptor‐specific. In an initial in vivo micro PET evaluation of the tracer using the same animal model, the bone metastasis was clearly visualized. These results suggest that ⁶⁸Ga‐DOTA‐E‐[c(RGDfK)]₂ is a promising PET tracer suitable for the imaging of αvβ3 and αvβ5 integrins in bone metastases. This novel PET tracer should be further evaluated concerning its usefulness for early detection of bone metastases and monitoring treatment response of these lesions. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and characterization of a porphyrazine–Gd(III) MRI contrast agent and in vivo imaging of a breast cancer xenograft model

Porphyrazines (Pz), or tetraazaporphyrins, are being studied for their potential use in detection and treatment of cancer. Here, an amphiphilic Cu–Pz–Gd(III) conjugate has been prepared via azide–alkyne Huisgen cycloaddition or ‘click’ chemistry between an azide functionalized Pz and alkyne functionalized DOTA–Gd(III) analog for use as an MRI contrast agent. This agent, Cu–Pz–Gd(III), is synthesized in good yield and exhibits solution‐phase ionic relaxivity (r₁ = 11.5 mm ^?1 s^?1) that is approximately four times higher than that of a clinically used monomeric Gd(III) contrast agent, DOTA–Gd(III). Breast tumor cells (MDA‐MB‐231) associate with Cu–Pz–Gd(III) in vitro, where significant contrast enhancement (9.336 ± 0.335 contrast‐to‐noise ratio) is observed in phantom cell pellet MR images. This novel contrast agent was administered in vivo to an orthotopic breast tumor model in athymic nude mice and MR images were collected. The average T₁ of tumor regions in mice treated with 50 mg kg^?1 Cu–Pz–Gd(III) decreased relative to saline‐treated controls. Furthermore, the decrease in T₁ was persistent relative to mice treated with the monomeric Gd(III) contrast agent. An ex vivo biodistribution study confirmed that Cu–Pz–Gd(III) accumulates in the tumors and is rapidly cleared, primarily through the kidneys. Differential accumulation and T₁ enhancement by Cu–Pz–Gd(III) in the tumor's core relative to the periphery offer preliminary evidence that this agent would find application in the imaging of necrotic tissue. Copyright © 2014 John Wiley & Sons, Ltd.     

Life‐threatening hemorrhage and prolonged wound healing are remarkable phenotypes manifested by complete plasminogen activator inhibitor‐1 deficiency in humans

Summary. Background: Plasminogen activator inhibitor‐1 (PAI‐1) is the primary physiological regulator of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) activity. A number of studies have shown that elevated levels of PAI‐1 are related to pathological states such as an increased risk of arterial thrombotic events and a poor prognosis for cancer patients; however, there are few reports about PAI‐1 deficiency in humans because the disorder is very rare. Objective: To understand the in vivo impact of a complete PAI‐1 deficiency, Serpine1^?/? mice were generated; a number of in vivo studies have been conducted to elucidate the function of PAI‐1 using Serpine1^?/? mice. The phenotypes demonstrated in Serpine1^?/? mice, however, were quite different from those in humans. Therefore, it is necessary to find out and analyze SERPINE1 deficiency in humans. Patient and methods: The patient is a 47‐year‐old woman who has had multiple episodes of major bleeding. Although most of the patient’s blood coagulation factors were functionally normal, her PAI‐1 antigen levels were undetectable. Therefore, DNA sequencing of the SERPINE1 gene were analyzed. Results: The proband had a homozygous 1‐bp duplication (C) at exon 3 (c.356dupC; p.Ile120AspfsX42). Both wild‐type PAI‐1 (42.7 kDa) and mutated (Mut) PAI‐1 (14.7 kDa) were expressed in COS‐1 cells, although the level of Mut PAI‐1 expressed in the cell lysates was much lower. Wild‐type PAI‐1 was observed in the culture supernatant, whereas no Mut PAI‐1 was detected in the supernatant. Conclusions: Considering the results of the present study, the translation of mouse studies to humans must be performed with great care.     

Gallium(III) complexes of NOTA‐bis (phosphonate) conjugates as PET radiotracers for bone imaging

Ligands with geminal bis(phosphonic acid) appended to 1,4,7‐triazacyclonone‐1,4‐diacetic acid fragment through acetamide (NOTAM^BP) or methylenephosphinate (NO2AP^BP) spacers designed for ⁶⁸Ga were prepared. Ga^III complexation is much faster for ligand with methylenephosphinate spacer than that with acetamide one, in both chemical (high reactant concentrations) and radiolabeling studies with no‐carrier‐added ⁶⁸Ga. For both ligands, formation of Ga^III complex was slower than that with NOTA owing to the strong out‐of‐cage binding of bis(phosphonate) group. Radiolabeling was efficient and fast only above 60 °C and in a narrow acidity region (pH ~3). At higher temperature, hydrolysis of amide bond of the carboxamide‐bis(phosphonate) conjugate was observed during complexation reaction leading to Ga–NOTA complex. In vitro sorption studies confirmed effective binding of the ⁶⁸Ga complexes to hydroxyapatite being comparable with that found for common bis(phosphonate) drugs such as pamindronate. Selective bone uptake was confirmed in healthy rats by biodistribution studies ex vivo and by positron emission tomography imaging in vivo. Bone uptake was very high, with SUV (standardized uptake value) of 6.19 ± 1.27 for [⁶⁸Ga]NO2AP^BP) at 60 min p.i., which is superior to uptake of ⁶⁸Ga–DOTA‐based bis(phosphonates) and [¹⁸F]NaF reported earlier (SUV of 4.63 ± 0.38 and SUV of 4.87 ± 0.32 for [⁶⁸Ga]DO3AP^BP and [¹⁸F]NaF, respectively, at 60 min p.i.). Coincidently, accumulation in soft tissue is generally low (e.g. for kidneys SUV of 0.26 ± 0.09 for [⁶⁸Ga]NO2AP^BP at 60 min p.i.), revealing the new ⁶⁸Ga complexes as ideal tracers for noninvasive, fast and quantitative imaging of calcified tissue and for metastatic lesions using PET or PET/CT. Copyright © 2014 John Wiley & Sons, Ltd.     

ImmunoPET Imaging of α<Subscript>v</Subscript>β<Subscript>6</Subscript> Expression Using an Engineered Anti-α<Subscript>v</Subscript>β<Subscript>6</Subscript> Cys-diabody Site-Specifically Radiolabeled with Cu-64: Considerations for Optimal Imaging with Antibody Fragments

Purpose

Increased expression of the α_vβ₆ integrin correlates with advanced tumor grade and poor clinical outcome, identifying α_vβ₆ as a prognostic indicator and an attractive target for molecular imaging. This work investigated the ability of a disulfide-stabilized [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody to image α_vβ₆ expression in vivo using a nu/nu mouse model bearing human melanoma xenografts and positron-emission tomography.

Procedures

Small-animal positron emission tomography (PET) imaging, quantitative ROI analysis, and ex vivo biodistribution were conducted to ascertain tumor uptake and organ distribution of the [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody. Immunohistochemical staining of tumors and mouse organs and immunoreactivity assays were utilized to correlate in vivo and ex vivo observations.

Results

PET imaging of the [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody revealed low tumor uptake at 24 h p.i. in DX3Puroβ₆ tumors (2.69 ± 0.45 %ID/g) with comparable results found in the DX3Puro tumors (2.24 ± 0.15 %ID/g). Quantitative biodistribution confirmed that DX3Puroβ₆ tumor uptake was highest at 24 h p.i. (4.63 ± 0.18 %ID/g); however, uptake was also observed in the stomach (4.84 ± 2.99 %ID/g), small intestines (4.50 ± 1.69 %ID/g), large intestines (4.73 ± 0.97 %ID/g), gallbladder (6.04 ± 1.88 %ID/g), and lungs (3.89 ± 0.69 %ID/g).

Conclusions

Small-animal PET imaging was successful in visualizing α_vβ₆-positive tumor uptake of the [⁶⁴Cu]NOTA-α_vβ₆ cys-diabody. Cys-diabody cross-reactivity was observed between human and murine α_vβ₆ and immunohistochemical staining confirmed the presence of an endogenous α_vβ₆ antigen sink, which led to suboptimal tumor contrast in this mouse model. Future investigations will focus on dose escalation studies to overcome the endogenous antigen sink while increasing DX3Puroβ₆ tumor uptake.

     

In Vivo Bacteriophage Peptide Display to Tailor Pharmacokinetics of Biological Nanoparticles

Purpose

Clinical use of most radiolabeled targeting agents has been limited because of the uptake and retention in kidney and/or liver. We hypothesized that bacteriophage (phage) display could be exploited to select for peptide sequences with fast clearance and low kidney uptake with the added ability to redirect phage clearance away from the reticuloendothelial system towards the kidney possessing rapid kidney clearance.

Procedures

In vivo phage display was performed to identify peptides displayed on phage that were excreted rapidly into the urine of mice. A novel in vitro assay using kidney cells, developed to predict in vivo kidney retention, and in vivo pharmacokinetic analyses were performed to characterize selected peptides/phage clones.

Results

Forty-three renal clearance clones (RCC) were identified. In vivo mixing experiments and in vitro kidney cell assays identified RCC1-02 as the lead compound. In vivo analysis of fluorescently labeled phage clones demonstrated the ability of RCC1-02 peptide to redirect the biodistribution of the large phage particle towards excretion via the kidney. Pharmacokinetic analysis of [¹¹¹In]-radiolabeled peptides revealed that kidney retention of the control ErBB-2-avid peptide, [¹¹¹In]DOTA-KCCYSL, at 2-h postinjection was 5.7?±?0.7 %ID/g. In comparison, [¹¹¹In]DOTA-RCC1-02 had kidney retention values of 1.66?±?0.43 %ID/g, respectively.

Conclusions

In vivo phage display can identify phage and corresponding peptides that rapidly clear the renal system. In the future, these peptides may be used to impart favorable pharmacokinetics onto a wide range of radioimaging or therapeutic macromolecules.     

Kidney volume quantification using contrast‐enhanced in vivo X‐ray micro‐CT in mice

The present study sought to establish a standard in vivo imaging procedure for mouse kidney anatomy evaluation using contrast‐enhanced high‐resolution X‐ray microtomography (micro‐CT). Micro‐CT estimation of kidney volume was compared with ex vivo measurement by micro‐CT and water displacement. Control values were obtained in four strains (BALB/c, C3H/HeN, 129/Sv and C57BL/6J) of healthy male and female mice aged 22 ± 2 weeks. An excellent correlation was found between in vivo and ex vivo kidney volumes (n = 26 mice; 52 kidneys; r = 0.96). In vivo measurement systematically overestimated ex vivo kidney volume by 28 ± 4%, while there was no significant difference between the ex vivo micro‐CT value and the true kidney volume on water displacement (2.3 ± 2.1%). In vivo kidney volume also correlated strongly with kidney weight and in vivo kidney length (n = 52 mice; 104 kidneys; r = 0.84, r = 0.92 respectively). Differences between strains were observed for kidney volume when comparing either kidney volume or kidney weight to body weight. In conclusion, this study demonstrated that contrast‐enhanced micro‐CT enables accurate in vivo measurement of kidney volume, length and thickness in mice. Reference parameters are reported for four strains. The technique provides a useful follow‐up research tool for mouse phenotyping and renal disease studies. Copyright © 2008 John Wiley & Sons, Ltd.     

COX‐1+/−COX‐2−/− genotype in mice is associated with shortened time to carotid artery occlusion through increased PAI‐1

Summary. Background: We found a high incidence of thrombotic deaths in COX‐1^+/?COX‐2^?/? mice and sought to define the mechanism of these events. The cyclooxygenase products thromboxane A₂ and prostacyclin are important in the regulation of coagulation but their role in fibrinolysis is largely unexplored. PAI‐1 blocks fibrinolysis by inhibiting plasminogen activator. Aim: Our objective was to explain the mechanism of increased thrombosis associated with the COX‐1^+/?COX‐2^?/? genotype. Methods: Carotid artery occlusion times were measured after photochemical injury. PAI‐1 levels were measured in the plasma by ELISA. PAI‐1 levels in the aorta were measured by RT‐PCR and Western blotting. Urinary metabolites of Thromboxane A₂ and prostacyclin were measured by ELISA. Results: The COX‐1^+/?COX‐2^?/? genotype is associated with a decreased time to occlusion in the carotid artery thrombosis model (30 ± 5 minutes vs 60 ± minutes in wild type, p<.001). The COX‐1^?/?COX‐2^+/+, COX‐1^+/?COX‐2^+/? and COX‐1^+/? COX‐2^+/+ all had occlusion times similar to wild type. COX‐1^+/+ COX‐2^?/? had a prolonged occlusion time. COX‐1^+/? COX‐2^?/? had increased PAI‐1 levels in the plasma and aorta and with a prolonged euglobulin lysis time (37.4 ± 10.2 hours vs 15.6 ± 9.8 hours in wild type, p<.004). The decreased time to occlusion in the COX‐1^+/?COX2^?/? mice was normalized by an inhibitory antibody to PAI‐1 whereas the antibody had no effect on the time to occlusion in wild type mice. Conclusion: The COX‐1^+/?COX‐2^?/? genotype is associated with a shortened time to occlusion in the carotid thrombosis model and the shortened time to occlusion is mediated through increased PAI‐1 levels resulting in decreased fibrinolysis.     

Plasminogen activator inhibitor‐1 inhibits factor VIIa bound to tissue factor

Summary. Background and objective: A growing body of experimental evidence supports broad inhibitory and regulatory activity of plasminogen activator inhibitor 1 (PAI‐1). The present study was designed to investigate whether PAI‐1 inhibits factor (F) VIIa complexed with tissue factor (TF), a well‐known procoagulant risk factor. Methods and results: The ability of PAI‐1 to inhibit FVIIa‐TF activity was evaluated in both clotting and factor X (FX) activation assays. PAI‐1 and its complex with vitronectin inhibit: (i) clotting activity of FVIIa‐TF (PAI‐1_IC50, 817 and 125 nm , respectively); (ii) FVIIa‐TF‐mediated FX activation (PAI‐1_IC50, 260 and 50 nm , respectively); and (iii) FVIIa bound to TF expressed on the surface of stimulated endothelial cells (PAI‐1_IC50, 260 and 120 nm , respectively). The association rate constant (k_a) for PAI‐1 inhibition of FVIIa‐TF was determined using a chromogenic assay. K_a for PAI‐1 inhibition of FVIIa bound to relipidated TF is 3.3‐fold higher than that for FVIIa bound to soluble TF (k_a = 0.09 ± 0.01 and 0.027 ± 0.03 μm ^?1 min^?1, respectively). Vitronectin increases k_a for both soluble and relipidated TF by 3.5‐ and 30‐fold, respectively (to 0.094 ± 0.020 and 2.7 ± 0.2 μm ^?1 min^?1). However, only a 3.5‐ to 5.0‐fold increase in the acylated FVIIa was observed on SDS PAGE in the presence of vitronectin for both relipidated and soluble TF, indicating fast formation of PAI‐1/vitronectin/FVIIa/relipidated TF non‐covalent complex. Conclusions: Our results demonstrate potential anticoagulant activity of PAI‐1 in the presence of vitronectin, which could contribute to regulation of hemostasis under pathological conditions such as severe sepsis, acute lung injury and pleural injury, where PAI‐1 and TF are overexpressed.     

Improved Radiosynthesis and Biological Evaluations of L- and D-1-[<Superscript>18</Superscript>F]Fluoroethyl-Tryptophan for PET Imaging of IDO-Mediated Kynurenine Pathway of Tryptophan Metabolism

Purpose

Tryptophan metabolism via indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway plays a role in immunomodulation and has been emerging as a plausible target for cancer immunotherapy. Imaging IDO-mediated kynurenine pathway of tryptophan metabolism with positron emission tomography (PET) could provide valuable information for noninvasive assessment of cancer immunotherapy response. In this work, radiotracer 1-(2-[¹⁸F]fluoroethyl)-L-tryptophan (1-L-[¹⁸F]FETrp) and its enantioisomer 1-D-[¹⁸F]FETrp were synthesized and evaluated for PET imaging of IDO-mediated kynurenine pathway of tryptophan metabolism.

Procedures

Enantiopure 1-L-[¹⁸F]FETrp and 1-D-[¹⁸F]FETrp were prepared by a nucleophilic reaction of N-boc-1-(2-tosylethyl) tryptophan tert-butyl ester with [¹⁸F]Fluoride, followed by acid hydrolysis in a GE Tracerlab FX-N module. In vitro cell uptake assays were performed with a breast cancer cell line MDA-MB-231. Small animal PET/computed tomography (CT) imaging was carried out in a mouse model bearing MDA-MB-231 xenografts.

Results

Automatic radiosynthesis of 1-L-[¹⁸F]FETrp and 1-D-[¹⁸F]FETrp was achieved by a one-pot two-step approach in 19.0 ± 7.0 and 9.0 ± 3.0 % (n = 3) decay-corrected yield with radiochemical purity over 99 %, respectively. In vitro cell uptake study indicated the uptake of 1-D-[¹⁸F]FETrp in MDA-MB-231 cells was 0.73 ± 0.07 %/mg of protein at 60 min, while, the corresponding uptake of 1-L-[¹⁸F]FETrp was 6.60 ± 0.77 %/mg. Further mechanistic assays revealed that amino acid transport systems L-tpye amino acid transporter (LAT) and alanine-, serine-, and cysteine-preferring (ASC), and enzyme IDO expression were involved in cell uptake of 1-L-[¹⁸F]FETrp. Small animal PET/CT imaging study showed the tumor uptake of 1-L-[¹⁸F]FETrp was 4.6 ± 0.4 % ID/g, while, the tumor uptake of 1-D-[¹⁸F]FETrp was low to 1.0 ± 0.2 % ID/g, which were confirmed by ex vivo biodistribution study.

Conclusions

We have developed a practical method for the automatic radiosynthesis of 1-L-[¹⁸F]FETrp and 1-D-[¹⁸F]FETrp. Our biological evaluation results suggest that 1-L-[¹⁸F]FETrp is a promising radiotracer for PET imaging of IDO-mediated kynurenine pathway of tryptophan metabolism in cancer.

     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

Syntheses and preliminary evaluation of [18F]AlF‐NOTA‐G‐TMTP1 for PET imaging of high aggressive hepatocellular carcinoma

The goal of this study is to evaluate a new ¹⁸F‐labeled imaging agent for diagnosing high metastatic (aggressive) hepatocellular carcinoma using positron emission tomography (PET). The new ¹⁸F‐labeled imaging agent [¹⁸F]AlF‐NOTA‐G‐TMTP1 was synthesized and radiolabeled with ¹⁸F using NOTA‐AlF chelation method. The tumor‐targeting characteristics of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was assessed in HepG2, SMCC‐7721, HCC97L and HCCLM3 xenografts. The total synthesis time was about 20 min with radiochemical yield of 25 ± 6%. The specific activity was about 11.1–14.8 GBq/µmol at the end of synthesis based on the amount of peptide used and the amount of radioactivity trapped on the C₁₈ column. The log P value of [¹⁸F]AlF‐NOTA‐G‐TMTP1 was ‐3.166 ± 0.022. [¹⁸F]AlF‐NOTA‐G‐TMTP1 accumulated in SMCC‐7721 and HCCLM3 tumors (high metastatic potential) in vivo and result in tumor/muscle (T/M) ratios of 4.5 ± 0.3 and 4.7 ± 0.2 (n = 4) as measured by PET at 40 min post‐injection (p.i.). Meanwhile, the tumor/muscle (T/M) ratios of HepG2 and HCC97L tumors (low metastatic potential) were1.6 ± 0.3 and 1.8 ± 0.4. The tumor uptake of [¹⁸F]AlF‐NOTA‐G‐TMTP1 could be inhibited 61.9% and 57.6% by unlabeled G‐TMTP1 in SMCC‐7721 and HCCLM3 xenografts at 40 min p.i., respectively. Furthermore, [¹⁸F]AlF‐NOTA‐G‐TMTP1 showed pretty low activity in the liver and intestines in all tumor bearing mice, such in vivo distribution pattern would be advantageous for the detection of hepatic carcinoma. Overall, [¹⁸F]AlF‐NOTA‐G‐TMTP1 may specifically target high metastatic or/and aggressive hepatocellular carcinoma with low background activity and, therefore, holds the potential to be used as an imaging agent for detecting tumor lesions within the liver area. Copyright © 2016 John Wiley & Sons, Ltd.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.