Accumulation In Non-target Organs Research Articles

Synthesis and Preclinical Evaluation of a 68Ga-Labeled Pyridine-Based Benzamide Dimer for Malignant Melanoma Imaging.

Benzamide (BZA), a small molecule that can freely cross cell membranes and bind to melanin, has served as an effective targeting group for melanoma theranostics. In this study, a novel pyridine-based BZA dimer (denoted as H-2) was labeled with 68Ga ([68Ga]Ga-H-2) for positron emission tomography (PET) imaging of malignant melanomas. [68Ga]Ga-H-2 was obtained with high radiochemical yield (98.0 ± 2.0%) and satisfactory radiochemical purity (>95.0%). The specificity and affinity of [68Ga]Ga-H-2 were confirmed in melanoma B16F10 cells and in vivo PET imaging of multiple tumor models (B16F10 tumors, A375 melanoma, and lung metastases). Monomeric [68Ga]Ga-H-1 was prepared as a control radiotracer to verify the effects of the molecular structure on pharmacokinetics. The values of the lipid-water partition coefficient of [68Ga]Ga-H-2 and [68Ga]Ga-H-1 demonstrated hydrophilicity with log P = -2.37 ± 0.07 and -2.02 ± 0.09, respectively. PET imaging and biodistribution showed a higher uptake of [68Ga]Ga-H-2 in B16F10 primary and metastatic melanomas than that in A375 melanomas. However, the relatively low uptake of monomeric [68Ga]Ga-H-1 in B16F10 tumors and high accumulation in nontarget organs resulted in poor PET imaging quality. This study demonstrates the synthesis and preclinical evaluation of the novel pyridine-based BZA dimer [68Ga]Ga-H-2 and indicates that the dimer tracer has promising applications in malignant melanoma-specific PET imaging because of its high uptake and long-time retention in malignant melanoma.

Development and preliminary evaluation of an integrin α2β1-targeted PET probe as a supplement and alternative of PSMA imaging for prostate cancer

An integrin α2β1-targeted PET probe (68Ga-IABtP) was developed to serve as a supplement and alternative of PSMA imaging for prostate cancer. 68Ga-IABtP was synthesized by labeling the precursor peptide with 68Ga with 93% labeling yield and 4.14 MBq/μg specific radioactivity. 68Ga-IABtP showed no specific uptake in LNCaP prostate cancer cell with low integrin α2β1 expression but significantly increased uptake in PC-3 prostate cancer cell with high integrin α2β1 expression, which could be specifically blocked by the integrin α2β1 monoclonal antibody. The efflux experiments demonstrated that 68Ga-IABtP could rapidly penetrate into PC-3 cell after cell binding, thereby prolonging the residence time in the tumor and allow enough time for probe clearance from the circulation and non-specific organs. The biodistribution study indicated that 68Ga-IABtP showed no specific accumulation in non-target organs and was quickly cleared from the kidney. The in vivo PET-CT imaging demonstrated that 68Ga-IABtP showed no specific uptake in LNCaP tumor but could specifically accumulate in the PC-3 tumor, and was rapidly cleared from spleen, intestine, kidney and liver, resulting in excellent contrast effect with low background signal and high target to non-target ratios.

Advances in Injectable In Situ-Forming Hydrogels for Intratumoral Treatment.

Chemotherapy has been linked to a variety of severe side effects, and the bioavailability of current chemotherapeutic agents is generally low, which decreases their effectiveness. Therefore, there is an ongoing effort to develop drug delivery systems to increase the bioavailability of these agents and minimize their side effects. Among these, intratumoral injections using in situ-forming hydrogels can improve drugs’ bioavailability and minimize drugs’ accumulation in non-target organs or tissues. This review describes different types of injectable in situ-forming hydrogels and their intratumoral injection for cancer treatment, after which we discuss the antitumor effects of intratumoral injection of drug-loaded hydrogels. This review concludes with perspectives on the future applicability of, and challenges for, the adoption of this drug delivery technology.

Clickable Radiocomplexes With Trivalent Radiometals for Cancer Theranostics: In vitro and in vivo Studies.

Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [90Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages.

Evaluation of a New 99mTc-labeled GnRH Analogue as a Possible Imaging Agent for Prostate Cancer Detection.

Prostate cancer is a serious threat to men's health so it is necessary to develop technics for early detection of this malignancy. The purpose of this research was the evaluation of a new99mTc-labeled GnRH analogue as an imaging probe for tumor targeting of prostate cancer. 99mTc-labeled-DLys6-GnRH analogue was prepared based on HYNIC as a chelating agent and tricine/ EDDA as coligands for labeling with 99mTc. HYNIC was coupled to epsilon amino group of DLys6 through aminobutyric acid (GABA) as a linker. Radiochemical purity and stability in normal saline and serum, were determined by TLC and HPLC methods. Furthermore, calculation of protein-binding and partition coefficient constant were carried out for 99mTc labeled peptide. The cellular experiments including receptor binding specificity and affinity were studied using three prostate cancer cell lines LN-CaP, DU-145 and PC-3. Finally, the animal assessment and SPECT imaging of radiolabeled GnRH analogue were evaluated on normal mice and nude mice bearing LN-CaP tumor. The GnRH conjugate was labeled with high radiochemical purity (~97%). The radiolabeled peptide showed efficient stability in the presence of normal saline and human serum. The in vitro cellular assays on three prostate cancer cell lines indicated that the radiotracer was bound to LN-CaP cells with higher affinity compared to DU-145 and PC-3 cells. The Kd values of 99mTc- HYNIC (tricine/ EDDA)-Gaba-D-Lys6GnRH were 89.39±26.71, 93.57±30.49 and107.3±18.82 in LN-CaP, PC-3 and DU-145 cells respectively. The biodistribution studies in normal mice and LN-CaP tumor-bearing nude mice showed similar results including rapid blood clearance and low radioactivity accumulation in non-target organs. High kidney uptake proved that the main excretion route of radiopeptide was through the urinary system. The tumor uptake was 1.72±0.45 %ID/g at 1h p.i. decreasing to 0.70±0.06%ID/g at 4h p.i. for 99mTc-HYNIC-Gaba-D-Lys6GnRH. The maximum tumor/ muscle ratio was 2.30 at 1h p.i. Pre-saturation of receptor using an excess of unlabeled peptide revealed that the tumor uptake was receptor mediated. The results of the SPECT image of LN-CaP tumor were in agreement with the biodistribution data. Based on this study, we suggest LN-CaP as a favorable cell line for in vivo studies on GnRH analogues. Moreover, this report shows that 99mTc-HYNIC (tricine/EDDA)-Gaba-D-Lys6GnRH may be a suitable candidate for further evaluation of prostate cancer.

Preclinical Evaluation of Radiolabeled Peptides for PET Imaging of Glioblastoma Multiforme.

In this study, we have compared four 68Ga-labeled peptides (three Arg-Gly-Asp (RGD) peptides and substance-P) with two 18F-tracers clinically approved for tumor imaging. We have studied in vitro and in vivo characteristics of selected radiolabeled tracers in a glioblastoma multiforme tumor model. The in vitro part of the study was mainly focused on the evaluation of radiotracers stability under various conditions. We have also determined in vivo stability of studied 68Ga-radiotracers by analysis of murine urine collected at various time points after injection. The in vivo behavior of tested 68Ga-peptides was evaluated through ex vivo biodistribution studies and PET/CT imaging. The obtained data were compared with clinically used 18F-tracers. 68Ga-RGD peptides showed better imaging properties compared to 18F-tracers, i.e., higher tumor/background ratios and no accumulation in non-target organs except for excretory organs.

Synthesis and Preclinical Characterization of the PSMA-Targeted Hybrid Tracer PSMA-I&F for Nuclear and Fluorescence Imaging of Prostate Cancer.

The prostate-specific membrane antigen (PSMA)–targeted radiotracers 68Ga/177Lu-PSMA-I&T and 99mTc-PSMA-I&S (for imaging and surgery) are currently successfully used for clinical PET imaging, radionuclide therapy, and radioguided surgery of metastatic prostate cancer. To additionally exploit the high sensitivity and spatial resolution of fluorescence imaging for improved surgical guidance, a PSMA-I&T–based hybrid tracer, PSMA-I&F (DOTAGA-k(Sulfo-Cy5)-y-nal-k-Sub-KuE), has been developed and evaluated. Methods: The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T, and their corresponding natGa-/68Ga- and natLu/177Lu counterparts was determined in LNCaP cells via competitive binding assays (IC50) and dual-tracer radioligand and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17 SCID and LNCaP xenograft–bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of 1 tumor-bearing animal as well as PSMA immunohistochemistry and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed. Results: Compared with the parent PSMA-I&T analogs, the PSMA affinities of PSMA-I&F and its natGa-/natLu-complexes remained high and unaffected by dye conjugation (7.9 < IC50 < 10.5 nM for all ligands). The same was observed for the internalization of 68Ga- and 177Lu-PSMA-I&F. In vivo, blood clearance of 68Ga- and 177Lu-PSMA-I&F was only slightly delayed by high plasma protein binding (94%–95%), and very low accumulation in nontarget organs was observed already at 1 h after injection. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5% ± 1.8 percentage injected dose per gram) and the kidneys (106% ± 23 percentage injected dose per gram). Tumor-to-background ratios of 2.1, 5.2, 9.6, and 9.6 for blood, liver, intestines, and muscle, respectively, at 1 h after injection led to excellent imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by immunohistochemistry. Conclusion: Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, 68Ga/177Lu-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T–based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma.

Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAb) might circumvent some of the limitations of radiolabeled full antibodies. In this study, a set of hCD20-targeting sdAbs was generated, and their capacity to bind hCD20 was evaluated in vitro and in vivo A lead sdAb, sdAb 9079, was selected on the basis of its specific tumor targeting and significant lower kidney accumulation compared with other sdAbs. SdAb 9079 was then radiolabeled with 68Ga and 177Lu for PET imaging and targeted therapy. The therapeutic potential of 177Lu-DTPA-sdAb was compared with that of 177Lu-DTPA-rituximab and unlabeled rituximab in mice bearing hCD20pos tumors. Radiolabeled with 68Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in nontarget organs, except kidneys. The tumor uptake of 177Lu-DTPA-sdAb 9079 after 1.5 h was 3.4 ± 1.3% ID/g, with T/B and T/M ratios of 13.3 ± 4.6 and 32.9 ± 15.6. Peak tumor accumulation of 177Lu-DTPA-rituximab was about 9 times higher, but concomitantly with high accumulation in nontarget organs and very low T/B and T/M ratios (0.8 ± 0.1 and 7.1 ± 2.4). Treatment of mice with 177Lu-DTPA-sdAb 9079 significantly prolonged median survival compared with control groups and was as effective as treatment with rituximab or its 177Lu-labeled variant. Taken together, sdAb 9079 displays promising features as a theranostic drug to treat CD20pos lymphomas. Mol Cancer Ther; 16(12); 2828-39. ©2017 AACR.

Multimodal Fluorescence and Bioluminescence Imaging Reveals Transfection Potential of Intratracheally Administered Polyplexes for Breast Cancer Lung Metastases.

Local delivery of anticancer agents or gene therapeutics to lung tumors can circumvent side effects or accumulation in non-target organs, but accessibility via the alveolar side of the blood-air barrier remains challenging. Polyplexes based on plasmid and linear polyethylenimine (LPEI) transfect healthy lung tissue when applied intravenously (i.v.) in the mouse, but direct delivery into the lungs results in low transfection of lung tissue. Nevertheless, LPEI could offer the potential to transfect lung tumors selectively, if accessible from the alveolar side. This study combined near infrared fluorescent protein 720 (iRFP720) and firefly luciferase as reporter genes for detection of tumor lesions and transfection efficiency of LPEI polyplexes, after intratracheal microspraying in mice bearing 4T1 triple negative breast cancer lung metastases. Simultaneous flow cytometric analysis of iRFP720 and enhanced green fluorescent protein expression in vitro demonstrated the potential to combine these reporter genes within transfection studies. Polyplex biophysics was characterized by single nanoparticle tracking analysis (NTA) to monitor physical integrity after microspraying in vitro. 4T1 cells were transduced with iRFP720-encoding lentivirus and evaluated by flow cytometry for stable iRFP720 expression. Growth of 4T1-iRFP720 cells was monitored in Balb/c mice by tomographic near infrared imaging, tissue and tumor morphology by computed tomography and magnetic resonance imaging. In 4T1-iRFP720 tumor-bearing mice, intratracheal administration of luciferase-encoding plasmid DNA by LPEI polyplexes resulted in successful tumor transfection, as revealed by bioluminescence imaging.

New neurotensin analogue radiolabeled by 99m-technetium as a potential agent for tumor identification.

It has been shown that more than 75% of ductal pancreatic adenocarcinomas overexpressed neurotensin (NT) receptors. Overexpression of NT receptors has been reported in various human tumor types. Hence, a non-invasive diagnosis and staging method could be very beneficial. In this work, we describe radiolabeling and evaluation of new neurotensin analogues to target neurotensin receptor-positive tumors such as pancreatic carcinoma. Radiolabeling was performed at 95°C for 10min using 99m Tc in the presence of tricine/EDDA exchange labeling. Radiochemical yield analysis involved ITLC and HPLC methods. A binding assay test was carried out in nine different concentrations of labeled neurotensin analogues in HT-29 cells. Radiopeptide-specific binding and internalization were studied in NT receptors expressing HT-29 cells. Biodistribution studies were performed in tumor-free BALB/c mice and HT-29 xenografted tumor-bearing nude mice. The peptide was efficiently labeled by 99m Tc with high radiochemical yields (>98%). The radioconjugate was thoroughly stable in the solution and human serum even for 24hr. The radiolabeled peptide showed high affinity (32.66±4.01nm) and specificity internalization (>%18 after 4hr) to HT-29 cells. The radiopeptide efficiently showed tumor size and location in tumor-bearing nude mice. In biodistribution, a receptor-specific uptake of radiopeptide was observed in neurotensin receptor-positive organs such as intestine. Uptake in the tumor was 4.59±0.23% ID/g after 2hr. Owing to excellent stability, high affinity, rapid blood clearance, low accumulation in non-target organs, and high uptake in tumor, the 99m Tc-HYNIC-peptide is a potential agent for targeting of NTR-overexpressing tumor cells in clinical surroundings. When successfully executed in the clinical surrounding, non-invasive imaging of NTR-positive tumors with 99m Tc-labeled new neurotensin analogues could facilitate therapy procedure and monitoring.

Synthesis and Evaluation of 131I-Skyrin as a Necrosis Avid Agent for Potential Targeted Radionuclide Therapy of Solid Tumors.

An innovative anticancer approach targeted to necrotic tissues, which serves as a noncancerous and generic anchor, may present a breakthrough. Necrosis avid agents with a flat conjugate aromatic structure selectively accumulate in necrotic tissues, but they easily form aggregates that undesirably distribute to normal tissues. In this study, skyrin, a dianthraquinone compound with smaller and distorted π-cores and thus decreased aggregates as compared with hypericin (Hyp), was designed to target necrosis for tumor therapy. Aggregation studies of skyrin by UV/vis spectroscopy showed a smaller self-association constant with skyrin than with Hyp. Skyrin was labeled by iodine-131 with a radiochemical purity of 98% and exhibited good stability in rat serum for 72 h. In vitro cell uptake studies showed significant difference in the uptake of 131I-skyrin by necrotic cells compared to normal cells (P < 0.05). Compared in rats with liver and muscle necrosis, radiobiodistribution, whole-body autoradiography, and SPECT/CT studies revealed higher accumulation of 131I-skyrin in necrotic liver and muscle (p < 0.05), but lower uptake in normal organs, relative to that of 131I-Hyp. In mice bearing H22 tumor xenografts treated with combretastatin A4 disodium phosphate, the highest uptake of 131I-skyrin was found in necrotic tumor. In conclusion, 131I-skyrin appears a promising agent with reduced accumulation in nontarget organs for targeted radionuclide therapy of solid tumors.

Irradiation parameters play a crucial role in the (n, γ) production of 170Tm suitable for clinical use in bone pain palliation

While neutron activation of natural Tm2O3 is the convenient strategy for producing 170Tm for use in palliative care of painful bone metastases, the coproduction of 171Tm (t ½ = 1.92 years) is as an impediment towards its clinical utility. The production strategy has been optimized to obtain 170Tm with adequate specific activity and radionuclidic purity. Preliminary clinical studies carried out using the 170Tm-labeled ethylenediaminetetramethylene phosphonic acid (170Tm-EDTMP) showed site specific localization of the radiopharmaceutical in skeleton with preferential accumulation in metastatic lesions along with almost no accumulation in non-target organs, a distribution pattern comparable to that of 99mTc-labeled methylene diphosphonate (99mTc-MDP).

Protein-functionalized PLGA nanoparticles of lamotrigine for neuropathic pain management.

Lamotrigine (LTG), a sodium and calcium channel blocker, has demonstrated efficacy for the treatment of neuropathic pain in multiple, randomized, controlled trials. However, its potential clinical applications in neuropathic pain are limited due to the risk of dose-dependent severe rashes associated with high dose and prompt dose escalation. Further, the poor pharmacokinetic profile due to non-selective distribution to organs other than brain reduces the efficacy of dosage regimen. Therefore, the aim of present investigation is to develop surface-engineered LTG nanoparticles (NPs) using transferrin and lactoferrin as ligand to deliver higher amount of drug to brain and improve the biodistribution and pharmacokinetic profile of drug with prolonged duration of action and reduced accumulation in non-target organs. The LTG NPs were prepared by nanoprecipitation and optimized by factorial design for high entrapment and optimized particle size. The optimized NPs were surface functionalized by conjugating with the lactoferrin (Lf) and transferrin (Tf) as ligands. The developed NPs were characterized for different physicochemical parameters and stability. The in vivo biodistribution showed preferential targeting to brain and reduced accumulation in non-target organs over a prolonged duration of time. Finally, partial sciatic nerve injury model was used to demonstrate the increased pharmacodynamic response as antinociceptive effect. Both biodistribution and pharmacodynamic study in mice confirmed that the approach used for LTG can help to increase clinical applications of LTG due to brain targeting and reduced side effects.

Liprosomes loading paclitaxel for brain-targeting delivery by intravenous administration: In vitro characterization and in vivo evaluation

In this study, a lipid–protein nanocomplex (liprosome) was evaluated for its potential use for brain-targeting drug delivery. Liprosome was fabricated with the desolvation–ultrasonication method and characterized in terms of particle size, size distribution, zeta potential, morphology, crystal state of the drug, and in vitro release. The in vivo distribution of paclitaxel loading lipid–protein nanocomplex (PTX-liprosome) and Taxol were compared after i.v. administration in mice. The prepared PTX-liprosome has a high entrapment efficiency (>90%), small particle size (approximately 110nm), and narrow distribution (P.I.<0.2). Transmission electron microscopy (TEM) indicated that liprosome had a spherical multilayer structure. X-ray photoelectron spectroscopy (XPS) showed that the conjugate of PTX and BSA was in the interior of the PTX-liprosome. Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD) demonstrated that the drug existed in a molecular or amorphous state. Fourier transform infrared spectroscopy (FTIR) suggested that the hydrophobic interactions, electrostatic interactions and hydrogen bonds among of the PTX, lipid and protein play an important role during the formation of the PTX-liprosome. The hemolysis test showed a good safety profile for the intravenous administration of liprosome. The result of the in vivo distribution suggested that liprosome increased the drug uptake by the brain tissue and decreased drug accumulation in non-target organs. Therefore, liprosome is a potential drug delivery system for transporting PTX to the brain.

Synthesis and biological evaluation of novel technetium-99m-labeled HYNIC-d-glucose as a potential tumor imaging agent

A conjugate of 6-hydrazinopyridine-3-carboxylic acid (HYNIC) with the d-Glucosamine Hydrochloride (DG) was synthesized though a multiple-step reaction. HYNIC-DG could be labeled successfully and efficiently with 99mTc using N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (tricine) and ethylenediamine-N,N′-diacetic acid (EDDA) or tricine and triphenylphosphine-3,3′,3″-trisulfonic acid trisodium salt (TPPTS) as co-ligands to form two 99mTc-HYNIC-DG complexes 99mTc-(t/E)HYNIC-DG and 99mTc-(t/T)HYNIC-DG in high yields (>95 %). The partition coefficient and electrophoresis results indicated they were very hydrophilic and electronegative. The biodistribution studies of two 99mTc-HYNIC-DG complexes in Kunming mice bearing S 180 tumor showed that 99mTc-(t/T)HYNIC-DG has more favorable characteristics than 99mTc-(t/E)HYNIC-DG. High tumor uptake, low or negligible accumulation in non-target organs, and good retention, suggesting 99mTc-(t/T)HYNIC-DG would be a novel potential tumor imaging agent.

Biological evaluation of 3-[18F]fluoro-α-methyl-d-tyrosine (d-[18F]FAMT) as a novel amino acid tracer for positron emission tomography

3-[(18)F]Fluoro-α-methyl-L-tyrosine (L-[(18)F]FAMT) is a useful amino acid tracer for positron emission tomography (PET) imaging of malignant tumors. Because D-amino acids are not well distributed in non-target organs and are rapidly excreted in urine, the D-isomer of [(18)F]FAMT could allow clear PET imaging of tumors early after administration. In this study, we prepared 3-[(18)F]fluoro-α-methyl-D-tyrosine (D-[(18)F]FAMT) and evaluated its usefulness. D-[(18)F]FAMT was synthesized according to the method for preparation of L-[(18)F]FAMT. The in vitro and in vivo stability of [(18)F]FAMT were evaluated by high-performance liquid chromatography. Cellular uptake of [(18)F]FAMT was evaluated using LS180 colon adenocarcinoma cells. Biodistribution studies were performed in LS180 tumor-bearing mice, and the tumors were imaged using a small-animal PET scanner. The radiolabeling yield of D-[(18)F]FAMT was approximately 10 %, similar to that of L-[(18)F]FAMT. Over 95 % of D-[(18)F]FAMT remained intact in mice until 60 min after administration. D-[(18)F]FAMT was gradually taken up by the LS180 cells. Tumor uptake of D-[(18)F]FAMT was competitively inhibited by pretreatment with α-methyl-L-tyrosine, a selective substrate for the system L-amino acid transporter 1 (LAT1), suggesting the involvement of LAT1 in tumor uptake of D-[(18)F]FAMT. In biodistribution studies, D-[(18)F]FAMT showed rapid clearance from the blood, marked accumulation and retention in the tumor, and lower accumulation in non-target organs, especially kidney and pancreas, compared to L-[(18)F]FAMT. The amount of D-[(18)F]FAMT in the tumor was also reduced, and tumor-to-blood ratio and tumor-to-muscle ratio of D-[(18)F]FAMT were similar to those of L-[(18)F]FAMT at every time point. PET imaging with D-[(18)F]FAMT did not provide a clear image of the tumor early after administration. However, D-[(18)F]FAMT provided higher tumor-to-background contrast than L-[(18)F]FAMT. D-[(18)F]FAMT showed rapid blood clearance, low accumulation in non-target organs, and tumor-selective imaging compared with L-[(18)F]FAMT. Thus, D-[(18)F]FAMT could potentially serve as a novel PET tracer for imaging malignant tumors.

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody.

With a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors. 5F7GGC Nb was radioiodinated with ¹²⁵I using Iodogen and with ¹³¹I using the residualizing agent N(ɛ)-(3-[¹³¹I]iodobenzoyl)-Lys⁵-N(α)-maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-D-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations. The radiochemical yields for Iodogen and [¹³¹I]IB-Mal-D-GEEEK labeling were 83.6±5.0% (n=10) and 59.6±9.4% (n=15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [¹³¹I]IB-Mal-D-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65±0.61% ID/g vs. 2.92±0.24% ID/g at 8h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios. Taken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [¹³¹I]IB-Mal-D-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (¹²⁹I) and PET imaging (¹²⁴I) of patients with HER2-expressing tumors.

Cu-64-Labeled Lactam Bridge-Cyclized α-MSH Peptides for PET Imaging of Melanoma

The purpose of this study was to examine and compare the melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (64)Cu-DOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSH(hex)}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex), were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSH(hex) was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSH(hex). The melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) and (64)Cu-DOTA-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex) displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex). The tumor uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) was between 12.39 ± 1.61 and 12.71 ± 2.68% ID/g at 0.5, 2, and 4 h postinjection. The accumulation of (64)Cu-NOTA-GGNle-CycMSH(hex) activity in normal organs was lower than 1.02% ID/g except for the kidneys 2, 4, and 24 h postinjection. The tumor/liver uptake ratios of (64)Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95, and 8.02, whereas the tumor/kidney uptake ratios of (64)Cu-NOTA-GGNle-CycMSH(hex) were 2.52, 3.60, and 5.74 at 2, 4, and 24 h postinjection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h postinjection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) as compared to (64)Cu-DOTA-GGNle-CycMSH(hex). High melanoma uptake coupled with low accumulation in nontarget organs suggested (64)Cu-NOTA-GGNle-CycMSH(hex) as a lead radiolabeled peptide for melanoma imaging and therapy.

Preclinical screening of anti‐HER2 nanobodies for molecular imaging of breast cancer

Accurate determination of tumor human epidermal growth factor receptor 2 (HER2)-status in breast cancer patients is possible via noninvasive imaging, provided adequate tracers are used. In this study, we describe the generation of a panel of 38 nanobodies, small HER2-binding fragments that are derived from heavy-chain-only antibodies raised in an immunized dromedary. In search of a lead compound, a subset of nanobodies was biochemically characterized in depth and preclinically tested for use as tracers for imaging of xenografted tumors. The selected compound, 2Rs15d, was found to be stable and to interact specifically with HER2 recombinant protein and HER2-expressing cells in ELISA, surface plasmon resonance, flow cytometry, and radioligand binding studies with low nanomolar affinities, and did not compete with anti-HER2 therapeutic antibodies trastuzumab and pertuzumab. Single-photon-emission computed tomography (SPECT) imaging quantification and biodistribution analyses showed that (99m)Tc-labeled 2Rs15d has a high tumor uptake in 2 HER2(+) tumor models, fast blood clearance, low accumulation in nontarget organs except kidneys, and high concomitant tumor-to-blood and tumor-to-muscle ratios at 1 h after intravenous injection. These values were dramatically lower for an irrelevant control (99m)Tc-nanobody and for (99m)Tc-2Rs15d targeting a HER2(-) tumor.

Nanospheres-Incorporated Implantable Hydrogel as a Trans-Tissue Drug Delivery System

The objective of this study is to investigate the anticancer efficacy of a drug delivery system comprised of gelatin hydrogel (jelly) containing cisplatin (CDDP)-loaded gelatin/poly(acrylic acid) nanoparticles by peritumoral implantation and to compare the treatment response between the implantation administration of the jelly and intravenous (i.v.) administration of the nanoparticles. It is found that the implantation of the jelly containing CDDP-loaded nanoparticles on tumor tissue exhibited significantly superior efficacy in impeding tumor growth and prolonging the lifetime of mice than that of i.v. injection of CDDP-loaded nanoparticles in a murine hepatoma H(22) cancer model. An in vivo biodistribution assay performed on tumor-bearing mice demonstrated that the jelly implant caused much higher concentration and retention of CDDP in tumor and lower CDDP accumulation in nontarget organs than that of i.v. injected nanoparticles. Immunohistochemical analysis demonstrated that the nanoparticles from the jelly can be distributed in tumor tissue not only by their diffusion but also by the vasculature in the implantation region into tumor interior, enabling CDDP to efficiently reach more viable cells of tumor compared with i.v. injected nanoparticles. Thus, nanoparticles for peritumoral chemotherapy are promising for higher treatment efficacy due to increased tumor-to-normal organ drug uptake ratios and improved drug penetration in tumors.