166Ho and 90Y labeled 6D2 monoclonal antibody for targeted radiotherapy of melanoma: Comparison with 188Re radiolabel

IntroductionAn approach to radioimmunotherapy (RIT) of metastatic melanoma is the targeting of melanin pigment with monoclonal antibodies (mAbs) to melanin radiolabeled with therapeutic radionuclides. The proof of principle experiments were performed using a melanin-binding antibody 6D2 of IgM isotype radiolabeled with a β emitter ¹⁸⁸Re and demonstrated the inhibition of tumor growth. In this study we investigated the efficacy of 6D2 antibody radiolabeled with two other longer lived β emitters ⁹⁰Y and ¹⁶⁶Ho in treatment of experimental melanoma, with the objective to find a possible correlation between the efficacy and half-life of the radioisotopes which possess high energy β (E_max > 1.5 MeV) emission properties.Methods6D2 was radiolabeled with longer lived β emitters ⁹⁰Y and ¹⁶⁶Ho in treatment of experimental melanoma in A2058 melanoma tumor-bearing nude mice. The immunoreactivity of the radiolabeled 6D2 mAb, its in vitro binding to the MNT1 human melanoma cells, the biodistribution and therapy in A2058 human melanoma bearing nude mice as well as dosimetry calculations were performed.ResultsWhen labeled with the longer lived ⁹⁰Y radionuclide, the 6D2 mAb did not produce any therapeutic effect in tumor bearing mice while the reduction of the tumor growth by ¹⁶⁶Ho-6D2 was very similar to the previously reported therapy results for ¹⁸⁸Re-6D2. In addition, ¹⁶⁶Ho-labeled mAb produced the therapeutic effect on the tumor without any toxic effects while the administration of the ⁹⁰Y-labeled radioconjugate was toxic to mice with no appreciable anti-tumor effect.Conclusions¹⁶⁶Ho-labeled mAb to melanin produced some therapeutic effect on the tumor without any toxic effects while the administration of the ⁹⁰Y-labeled radioconjugate was toxic to mice with no appreciable anti-tumor effect. We concluded that the serum half-life of the 6D2 carrier antibody matched well the physical half-life of ¹⁶⁶Ho to deliver the tumoricidal absorbed dose to the tumor. Further investigation of this radionuclide for RIT of melanoma is warranted.     

Methodology for labeling proteins and peptides with lead-212 (212Pb)

IntroductionAlpha particles possess an exquisite degree of cytotoxicity when employed for targeted α-particle therapy (TAT) or radioimmunotherapy (RIT). ²¹²Pb, which acts as an in vivo generator of the α-emitting nuclide ²¹²Bi has shown great promise in pre-clinical studies when used to label the HER2 binding antibody, trastuzumab. Currently, the first RIT clinical trial employing ²¹²Pb radiolabeled trastuzumab is in progress. This report provides detailed current protocol operations and steps that were generated for use in the clinical trial as well as the relevant pre-clinical experimentation, and describes in detail the labeling of proteins or peptides with ²¹²Pb as provided via a ²²⁴Ra based generator system.Methods²¹²Pb was eluted from the ²²⁴Ra/²¹²Pb generator using hydrochloric acid (2 M). The generator eluate was evaporated and digested with nitric acid (8 M) followed by extraction of the ²¹²Pb with dilute nitric acid (0.1 M). The dilute nitric acid solution of ²¹²Pb was used to label the immunoconjugate Trastuzumab-TCMC (2-(4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)-cyclododecane) at pH 5.5.ResultsElution of ²¹²Pb from the generator was efficient yielding > 90% of available ²¹²Pb. Trastuzumab-TCMC was efficiently labeled with a radiochemical yield of 94% ± 4% (n = 7) by ITLC and an isolated yield of 73% ± 3% (n = 7).ConclusionsThe results show the feasibility of generating radioimmunoconjugates and peptide conjugates for use as in vivo α generator systems in the clinic. The technology holds promise in applications involving the treatment of minimal disease such as micrometastases and residual tumor after surgical debulking, hematological cancers, infections, and compartmental cancers, such as ovarian cancer.     

Comparative analysis of multiple myeloma treatment by CD138 antigen targeting with bismuth-213 and Melphalan chemotherapy

IntroductionMultiple myeloma (MM) is a B-cell malignancy of terminally differentiated plasma cells within the bone marrow. Despite intense research to develop new treatments, cure is almost never achieved. Alpha-radioimmunotherapy (RIT) has been shown to be effective in vivo in a MM model. In order to define where alpha-RIT stands in MM treatment, the aim of this study was to compare Melphalan, MM standard treatment, with alpha-RIT using a [213Bi]-anti-mCD138 antibody in a syngeneic MM mouse model.MethodsC57BL/KaLwRij mice were grafted with 1 × 10⁶ 5T33 murine MM cells. Luciferase transfected 5T33 cells were used for in vivo localization. The first step of the study was to assess the dose-response of Melphalan 21 days after engraftment. The second step consisted in therapeutic combination: Melphalan followed by RIT at day 22 or day 25 after engraftment. Toxicity (animal weight, blood cell counts) and treatment efficacy were studied in animals receiving no treatment, injected with Melphalan alone, RIT alone at day 22 or day 25 (3.7 MBq of [213Bi]-anti-CD138) and Melphalan combined with alpha-RIT.ResultsFifty percent of untreated mice died by day 63 after MM engraftment. In mice treated with Melphalan alone, only the 200 μg dose improved median survival. No animal was cured after Melphalan treatment whereas 60% of the mice survived with RIT alone at day 22 after tumor engraftment with only slight and reversible hematological radiotoxicity. No therapeutic effect was observed with alpha-RIT 25 days after engraftment. Melphalan and alpha-RIT combination does not improve overall survival compared to RIT alone, and results in increased leukocyte and red blood cell toxicity.ConclusionsAlpha-RIT seems to be a good alternative to Melphalan. Association of these two treatments provides no benefit. The perspectives of this work would be to evaluate RIT impact on the regimens incorporating the novel agents bortezomide, thalidomide and lenalidomide.     

The human polynucleotide kinase/phosphatase (hPNKP) inhibitor A12B4C3 radiosensitizes human myeloid leukemia cells to Auger electron-emitting anti-CD123 111In-NLS-7G3 radioimmunoconjugates

IntroductionLeukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131^- phenotype uniquely displayed by LSCs.MethodsThe surviving fraction (SF) of CD123⁺/CD131^- AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33–266 nmols/L; 0.74 MBq/μg) or to γ-radiation (0.25-5 Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16–66 nmols/L) or with γ-radiation (0.25–2 Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation.ResultsBinding of ¹¹¹In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1 μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131^- epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25 Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16–66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25–0.5 Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6 Gy to AML-5 cells.ConclusionsWe conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.     

99mTc-labeled SWL specific peptide for targeting EphA2 receptor

IntroductionEphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, ^99mTc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated.MethodsThe SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by ^99mTc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with ^99mTc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution.ResultsImmunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. ^99mTc-HYNIC-SWL displayed high binding affinity with A549 cells (K_D = 2.6 ± 0.7 nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44 ± 0.12 %ID/g) than in OCM-1 tumors (0.43 ± 0.20 %ID/g) at 1 h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58 ± 0.20 %ID/g) was seen. These data suggest that ^99mTc-HYNIC-SWL specifically targets EphA2 in tumors.ConclusionsThe expression of EphA2 can be noninvasively investigated using ^99mTc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of ^99mTc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging.     

Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model

IntroductionDysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using ¹⁸ F-FDG and ¹⁸ F-FLT PET.MethodsU-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 μg) were administered, and mice were evaluated with ¹⁸ F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 μg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using ¹⁸ F-FDG and ¹⁸ F-FLT PET.ResultsIn the dose response study, rilotumumab at doses of 300 and 500 μg was similarly effective against tumor growth. Treatment with 300 and 500 μg rilotumumab inhibited ¹⁸ F-FDG accumulation with significant decreases of ? 37% and ? 40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 μg rilotumumab inhibited ¹⁸ F-FDG and ¹⁸ F-FLT accumulation with a maximum %ID/g of ? 41% and ? 64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed.ConclusionsRilotumumab inhibited ¹⁸ F-FDG and ¹⁸ F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate ¹⁸ F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.     

A systematic review of yttrium-90 radioembolization for unresectable liver metastases of melanoma

PurposeTo assess the effectiveness of yttrium-90 (⁹⁰Y) radioembolization in the treatment of unresectable liver metastases of melanoma.MethodsPubMed and EMBASE were systemically searched for all English language studies related to ⁹⁰Y radioembolization for unresectable liver metastases of melanoma, including clinical trials, observational studies, and abstracts from conferences, published between January 1991 and March 2016.ResultsA total of 12 reports (7 observational studies and 5 abstracts from conferences) involving 255 patients were included in the analysis. The primary sites of melanoma were cutaneous (n = 22; 8.6%), ocular (n = 197; 77.3%), rectal (n = 3; 1.2%), and unknown (n = 33; 12.9%). The median disease control rate at 3 months was 73.6% (range, 58.3%–88.9%). Among the 207 patients for whom tumor response at 3 months was reported, complete response was seen in 1.0% (2/207), partial response was seen in 19.3% (40/207), stable disease was seen in 46.9% (97/207), and progressive disease was seen in 32.9% (68/207). The median survival was 10 months (range, 7–13.4 months), and the median 1-year survival rate was 34.6% (range, 23%–80%). Complications of ⁹⁰Y radioembolization were reported in 13 cases. The most common side effects were fatigue (median, 36.1%), abdominal pain (median, 17.8%), and nausea (median, 15.0%).Conclusions⁹⁰Y radioembolization is a promising alternative therapy for the treatment of unresectable liver metastases of melanoma, with encouraging effects on disease control and survival. Some complications can occur, and side effects are frequent but mild.     

Biologic correlation between glucose transporters,hexokinase-II,Ki-67 and FDG uptake in malignant melanoma

IntroductionThe purpose of this study was to investigate the correlative association between tumoral 2-deoxy-2-[¹⁸ F]-fluoro-D-glucose (FDG) uptake, and the expressions of glucose transporter 1 (GLUT-1), glucose transporter 3 (GLUT-3), hexokinase II (HK-2), and Ki-67 expression in malignant melanoma.MethodsNineteen patients with histologically proven malignant melanoma and pretreatment FDG PET/CT performance were involved in this preliminary study. For semi-quantitative analysis of FDG PET/CT, maximal standardized uptake values (SUVmax) were estimated. Immunohistochemical staining of tumor sections was performed for GLUT-1, GLUT-3, and HK-2, and for the cell proliferation maker Ki-67. Especially, by combining proportions and intensity of immunochemical staining, we evaluated modified immunohistologic scores of GLUT-1 and GLUT-3.ResultsThe SUVmax of malignant melanoma lesions ranged from 2 to 18.7 (average; 9.1 ± 5.4). Comparison between nodal and extranodal lesions revealed no significant difference of SUVmax (p = 0.97). GLUT-1 staining showed the most positive expression level (89.5%, 17/19) among the diverse immunohistochemical markers. There were significant relationships between FDG uptake of malignant melanoma and GLUT-1 proportion (p < 0.0001), GLUT-1 intensity (p < 0.0001), GLUT-3 proportion (p = 0.031), GLUT-3 intensity (p = 0.009), GLUT-1 immunohistologic scores (p < 0.0001), and GLUT-3 immunohistologic scores (p = 0.028). HK-2 was not expressed in all melanoma samples. Although Ki-67 expression showed a high grade in all staining, there was no significant link between FDG uptake and Ki-67 grades (p = 0.38).ConclusionsThe data in this preliminary study indicate that FDG uptake in malignant melanoma is determined by GLUT-1 and GLUT-3, whereas HK-2 and Ki-67 play no role in FDG uptake of malignant melanoma.     

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody

IntroductionWith 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.Methods5F7GGC Nb was radioiodinated with ¹²⁵I using Iodogen and with ¹³¹I using the residualizing agent N^?-(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.ResultsThe 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 8 h), 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.ConclusionsTaken 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.     

Tracer level radiochemistry to clinical dose preparation of 177Lu-labeled cyclic RGD peptide dimer

AimIntegrin α_vβ₃ plays a significant role in angiogenesis during tumor growth and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine(R)-glycine(G)-aspartic acid(D) tripeptide sequence. The over-expression of integrin α_vβ₃ during tumor growth and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. Considering the advantages of ¹⁷⁷Lu for targeted radiotherapy and enhanced tumor targeting capability of cyclic RGD peptide dimer, an attempt has been made to optimize the protocol for the preparation of clinical dose of ¹⁷⁷Lu labeled DOTA-E[c(RGDfK)]₂ (E = Glutamic acid, f = phenyl alanine, K = lysine) as a potential agent for targeted tumor therapy.Methods¹⁷⁷Lu was produced by thermal neutron bombardment on enriched Lu₂O₃ (82% in ¹⁷⁶Lu) target at a flux of 1 × 10¹⁴ n/cm².s for 21 d. Therapeutic dose of ¹⁷⁷Lu-DOTA-E[c(RGDfK)]₂ (7.4 GBq) was prepared by adding the aqueous solution of the ligand and ¹⁷⁷LuCl₃ to 0.1 M NH₄OAC buffer containing gentisic acid and incubating the reaction mixture at 90 °C for 30 min. The yield and radiochemical purity of the complex was determined by HPLC technique. Parameters, such as, ligand-to-metal ratio, pH of the reaction mixture, incubation time and temperature were varied using tracer quantity of ¹⁷⁷Lu (37 MBq) in order to arrive at the optimized protocol for the preparation of clinical dose. Biological behavior of the radiotracer prepared was studied in C57/BL6 mice bearing melanoma tumors.Results¹⁷⁷Lu was produced with a specific activity of 950 ± 50 GBq/mg (25.7 ± 1.4 Ci/mg) and radionuclidic purity of 99.98%. A careful optimization of several parameters showed that ¹⁷⁷Lu-DOTA-E[c(RGDfK)]₂ could be prepared with adequately high radiochemical purity using a ligand-to-metal ratio ~ 2. Based on these studies therapeutic dose of the agent with 7.4 GBq of ¹⁷⁷Lu was formulated in ~ 63 GBq/μM specific activity with high yield (98.2 ± 0.7%), radiochemical purity and in vitro stability. Biodistribution studies carried out in C57/BL6 mice bearing melanoma tumors revealed specific accumulation of the radiolabeled conjugate in tumor (3.80 ± 0.55% ID/g at 30 min p.i.) with high tumor to blood and tumor to muscle ratios. However, the uptake of the radiotracer in the tumor was found to be reduced to 1.51 ± 0.32 %ID/g at 72 h p.i.ConclusionsThe present work successfully demonstrates the formulation of an optimized protocol for the preparation of ¹⁷⁷Lu labeled DOTA-E[c(RGDfK)]₂ for PRRT applications using ¹⁷⁷Lu produced by direct neutron activation in a medium flux research reactor.     

Preclinical evaluation of NETA-based bifunctional ligand for radioimmunotherapy applications using 212Bi and 213Bi: Radiolabeling,serum stability,and biodistribution and tumor uptake studies

IntroductionDespite the great potential of targeted α-radioimmunotherapy (RIT) as demonstrated by pre-clinical and clinical trials, limited progress has been made on the improvement of chelation chemistry for ²¹²Bi and ²¹³Bi. A new bifunctional ligand 3p-C-NETA was evaluated for targeted α RIT using ²¹²Bi and ²¹³Bi.MethodsRadiolabeling of 3p-C-NETA with ^205/6Bi, a surrogate of ²¹²Bi and ²¹³Bi, was evaluated at pH 5.5 and room temperature. In vitro stability of the ^205/6Bi-3p-C-NETA-trastuzumab conjugate was evaluated using human serum (pH 7, 37 °C). Immunoreactivity and specific activity of the ^205/6Bi-3p-C-NETA-trastuzumab conjugate were measured. An in vivo biodistribution study was performed to evaluate the in vivo stability and tumor targeting properties of the ^205/6Bi-3p-C-NETA-trastuzumab conjugate in athymic mice bearing subcutaneous LS174T tumor xenografts.ResultThe 3p-C-NETA-trastuzumab conjugate was extremely rapid in complexing with ^205/6Bi, and the corresponding ^205/6Bi-3p-C-NETA-trastuzumab was stable in human serum. ^205/6Bi-3p-C-NETA-trastuzumab was prepared with a high specific activity and retained immunoreactivity. ^205/6Bi-3p-C-NETA-trastuzumab conjugate displayed excellent in vivo stability and targeting as evidenced by low normal organ and high tumor uptake.ConclusionThe results of the in vitro and in vivo studies indicate that 3p-C-NETA is a promising chelator for RIT applications using ²¹²Bi and ²¹³Bi. Further detailed in vivo evaluations of 3p-C-NETA for targeted α RIT are warranted.     

Heptamethine cyanine based 64Cu-PET probe PC-1001 for cancer imaging: Synthesis and in vivo evaluation

PurposeDevelopment of a heptamethine cyanine based tumor-targeting PET imaging probe for noninvasive detection and diagnosis of breast cancer.MethodsTumor-specific heptamethine–cyanine DOTA conjugate complexed with Cu-64 (PC-1001) was synthesized for breast cancer imaging. In vitro cellular uptake studies were performed in the breast cancer MCF-7 and noncancerous breast epithelial MCF-10A cell lines to establish tumor specificity. In vivo time-dependent fluorescence and PET imaging of breast tumor xenografts in mice were performed. Blood clearance, biodistribution, and tumor-specific uptake and plasma binding of PC-1001 were quantified. Tumor histology (H&E staining) and fluorescence imaging were examined.ResultsPC-1001 displayed similar fluorescence properties (ε = 82,880 cm^? 1 M^? 1, E_x/E_m = 750/820 nm) to the parental dye. Time-dependent cellular accumulation indicated significantly higher probe uptake (> 2-fold, 30 min) in MCF-7 than MCF-10A cells and the uptake was observed to be mediated by organic anion transport peptides (OATPs) system. In vivo studies revealed that PC-1001 has desirable accumulation profile in tumor tissues, with tumor versus muscle uptake of about 4.3 fold at 24 h and 5.8 fold at 48 h post probe injections. Blood half-life of PC-1001 was observed to be 4.3 ± 0.2 h. Microscopic fluorescence imaging of harvested tumor indicated that the uptake of PC-1001 was restricted to viable rather than necrotic tumor cells.ConclusionsA highly efficient tumor-targeting PET/fluorescence imaging probe PC-1001 is synthesized and validated in vitro in MCF-7 breast cancer cells and in vivo in mice breast cancer xenograft model.     

Enhancing the efficiency of 5-aminolevulinic acid-mediated photodynamic therapy using 5-fluorouracil on human melanoma cells

Background5-Aminolevulinic acid-mediated photodynamic therapy (ALA–PDT) is an effective and noninvasive modality for treatment of several types of non-melanoma skin cancers. This in-vitro study attempted to know whether the killing effect of ALA–PDT on the human melanoma cells (Mel-Rm cell line) could be increased by the presence of 5-fluorouracil (5-FU).MethodsTo evaluate the effect of ALA–PDT in combination with 5-FU on viability of human melanoma Mel-Rm cells, the cells incubated with 5-ALA and 5-FU for 3 h in nontoxic concentrations, and subsequently illuminated with a 630 nm light-emitting diode array. The cells viability and cytotoxicity determined by mitochondrial activity and lactate dehydrogenase assays.ResultsCombination of ALA–PDT and 5-FU (FU–ALA–PDT) showed a considerable growth inhibition according to the results of MTT assay compared to ALA–PDT. The results of LDH assay also showed a cytotoxicity effect in ALA–PDT; however, the FU–ALA–PDT showed no significantly enhancement in cytotoxicity compared to ALA–PDT using LDH assay.ConclusionThe Mel-Rm cells incubation with 5-FU before PDT enhances the efficiency of 5-Aminolevulinic acid-mediated photodynamic therapy.     

A comparison of Re-188-MN-16ET-lipiodol and transcatheter arterial chemoembolization in the treatment of hepatoma: An animal study

IntroductionIn patients with unresectable HCC, transcatheter arterial chemoembolization (TACE) is a widely used treatment. Recently, as an alternative treatment modality for HCC, transcatheter arterial embolization with radioisotopes has been investigated. In this study, we compared the therapeutic efficacy of an intra-hepatic arterial injection of Re-188-MN-16ET-lipiodol and the TACE method in rats with liver tumors.MethodsTwelve male rats bearing hepatic tumors were divided into three groups to evaluate the efficacy of treatment (four in each group). Group 1 received an intra-hepatic arterial injection of 0.2 mCi of Re-188-MN-16ET-lipiodol; group 2 received epirubicin (0.5 mg/kg) and 0.1 ml of lipiodol emulsion; group 3 received 0.1 ml of normal saline and served as the control group. Tumor size was measured by liver sonography before injection, at two weeks, four weeks and eight weeks after injection. Survival time was calculated from the day of treatment to 56 days after treatment by the life-table method. The response to treatment and the survival time in each group were evaluated and compared.ResultsAll rats treated with Re-188 MN-16ET-lipiodol showed good response to the therapy. Their tumor size decreased and all rats survived over eight weeks. All rats treated with epirubicin plus lipiodol survived over 8 weeks; however, two rats (50%) showed increased tumor size in the 8th week. As for the control group (rats treated with normal saline), all rats survived less than 37 days with continuous tumor growth.ConclusionResults showed that Re-188-MN-16ET-lipiodol can be a potential therapeutic pharmaceutical for the treatment of liver tumors.     

Uptake of O-(2-[18F]fluoroethyl)-L-tyrosine in reactive astrocytosis in the vicinity of cerebral gliomas

PET using O-(2-[¹⁸F]fluoroethyl)-L-tyrosine (¹⁸F-FET) allows improved imaging of tumor extent of cerebral gliomas in comparison to MRI. In experimental brain infarction and hematoma, an unspecific accumulation of ¹⁸F-FET has been detected in the area of reactive astrogliosis which is a common cellular reaction in the vicinity of cerebral gliomas. The aim of this study was to investigate possible ¹⁸F-FET uptake in the area of reactive gliosis in the vicinity of untreated and irradiated rat gliomas.MethodsF98-glioma cells were implanted into the caudate nucleus of 33 Fisher CDF rats. Sixteen animals remained untreated and in 17 animals the tumor was irradiated by Gamma Knife 5–8 days after implantation (2/50 Gy, 3/75 Gy, 6/100 Gy, 6/150 Gy). After 8–17 days of tumor growth the animals were sacrificed following injection of ¹⁸F-FET. Brains were removed, cut in coronal sections and autoradiograms of ¹⁸F-FET distribution were produced and compared with histology (toluidine blue) and reactive astrogliosis (GFAP staining). ¹⁸F-FET uptake in the tumors and in areas of reactive astrocytosis was evaluated by lesion to brain ratios (L/B).ResultsLarge F98-gliomas were present in all animals showing increased ¹⁸F-FET-uptake which was similar in irradiated and non-irradiated tumors (L/B: 3.9 ± 0.8 vs. 4.0 ± 1.3). A pronounced reactive astrogliosis was noted in the vicinity of all tumors that showed significantly lower ¹⁸F-FET-uptake than the tumors (L/B: 1.5 ± 0.4 vs. 3.9 ± 1.1). The area of ¹⁸F-FET-uptake in the tumor was congruent with histological tumor extent in 31/33 animals. In 2 rats irradiated with 150 Gy, however, high ¹⁸F-FET uptake was noted in the area of astrogliosis which led to an overestimation of the tumor size.ConclusionsReactive astrogliosis in the vicinity of gliomas generally leads to only a slight ¹⁸F-FET-enrichment that appears not to affect the correct definition of tumor extent for treatment planning.     

Evaluation of two methods for concentrating perrhenate (188Re) eluates obtained from 188W–188Re generator

Two novel and simple methods for post-elution concentration (PEC) of ¹⁸⁸Re-perrhenate, using (i) a single diethyl amino ethyl (DEAE) cellulose anion exchanger column and (ii) a combination of Dowex-1×8 and AgCl columns, are described here. In the first system, 20–25 ml of ¹⁸⁸Re-perrhenate in acidic ammonium acetate was trapped in the small anion exchanger column of DEAE cellulose which was subsequently recovered in 4 ml of normal saline. In the second method, ¹⁸⁸Re-perrhenate eluate in 20–40 ml normal saline was trapped on a small strong base anion exchanger column, Dowex-1×8, and recovered in 5 ml of 0.2 M NaI solution and finally processed over 1 g AgCl column so as to obtain it in physiological saline solution in a final volume of 6.5 ml including 1.5 ml de-ionized water washings. In both the methods, the radiochemical purity of ¹⁸⁸Re-perrhenate was >98% and ¹⁸⁸W breakthrough was less than 10⁻³%.     

Monitoring tumor response with [18F]FMAU in a sarcoma-bearing mouse model after liposomal vinorelbine treatment

ObjectivePrevious studies have shown that the accumulation level of FMAU in tumor is proportional to its proliferation rate. This study demonstrated that 2′-deoxy-2′-[¹⁸F]fluoro-β-d-arabinofuranosyluracil ([¹⁸F]FMAU) is a promising PET probe for noninvasively monitoring the therapeutic efficacy of 6% PEGylated liposomal vinorelbine (lipo-VNB) in a subcutaneous murine NG4TL4 sarcoma mouse model.MethodsFemale syngenic FVB/N mice were inoculated with NG4TL4 cells in the right flank. After tumor size reached 150 ± 50 mm³ (day 0), lipo-VNB (5 mg/kg) was intravenously administered on days 0, 3 and 6. To monitor the therapeutic efficacy of lipo-VNB, [¹⁸F]FMAU PET was employed to evaluate the proliferation rate of tumor, and it was compared with that observed from [¹⁸F]FDG/[¹⁸F]fluoroacetate PET. The expression of proliferating cell nuclear antigen (PCNA) in tumor during treatment was determined by semiquantitative analysis of immunohistochemical staining.ResultsA significant inhibition (p < 0.001) in tumor growth was observed on day 3 after a single dose treatment. The tumor-to-muscle ratio (T/M) derived from [¹⁸F]FMAU-PET images of lipo-VNB-treated group declined from 2.33 ± 0.16 to 1.26 ± 0.03 after three doses of treatment, while that of the control remained steady. The retarded proliferation rate of lipo-VNB-treated sarcoma was confirmed by PCNA immunohistochemistry staining. However, both [¹⁸F]FDG and [¹⁸F]fluoroacetate microPET imaging did not show significant difference in T/M between the therapeutic and the control groups throughout the entire experimental period.ConclusionLipo-VNB can effectively impede the growth of NG4TL4 sarcoma. [¹⁸F]FMAU PET is an appropriate modality for early monitoring of the tumor response during the treatment course of lipo-VNB.     

Detection of activated platelets in a mouse model of carotid artery thrombosis with 18F-labeled single-chain antibodies

IntroductionActivated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel ¹⁸F PET radiotracer based on this antibody.MethodsScFv_anti-LIBS and control antibody mut-scFv were reacted with N-succinimidyl-4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl₃ induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied.ResultsAfter incubation with increasing concentrations of ¹⁸F-scFv_anti-LIBS clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled ¹⁸F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6).ConclusionsOur results show that the novel antibody radiotracer ¹⁸F-scFv_anti-LIBS is useful for the sensitive detection of activated platelets and thrombosis.Advances in knowledge and implications for patient careWe describe the first ¹⁸F variant of a scFv_anti-LIBS against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.     

213Bi-anti-EGFR radioimmunoconjugates and X-ray irradiation trigger different cell death pathways in squamous cell carcinoma cells

IntroductionTreatment of patients with squamous cell carcinoma of head and neck is hampered by resistance of tumor cells to irradiation. Additional therapies enhancing the effect of X-ray irradiation may be beneficial. Antibodies targeting EGFR have been shown to improve the efficacy of radiation therapy. Therefore, we analyzed cytotoxicity of ²¹³Bi-anti-EGFR immunoconjugates in combination with X-ray irradiation.MethodsThe monoclonal anti-EGFR antibody matuzumab was coupled to CHX-A”-DTPA forming stable complexes with ²¹³Bi. Cytotoxicity of X-ray radiation, of treatment with ²¹³Bi-anti-EGFR monoclonal antibodies (MAb) or of a combined treatment regimen was assayed using cell proliferation and colony formation assays in UD-SCC5 cells. Key proteins of cell-cycle arrest and cell death were examined by Western blot analysis. Cell cycle analysis was performed by flow cytometry. DNA double-strand breaks were detected via γH2AX and quantified using Definiens™ software.ResultsIrradiation with X-rays or treatment with ²¹³Bi-anti-EGFR-MAb resulted in median lethal dose (LD50) values of 12 Gy or 130 kBq/mL, respectively. Treatment with 37 kBq/mL of ²¹³Bi-anti-EGFR-MAb or 2 Gy of X-rays had only little effect on colony formation of UD-SCC5 cells. In contrast, a combined treatment regimen (37 kBq/mL plus 2 Gy) significantly decreased colony formation and enhanced the formation of DNA double-strand breaks. As revealed by flow cytometry, radiation treatments caused accumulation of cells in the G0/G1 phase. Both treatment with ²¹³Bi-anti-EGFR immunoconjugates and application of the combined treatment regimen triggered activation of genes of signaling pathways involved in cell-cycle arrest and induction of apoptosis like p21/Waf, GADD45, Puma and Bax, which were only marginally modulated by X-ray irradiation of cells.Conclusions²¹³Bi-anti-EGFR-MAb enhances cytotoxicity of X-ray irradiation in UD-SCC5 cells most probably due to effective induction of DNA double-strand breaks. Induction of genes involved in cell-cycle arrest and cell death is almost exclusively due to ²¹³Bi-anti-EGFR-MAb and seems to be independent of p53 function.     

Diversity of RGD radiotracers in monitoring antiangiogenesis of flavopiridol and paclitaxel in ovarian cancer xenograft-bearing mice

IntroductionAlthough encouraging results had been shown in antiangiogenesis therapy monitoring, the underlying mechanism of RGD radiotracer accumulation needs to be further illustrated. This study was aimed to investigate the diversity of RGD radiotracers in monitoring antiangiogenic agent's effects and the underlying mechanism in ovarian cancer-bearing mice with a new agent flavopiridol compared with paclitaxel.MethodsOvarian cancer SKOV-3 xenograft-bearing mice were established and divided into three groups, flavopiridol, paclitaxel and control. Flavopiridol (5 mg/kg body weight) and paclitaxel (20 mg/kg body weight) were administered every 3 days for 16 days. Tumor growth and proliferation were monitored by caliper measurements and immunofluorescence staining. Antiangiogenic effects were determined by tumor microvessel density (MVD) in vivo and by endothelial cell tube formation assay in vitro, respectively. ^99mTc-3P-RGD₂ was prepared, and its biodistribution studies were carried out. The effect of antiangiogenesis therapy on integrin αvβ3 expression was studied by immunohistochemical staining and flow cytometry.ResultsBoth paclitaxel and flavopiridol therapy could apparently inhibit tumor growth and proliferation, and antiangiogenic effects of therapy were validated in vivo and in vitro. However, compared with the control group, ID%/g tumor uptake of ^99mTc-3P-RGD₂ showed a significant decrease at 2 hours (by 39.96% ± 8.23%, P = 0.044) and at 4 hours (by 35.76% ± 11.42%, P = 0.024) post injection in the paclitaxel-treated group, but a slight increase of tumor uptake in the flavopiridol-treated group at 2 hours (by 4.42% ± 0.24%, p = 0.898) and at 4 hours (by 12.2% ± 1.84%, P = 0.702). The further studies indicated flavopiridol therapy has a dual-effect, reducing integrin αvβ3 expression on endothelial cells due to the reduction of tumor MVD and up-regulating the integrin αvβ3 expression on tumor cells.ConclusionsThere is diversity in evaluating antiangiogenic response when using ^99mTc-3P-RGD₂, which may be an important reminder in future clinical applications of RGD radiotracers as a strategy for antiangiogenesis therapy response monitoring.