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.     

Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy

IntroductionIn spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium(¹⁸⁸Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy.MethodsMice bearing A2058 melanoma xenografts were treated with either 1.5 mCi ¹⁸⁸Re-6D2 antibody, saline, unlabeled 6D2 antibody or ¹⁸⁸Re-labeled non-specific IgM.ResultsOn Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P < 0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers — chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells.ConclusionsThese results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5 + and JARID1B + cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers.     

Determining efficacy of breast cancer therapy by PET imaging of HER2 mRNA

IntroductionMonitoring the effectiveness of therapy early and accurately continues to be challenging. We hypothesize that determination of Human Epidermal Growth Factor Receptor 2 (HER2) mRNA in malignant breast cancer (BC) cells by positron emission tomography (PET) imaging, before and after treatment, would reflect therapeutic efficacy.MethodWT4340, a peptide nucleic acid (PNA) 12-mer complementary to HER2 mRNA was synthesized together with -CSKC, a cyclic peptide, which facilitated internalization of the PNA via IGFR expressed on BC cells, and DOTA that chelated Cu-64. Mice (n = 8) with BT474 ER +/HER2+ human BC received doxorubicin (DOX, 1.5 mg/kg) i.p. once a week for six weeks. Mice (n = 8) without DOX served as controls. All mice were PET imaged with F-18-FDG and 48 h later with Cu-64-WT4340. PET imaging were performed before and 72 h after each treatment. Standardized uptake values (SUVs) were determined and percent change calculated. Animal body weight (BW) and tumor volume (TV) were measured.ResultsSUVs for Cu-64-WT4340 after DOX treatment declined by 54% ± 17% after the second dose, 41% ± 15% after the fourth dose, and 29% ± 7% after the sixth dose, compared with 42% ± 22%, 31% ± 18%, and 13% ± 9% (p < 0.05) for F-18-FDG. In untreated mice, the corresponding percent SUVs for Cu-64-WT4340 were 145% ± 82%, 165% ± 39%, and 212% ± 105% of pretreatment SUV, compared with 108% ± 28%, 151% ± 8%, and 152% ± 35.5%, (p < 0.08) for F-18-FDG. TV in mice after second dose was 114.15% ± 61.83%, compared with 144.7% ± 64.4% for control mice. BW of DOX-treated mice was 103.4% ± 7.6% of pretreatment, vs. 100.1% ± 4.3% for control mice.ConclusionTherapeutic efficacy was apparent sooner by molecular PET imaging than by determination of reduction in TV.     

PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists

IntroductionThe gastrin-releasing peptide receptor (GRPR) was shown to be expressed with high density on several types of cancers. Radiolabeled peptides for imaging and targeted radionuclide therapy have been developed. In this study, we evaluated the potential of statine-based bombesin antagonists, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) through oligoethyleneglycol spacers, labeled with ¹⁷⁷Lu and we determined the effect of polyethyleneglycol (PEG) spacer length on in vitro and in vivo properties.MethodsThe bombesin antagonists were synthesized on solid phase using Fmoc chemistry; the spacers Fmoc-dPEG_x-OH (x = 2, 4, 6 and 12) and the DOTA(tBu)₃ were coupled using a standard procedure. The peptides were labeled with ¹⁷⁷Lu and evaluated in vitro (lipophilicity, serum stability, internalization and binding affinity assays). Biodistribution studies were performed in PC-3 tumor-bearing nude mice.ResultsThe solid-phase synthesis was straightforward with an overall yield ranging from 30% to 35% based on the first Fmoc cleavage. The hydrophilicity increased with spacer length (logD: − 1.95 vs − 2.22 of PEG₂ and PEG₁₂ analogs, respectively). There is a tendency of increased serum stability by increasing the spacer length (T_1/2 = 246 ± 4 and 584 ± 20 for PEG₂ and PEG₆ analogs, respectively) which seems to reverse with the PEG₁₂ analog. The IC₅₀ values are similar with the only significant difference of the PEG₁₂ analog. The ¹⁷⁷Lu-labeled PEG₄ and PEG₆ conjugates showed similar pharmacokinetic with high tumor uptake and excellent tumor-to-kidney ratios (7.8 and 9.7 at 4 h for the PEG₄ and PEG₆ derivatives, respectively). The pancreas uptake was relatively high at 1 h but it shows fast washout (0.46% ± 0.02% IA/g and 0.29% ± 0.08% IA/g already at 4 h).ConclusionAmong all the studied analogs the PEG₄ and PEG₆ showed significantly better properties. The very high tumor-to-non-target organ ratios, in particular tumor-to-kidney ratios, already at early time point will be important in regard to safety concerning kidney toxicity.     

Synthesis and evaluation of 11C-labeled naphthalene derivative as a novel non-peptidergic probe for the β-secretase (BACE1) imaging in Alzheimer's disease brain

IntroductionAs a first trial for in vivo imaging of β-secretase (BACE1) in Alzheimer's disease brain, we applied a novel non-peptidergic small molecule which has high affinity to the enzyme, naphthalene-1-carboxylic acid (3′-chloro-4′-fluoro-4-piperazin-1-yl-biphenyl-3-yl)amide (NCFB) into positron emission tomography (PET) probe. In the current study, N-¹¹C-methylated compound of NCFB, [¹¹C]Me-NCFB was synthesized and evaluated for the visualization of BACE1 in brain.MethodsBACE1 inhibitory constant was measured by FRET assay. [¹¹C]Me-NCFB was synthesized from NCFB with [¹¹C]methyl triflate. To evaluate properties of [¹¹C]Me-NCFB, log P value, stability in mouse plasma and brain uptake index were measured. The biodistribution in 6-week-old ddY mice was also studied.ResultsBACE1 inhibitory constant showed an affinity of Me-NCFB to the enzyme (IC₅₀ = 2.3 ± 0.80 μM). [¹¹C]Me-NCFB was synthesized in a 3.0% ± 0.55% decay-corrected radiochemical yield. [¹¹C]Me-NCFB showed high lipophilicity, high stability in mouse plasma and blood–brain barrier (BBB) permeability. Injected to 6-week-old ddY mice, [¹¹C]Me-NCFB penetrated BBB and was retained in the brain (0.79% ± 0.22% ID/g at 2 min and 0.75% ± 0.08% ID/g at 60 min after injection, respectively), moreover, rapid blood clearance was observed.Conclusion[¹¹C]Me-NCFB could have a potential as a PET probe for the imaging of BACE1 in the brain.     

68Ga-DOTA-NGR as a novel molecular probe for APN-positive tumor imaging using MicroPET

Aminopeptidase N (APN) is selectively expressed on many tumors and the endothelium of tumor neovasculature, and may serve as a promising target for cancer diagnosis and therapy. Asparagine–glycine–arginine (NGR) peptides have been shown to bind specifically to the APN receptor and have served as vehicles for the delivery of various therapeutic drugs in previous studies. The purpose of this study was to synthesize and evaluate the efficacy of a ⁶⁸Ga-labeled NGR peptide as a new molecular probe that binds to APN.MethodsNGR peptide was conjugated with 1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’-tetraacetic acid (DOTA) and labeled with ⁶⁸Ga at 95 °C for 10 min. In vitro uptake and binding analysis was performed with A549 and MDA-MB231 cells. Biodistribution of ⁶⁸Ga-DOTA-NGR was determined in normal mice by dissection method. ⁶⁸Ga-DOTA-NGR PET was performed in A549 and MDA-MB231 xenografts, and included dynamic and static imaging. APN expression in tumors and new vasculatures was analyzed by immunohistochemistry.ResultsThe radiochemical purity of ⁶⁸Ga-DOTA-NGR was 98.0% ± 1.4% with a specific activity of about 17.49 MBq/nmol. The uptake of ⁶⁸Ga-DOTA-NGR in A549 cells increased with longer incubation times, and could be blocked by cold DOTA-NGR, while no specific uptake was found in MDA-MB231 cells. In vivo biodistribution studies showed that ⁶⁸Ga-DOTA-NGR was mainly excreted from the kidney, and rapidly cleared from blood and nonspecific organs. MicroPET imaging showed that high focal accumulation had occurred in the tumor site at 1 h post-injection (pi) in A549 tumor xenografts. A significant reduction of tumor uptake was observed following coinjection with a blocking dose of DOTA-NGR, whereas only mild uptake was found in MDA-MB231 tumor xenografts. Tumor uptake, measured as the tumor/lung ratio, increased with time peaking at 12.58 ± 1.26 at 1.5 h pi. Immunohistochemical staining confirmed that APN was overexpressed on A549 cells and neovasculature.Conclusions⁶⁸Ga-DOTA-NGR was easily synthesized and showed favorable biodistribution and kinetics. ⁶⁸Ga-DOTA-NGR could also specifically bind to the APN receptor in vitro and in vivo, and might be a potential molecular probe for the noninvasive detection of APN-positive tumors and neovasculature.     

Reactor production and electrochemical purification of 169Er: A potential step forward for its utilization in in vivo therapeutic applications

IntroductionThe aim of the present study was to develop and demonstrate a viable method for the reactor production of ¹⁶⁹Er with acceptable specific activity using moderate flux reactor and its purification from ¹⁶⁹Yb following electrochemical pathway based on mercury-pool cathode to avail ¹⁶⁹Er in radionuclidically pure form essential for its therapeutic use.MethodsErbium-169 was produced in reactor by neutron bombardment of isotopically enriched (98.2% in ¹⁶⁸Er) erbium target at a thermal neutron flux of ~ 8 × 10¹³ n.cm^- 2.s^- 1 for 21 d. A thorough optimization of irradiation parameters including neutron flux, irradiation time and target cooling time was carried out. The influence of different experimental parameters for the quantitative removal ¹⁶⁹Yb from ¹⁶⁹Er was investigated, optimized and based on the results; a two-cycle electrochemical separation procedure was adopted. The suitablility of purified ¹⁶⁹Er for application in radiation synovectomy and bone pain palliation was ascertained by carrying out radiolabeling studies with hydroxypaptite (HA) particles and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaminomethylene phosphonic acid (DOTMP), respectively.ResultsThermal neutron irradiation of 10 mg of isotopically enriched (98.2% in ¹⁶⁸Er) erbium target at a flux of ~ 8 × 10¹³ n.cm^- 2.s^- 1 for 21 d followed by a two-step electrochemical separation of ¹⁶⁹Yb impurity yielded ~ 3.7 GBq (100 mCi) of ¹⁶⁹Er with a specific activity of ~ 370 MBq/mg (10 mCi/mg) and radionuclidic purity of > 99.99%. The reliability of this approach was amply demonstrated by performing several production batches, where the performance of each batch remained consistent. The utility of the purified ¹⁶⁹Er was demonstrated in the radiolabeling studies with HA particles and DOTMP, wherein both the radiolabeled products were obtained with high radiolabeling yield (> 99%).ConclusionsA viable strategy for the batch production and purification of ¹⁶⁹Er, suitable for therapeutic applications, has been developed and demonstrated.     

Low-level γ-ray spectrometry at the underground laboratory Garching

Two low-background setups for material screening based on HPGe detectors were built in the Garching Underground Laboratory with an overburden of ~10 m.w.e. They include several layers of passive shielding as well as an active muon veto. The first setup (GEM) comprises a 150% efficiency HPGe detector which can optionally be surrounded by a NaI(Tl) scintillation detector that serves as anti-Compton veto. The second setup (LoAx) consists of two smaller HPGe detectors which are arranged face-to-face to cover a larger solid angle around the sample and to allow coincidence measurements.For a 5.6 kg piece of copper after 11 days of measurement we have reached a sensitivity for ²²⁶Ra and ²²⁸Ra/²²⁸Th of ~5 mBq kg⁻¹ with the GEM setup. In the LoAx setup we have achieved limits of less than 100 mBq kg⁻¹ for ²³⁴Th and ²¹⁰Pb with a 156 g sample of PPO wavelength shifter after 18 days of measurement.     

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.     

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.     

Distribution of naturally occurring radionuclides activity concentration in East Malaysian marine sediment

Studies of naturally occurring radioactive materials (NORM) distribution of ²²⁶Ra, ²²⁸Ra and ⁴⁰K in East Malaysia were carried out as part of a marine coastal environment project. The results of measurements will serve as baseline data and background reference level for Malaysia coastlines. Sediments from 21 coastal locations and 10 near shore locations were collected for analyses. The samples were dried, finely ground, sealed in a container and stored for a minimum of 30 days to establish secular equilibrium between ²²⁶Ra and ²²⁸Ra and their respective radioactive progenies. They were counted using a high-purity germanium (HPGe) spectrometer covering the respective progeny energy peak. For ⁴⁰K, the presence of this was measured directly via its 1460 keV energy peak. The concentration of ²²⁶Ra, ²²⁸Ra and ⁴⁰K in samples obtained from coastal Sarawak ranged between 23 and 41 (mean 30±2) Bq/kg, 27 and 45 (mean 39±4) Bq/kg and 142 and 680 (mean 462±59) Bq/kg, respectively. Meanwhile, the concentration of ²²⁶Ra, ²²⁸Ra and ⁴⁰K for samples obtained from coastal Sabah ranged between 16 and 30 (mean 23±2) Bq/kg, 23 and 45 (mean 35±4) Bq/kg and 402 and 842 (mean 577±75) Bq/kg, respectively. For the Sarawak near shore stations, the concentration of ²²⁶Ra, ²²⁸Ra and ⁴⁰K ranged between 11 and 36 (mean 22±2) Bq/kg, 21 and 65 (mean 39±5) Bq/kg and 149 and 517 (mean 309±41) Bq/kg, respectively. Meanwhile, the concentration of ²²⁶Ra, ²²⁸Ra and ⁴⁰K for samples obtained from Sabah ranged between 9 and 31 (mean 14±2) Bq/kg, 10 and 48 (mean 21±3) Bq/kg and 140 and 580 (mean 269±36) Bq/kg, respectively. The calculated external hazard values of between 0.17 and 0.33 (less than unity) showed that there is little risk of external hazard to the workers handling the sediments.     

Radium-226 concentration in spring water sampled in high radon regions

Water ²²⁶Ra concentration in springs was measured in regions with high indoor radon: Ural, North Caucasus (Russia), Niska Banja (Serbia), Piestany (Slovakia), and Issyk-Kul (Kyrgyzstan). This paper presents the results for ²²⁶Ra concentration above 0.03 Bq l^–1. Radium in water could indicate indoor radon problem in the region and water investigation is useful at the initial stage of radon survey. Even low ²²⁶Ra concentration in water (0.1–0.6 Bq l^–1) caused high ²²⁶Ra activity in travertine (up to 1500 Bq kg^?1), which resulted in indoor radon concentration above 2000 Bq m^?3 (Niska Banja).     

Optimization of reaction conditions for the radiolabeling of DOTA and DOTA-peptide with 44m/44Sc and experimental evidence of the feasibility of an in vivo PET generator

IntroductionAmong the number of generator systems providing radionuclides with decay parameters promising for imaging and treatment applications, there is the ⁴⁴Ti (T_1/2 = 60 years)/⁴⁴Sc (T_1/2 = 3.97 h) generator. This generator provides a longer-lived daughter for extended PET/CT measurements compared to the chemically similar system ⁶⁸Ge/⁶⁸Ga. Scandium also exists as ⁴⁷Sc, a potential therapeutic radionuclide. It is possible to produce ⁴⁴Sc in a cyclotron using, for example, the ⁴⁴Ca (d, n) ⁴⁴Sc nuclear reaction. In that case, the isomeric state ^44mSc (T_1/2 = 58.6 h) is co-produced and may be used as an in vivo ^44mSc/⁴⁴Sc generator. The aim of this study is to evaluate the feasibility of this in vivo ^44mSc/⁴⁴Sc generator and to demonstrate that the daughter radionuclide stays inside the chelator after decay of the parent radionuclide. Indeed, the physico-chemical process occurring after the primary radioactive decay (EC, IT, Auger electron …) has prevented in many cases the use of in-vivo generator, because of the post-effect as described in the literature.MethodsThe DOTA macrocyclic ligand forms stable complexes with many cations and has been shown to be the most suitable chelating moiety for scandium. Initially, the radiolabeling of DOTA and a DOTA-peptide (DOTATATE) with Sc was performed and optimized as a function of time, pH, metal-to-ligand ratio and temperature. Next, the physico-chemical processes that could occur after the decay (post-effect) were studied. ^44mSc(III)-labeled DOTA-peptide was quantitatively adsorbed on a solid phase matrix through a hydrophobic interaction. Elutions were then performed at regular time intervals using a DTPA solution at various concentrations. Finally, the radiolabelled complex stability was studied in serum.ResultsRadiolabeling yields ranged from 90% to 99% for metal-to-ligand ratio ranging from 1:10 to 1:500 for DOTA or DOTATATE respectively. The optimum physico-chemical parameters were pH = 4–6, t = 20 min, T = 70 °C. Then, the ^44mSc-DOTATATE complex, radiolabeled at 98%, was adsorbed through a hydrophobic interaction to a solid phase. Unlabeled scandium was completely eluted from the column whereas the Sc-DOTATATE complex was 100% retained. The release of ⁴⁴Sc from the complex due to decay was less than 1% over 2 periods of ^44mSc, independent of the DTPA concentration used for elution. ^44mSc/⁴⁴Sc-DOTATATE was stable in serum over 72 h.ConclusionsThe results indicate that the decay of ^44mSc to ⁴⁴Sc does not affect the integrity of the radiolabeled compound. Thus the ^44mSc/⁴⁴Sc generator is chemically valid and stable in serum. It could be used for PET imaging as an in-vivo generator increasing the life time of the scandium and allowing the use of antibody as labelled compound. Further in-vivo biological evaluations should complete this work.     

Test–retest repeatability of quantitative cardiac 11C-meta-hydroxyephedrine measurements in rats by small animal positron emission tomography

IntroductionThe norepinephrine analogue ¹¹C-meta-hydroxyephedrine (HED) has been used to interrogate sympathetic neuronal reuptake in cardiovascular disease. Application for longitudinal studies in small animal models of disease necessitates an understanding of test–retest variability. This study evaluated the repeatability of multiple quantitative cardiac measurements of HED retention and washout and the pharmacological response to reuptake blockade and enhanced norepinephrine levels.MethodsSmall animal PET images were acquired over 60 min following HED administration to healthy male Sprague Dawley rats. Paired test and retest scans were undertaken in individual animals over 7 days. Additional HED scans were conducted following administration of norepinephrine reuptake inhibitor desipramine or continuous infusion of exogenous norepinephrine. HED retention was quantified by retention index, standardized uptake value (SUV), monoexponential and one-compartment washout. Plasma and cardiac norepinephrine were measured by high performance liquid chromatography.ResultsTest retest variability was lower for retention index (15% ± 12%) and SUV (19% ± 15%) as compared to monoexponential washout rates (21% ± 13%). Desipramine pretreatment reduced myocardial HED retention index by 69% and SUV by 85%. Chase treatment with desipramine increased monoexponential HED washout by 197% compared to untreated controls. Norepinephrine infusion dose-dependently reduced HED accumulation, reflected by both retention index and SUV, with a corresponding increase in monoexponential washout. Plasma and cardiac norepinephrine levels correlated with HED quantitative measurements.ConclusionThe repeatability of HED retention index, SUV, and monoexponential washout supports its suitability for longitudinal PET studies in rats. Uptake and washout of HED are sensitive to acute increases in norepinephrine concentration.     

An improved generator for the production of 212Pb and 212Bi from 224Ra

We have developed an improved generator for the production of the alpha emitting radionuclide ²¹²Bi and its parent, ²¹²Pb. These radionuclides are well suited to use as radiotherapeutic agents due to their relatively short half lives and appropriate particle particle emissions. The parent, ²²⁴Ra, is available from a long-lived parent and can be isolated on a generator which produces the daughters in good yield and low breakthrough. The ²¹²Bi can be eluted by itself or in equilibrium with its parent.     

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.     

PET quantification with a histogram derived total activity metric: Superior quantitative consistency compared to total lesion glycolysis with absolute or relative SUV thresholds in phantoms and lung cancer patients

IntroductionThe increasing use of molecular imaging probes as biomarkers in oncology emphasizes the need for robust and stable methods for quantifying tracer uptake in PET imaging. The primary motivation for this research was to find an accurate method to quantify the total tumor uptake. Therefore we developed a histogram-based method to calculate the background subtracted lesion (BSL) activity and validated BSL by comparing the quantitative consistency with the total lesion glycolysis (TLG) in phantom and patient studies.MethodsA thorax phantom and a PET-ACR quality assurance phantom were scanned with increasing FDG concentrations. Volumes of interest (VOIs) were placed over each chamber. TLG was calculated with a fixed threshold at SUV 2.5 (TLG_2.5) and a relative threshold at 42% of SUV_max (TLG_42%). The histogram for each VOI was built and BSL was calculated. Comparison with the total injected FDG activity (TIA) was performed using concordance correlation coefficients (CCC) and the slope (a). Fifty consecutive patients with FDG-avid lung tumors were selected under an IRB waiver. TLG_42%, TLG_2.5 and BSL were compared to the reference standard calculating CCC and the slope.ResultsIn both phantoms, the CCC for lesions with a TIA ≤ 50 ml*SUV between TIA and BSL was higher and the slope closer to 1 (CCC = 0.933, a = 1.189), than for TLG_42% (CCC = 0.350, a = 0.731) or TLG_2.5 (CCC = 0.761, a = 0.727). In 50 lung lesions BSL had a slope closer to 1 compared to the reference activity than TLG_42% (a = 1.084 vs 0.618 – for high activity lesions) and also closer to 1 than TLG_2.5 (a = 1.117 vs 0.548 – for low activity lesions).ConclusionThe histogram based BSL correlated better with TIA in both phantom studies than TLG_2.5 or TLG_42%. Also in lung tumors, the BSL activity is overall more accurate in quantifying the lesion activity compared to the two most commonly applied TLG quantification methods.     

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: Influence of isomeric substitution on radiolabeling and target cell residualization

IntroductionN-succinimidyl 4-guanidinomethyl-3-[^⁎I]iodobenzoate ([^⁎I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [¹³¹I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[¹³¹I]iodobenzoate (iso-[¹³¹I]SGMIB) wherein this bulky group was moved from ortho to meta position.MethodsBoc₂-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc₂-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors — trastuzumab (Tras) and a nanobody 5 F7 (Nb) — were labeled using iso-[^⁎I]SGMIB and [^⁎I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed.ResultsWhen the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc₂-iso-[¹³¹I]SGMIB were significantly higher than those for Boc₂-[¹³¹I]SGMIB (70.7 ± 2.0% vs 56.5 ± 5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[¹³¹I]SGMIB than with [¹³¹I]SGMIB (Nb, 33.1 ± 7.1% vs 28.9 ± 13.0%; Tras, 45.1 ± 4.5% vs 34.8 ± 10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5 F7 Nb indicated similar residualizing capacity over 6 h; however, at 24 h, radioactivity retained intracellularly for iso-[¹³¹I]SGMIB-Nb was lower than for [¹²⁵I]SGMIB-Nb (46.4 ± 1.3% vs 56.5 ± 2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[¹²⁵I]SGMIB and [¹³¹I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [¹³¹I]SGMIB-Tras.ConclusionGiven the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.     

Cation exchange separation of 61Cu2+ from natCo targets and preparation of 61Cu–DOTA–HSA as a blood pool agent

An improved method for isolation of ⁶¹Cu²⁺ from a ^natCo target using cation exchange was developed. ⁶¹Cu²⁺ was eluted from a cation exchange resin column by 0.2 M HCl with 90% acetone, while Co²⁺ remained on the column. The whole separation process was completed within 50 min at more than 72% yield. The Co²⁺ impurity level in ⁶¹Cu²⁺ solution was reduced to less than 0.1 ppm. Highly pure ⁶¹Cu²⁺ solution was then applied to prepare ⁶¹Cu–1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)–human serum albumin (HSA) which showed good blood pool imaging properties.