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.     

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.     

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

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

Copper-64 radiolabeling and biological evaluation of bifunctional chelators for radiopharmaceutical development

IntroductionThe development of novel bifunctional chelates for attaching copper-64 to biomolecules has been an active area of research for several years. However, many of these ⁶⁴Cu-chelates have poor in vivo stability or harsh radiolabeling conditions.MethodsIn this study, two triazacyclononane analogs; C-NE3TA (4-carboxymethyl-7-[2-(carboxymethyl-amino)-3-(4-nitro-phenyl)-propyl]-[1,4,7]triazo-nan-1-yl-acetic acid) and N-NE3TA (4-carboxymethyl-7-[2-[carboxymethyl-(4-nitro-benzyl)-amino]-ethyl]-[1,4,7]triazonan-1-yl-acetic acid) were evaluated for their labeling efficiency with ⁶⁴Cu at room temperature and evaluated in vitro and in vivo. In vitro studies included complexation kinetics with Cu(II) using a spectrophotometric method and rat serum stability, while the in vivo biodistribution was evaluated using SCID mice.ResultsC-NE3TA and N-NE3TA were labeled at > 95% efficiency up to ~ 3.4 Ci/μmol. Both C-NE3TA and N-NE3TA formed complexes with Cu(II) almost immediately, with the Cu(II) complexation by C-NE3TA being faster than the formation of Cu(II)-N-NE3TA. Both ⁶⁴Cu-N-NE3TA and ⁶⁴Cu-C-NE3TA were 96.1% and 90.5% intact after 48 h incubation in rat serum, respectively. This is compared to ⁶⁴Cu complexes of the control chelators, p-NH₂-Bn-DOTA and p-NH₂-Bn-NOTA, with 93.9% and 97.9% retention of ⁶⁴Cu in the complex, respectively. In vivo evaluation of ⁶⁴Cu-N-NE3TA and ⁶⁴Cu-C-NE3TA demonstrates good clearance from normal tissues except for the liver, where 59% and 51% of the radioactivity is retained at 24 h compared to 1 h for ⁶⁴Cu-N-NE3TA and ⁶⁴Cu-C-NE3TA, respectively. This compares to 78% and 3% retention for ⁶⁴Cu-p-NH₂-Bn-DOTA and ⁶⁴Cu-p-NH₂-Bn-NOTA.ConclusionsThese studies demonstrate that while N-NE3TA and C-NE3TA appear to be superior chelators for ⁶⁴Cu than p-NH₂-Bn-DOTA, they are not better than p-NH₂-Bn-NOTA. Nevertheless, it may still be interesting to evaluate these chelators after conjugation to biomolecules.     

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.     

Effects of photodynamic therapy with talaporfin sodium on squamous cell carcinoma and sarcoma cells

BackgroundPhotodynamic therapy (PDT) uses a photosensitizer and light to destroy abnormal cells. Talaporfin sodium (NPe6) is a second-generation photosensitizer.MethodsWe evaluated the toxic effects of different combinations of laser and NPe6 doses on squamous cell carcinoma (KLN205) and sarcoma (Meth A) cell lines. The cells were incubated with 0, 5, 10, or 30 μg/mL NPe6 for 24 h. The cells were then irradiated with 0, 5, 15, or 30 mW/cm² of laser power, and 0, 1, 5, 10, or 20 J/cm² of laser energy. Cell viability was evaluated after 24 h. We also evaluated the cytotoxic effects of continuous wave or square-wave modulated laser irradiations (2, 5, or 10 Hz, 50% duty) on Meth A cells.ResultsThe median lethal doses of NPe6 against the KLN205 and Meth A cells after treatment at a fluence rate of 15 mW/cm² and a light dose of 20 J/cm² were 18.6 and 5.0 μg/mL, respectively. Meth A cells were more sensitive to PDT than KLN205 cells. There was no significant difference between the effects of continuous wave and square-wave modulated lasers on Meth A cell viability.ConclusionsNPe6 PDT induced cell death in a dose-dependent manner in KLN205 and Meth A cells. More work is required to evaluate the cytotoxic effects of square-wave modulated laser therapy at low light doses.     

Comparative biodistributions and dosimetry of [177Lu]DOTA-anti-bcl-2-PNA-Tyr3-octreotate and [177Lu]DOTA-Tyr3-octreotate in a mouse model of B-cell lymphoma/leukemia

IntroductionThe B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene in non-Hodgkin’s lymphoma (NHL) is a dominant inhibitor of apoptosis. We developed a ¹⁷⁷Lu-labeled bcl-2 antisense peptide nucleic acid (PNA)–peptide conjugate designed for dual modality NHL therapy, consisting of a radiopharmaceutical capable of simultaneously down-regulating apoptotic resistance and delivering cytotoxic internally emitted radiation.MethodsDOTA-anti-bcl-2-Tyr³-octreotate was synthesized, labeled with ¹⁷⁷Lu, and purified using RP-HPLC. The PNA–peptide conjugate was evaluated in Mec-1 NHL-bearing mice and compared to [¹⁷⁷Lu]DOTA-Tyr³-octreotate in biodistribution and excretion studies. These data were then used to generate in vivo dosimetry models.ResultsThe PNA–peptide conjugate was readily prepared and radiolabeled in high yield and radiochemical purity. An in vivo blocking study determined that administration of 50 μg of non-radioactive PNA–peptide was the optimal mass for maximum delivery to the tumor. Based on that result, a dosing regimen of ¹⁷⁷Lu-PNA–peptide, for radiologic effect, followed by the optimal mass of non-radioactive compound, for antisense effect, was designed. Using that dosing regimen, biodistribution of the PNA–peptide showed uptake in the tumor with minimal washout over a 4-day period. Uptakes in receptor-positive normal organs were low and displayed nearly complete washout by 24 h. Dosimetry models showed that the tumor absorbed dose of the PNA–peptide conjugate was approximately twice that of the peptide-only conjugate.ConclusionsBiodistribution data showed specific tumor targeting of the ¹⁷⁷Lu-labeled PNA–peptide compound with minimal receptor-positive normal tissue uptake when compared to [¹⁷⁷Lu]DOTA-Tyr³-octreotate. In vivo dosimetry models predicted a more favorable tumor absorbed dose from [¹⁷⁷Lu]DOTA-anti-bcl-2-Tyr³-octreotate.     

In vitro and in vivo evaluation of novel ligands for radioimmunotherapy

Novel ligands cis-2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-1-piperidineacetic acid (PIP-DTPA), cis-[(1R,11S)-6,9,15-Tris-carboxymethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadec-3-yl]-acetic acid (PIP-DOTA), cis-{2,7-bis-[bis-carboxymethyl-amino)-methyl]-azepan-1-yl}-acetic acid (AZEP-DTPA), [2-(4,7-bis-carboxymethyl-[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]-tetraacetic acid (NETA) and [{4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perhydro-1,4,7-triazonin-1-yl}-acetic acid (NPTA) are investigated as potential chelators of 177Lu, 90Y, 212Pb and 213Bi for radioimmunotherapy (RIT). The new ligands are radiolabeled with 177Lu, 86/88/90Y, 203Pb and 205/6Bi, and in vitro stability and in vivo stability of the radiolabeled complexes are assessed in human serum and athymic mice, respectively. In vitro studies indicate that all radiolabeled complexes with the exception of 90Y-AZEP-DTPA are stable in serum for 5-11 days. All new ligands examined herein are found to tightly hold 177Lu in vivo. Piperidine-backboned DTPA (PIP-DTPA) complexes radiolabeled with all radioisotopes examined display excellent in vivo stability, that is, excretion without dissociation. The azepane-backboned DTPA derivative, AZEP-DTPA, appears ineffective in binding all but 177Lu in vivo. NETA and NPTA radiolabeled with 86Y or 177Lu exhibit rapid blood clearance and low organ uptakes. Significant accretion in the kidney, femur and/or liver is observed with 203Pb-labeled AZEP-DTPA, PIP-DOTA and NPTA. Both 203Pb-PIP-DOTA and 205/6Bi-PIP-DOTA result in moderate to high renal accumulation of radioactivity. NETA exhibits improved renal accumulation with respect to PIP-DOTA for 205/6Bi but also shows significant liver uptake. Of all ligands studied, only PIP-DTPA appears to effectively bind 203Pb and 205/6Bi in vivo. PIP-DTPA, PIP-DOTA, NETA and NPTA all show strong evidence of rapid blood clearance and low organ uptake for 177Lu and 90Y. Serum stability and in vivo biodistribution results suggest PIP-DTPA as a potential chelating agent with broad applicability for use in 177Lu, 90Y, 212Pb and 213Bi RIT.     

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.     

11C-Labeling of a potent hydroxyethylamine BACE-1 inhibitor and evaluation in vitro and in vivo

IntroductionThe enzyme β-secretase 1 (BACE-1) is associated with the catalytic cleavage of amyloid precursor protein (APP) which leads to the production of amyloid-β, an amyloidogenic peptide that forms insoluble fibrils and is linked to neurodegeneration and Alzheimer's disease (AD). A PET-radioligand for the quantification of BACE-1 would be useful for the understanding of AD. In this report, we describe the synthesis and carbon-11 radiolabeling of a potent hydroxyethylamine BACE-1 enzyme inhibitor (BSI-IV) and its evaluation in vitro and in vivo.Methods¹¹[C]-N¹-((2S,3R)-4-(cyclopropylamino)-3-hydroxy-1-phenylbutan-2-yl)-5-(N-methylmethyl-sulfonamido)-N³-((R)-1-phenylethyl)isophthalamide, a β-secretase inhibitor, denoted here as [¹¹C]BSI-IV was synthesized through a palladium-mediated aminocarbonylation with an aryl halide precursor (I or Br) and [¹¹C]CO. The effect of different palladium/ligand-complexes on radiochemical yield in the carbonylative reaction was investigated. The binding of the labeled compound to BACE-1 enzyme was studied in vitro by frozen section autoradiography from brains of healthy rats. Dynamic small animal PET-CT studies and ex vivo biodistribution were performed in male rats.ResultsThe halide precursors were synthesized in six steps starting from methyl-3-nitrobenzoate with an overall yield of 21–26%. [¹¹C]BSI-IV was obtained in 29 ± 12% decay corrected radiochemical yield (n = 12) with a specific activity of 790 ± 155 GBq/μmol at the end of synthesis with a radiochemical purity of > 99%. The preclinical studies showed that [¹¹C]BSI-IV has a rapid metabolism in rat with excretion to the small intestines.Conclusion¹¹[C]BSI-IV was obtained in sufficient amount and purity to enable preclinical investigation. The preclinical studies showed low specific binding in vitro and fast clearance in vivo and a low uptake in the brain. These findings suggests that [¹¹C]BSI-IV has limited use as a PET-ligand for the study of BACE-1 or AD.     

Coinciding exercise with peak serum caffeine does not improve cycling performance

ObjectivesTo investigate whether coinciding peak serum caffeine concentration with the onset of exercise enhances subsequent endurance performance.DesignRandomised, double-blind, crossover.MethodsIn this randomised, placebo-controlled, double-blind crossover study, 14 male trained cyclists and triathletes (age 31 ± 5 year, body mass 75.4 ± 5.7 kg, VO₂max 69.5 ± 6.1 mL kg^?1 min^?1 and peak power output 417 ± 35 W, mean ± SD) consumed 6 mg kg^?1 caffeine or a placebo either 1 h (C_1 h) prior to completing a 40 km time trial or when the start of exercise coincided with individual peak serum caffeine concentrations (C_peak). C_peak was determined from a separate ‘caffeine profiling’ session that involved monitoring caffeine concentrations in the blood every 30 min over a 4 h period.ResultsFollowing caffeine ingestion, peak serum caffeine occurred 120 min in 12 participants and 150 min in 2 participants. Time to complete the 40 km time trial was significantly faster (2.0%; p = 0.002) in C_1 h compared to placebo. No statistically significant improvement in performance was noted in the C_peak trial versus placebo (1.1%; p = 0.240). Whilst no differences in metabolic markers were found between C_peak and placebo conditions, plasma concentrations of glucose (p = 0.005), norepinephrine and epinephrine (p ≤ 0.002) were higher in the C_1 h trial 6 min post-exercise versus placebo.ConclusionsIn contrast to coinciding peak serum caffeine concentration with exercise onset, caffeine consumed 60 min prior to exercise resulted in significant improvements in 40 km time trial performance. The ergogenic effect of caffeine was not found to be related to peak caffeine concentration in the blood at the onset of endurance exercise.     

Efficacy of erythrosine and cyanidin-3-glucoside mediated photodynamic therapy on Porphyromonas gingivalis biofilms using green light laser

BackgroundThe purpose of this in vitro study was to evaluate the efficacy of erythrosine and cyanidin-3-glucoside as photosensitizers in PDT for the elimination of Porphyromonas gingivalis (P. gingivalis) biofilms.MethodsP. gingivalis biofilms were prepared from a chronic periodontitis subject. Erythrosine and cyanidin-3-glucoside were prepared and randomly allocated as follows: 110, 220, 330, and 440 μM erythrosine; 101, 202, 303, and 404 μM anthocyanin; and 440 μM erythrosine + 404 μM cyanidin-3-glucoside. There were 18 PDT experimental groups (non-irradiated/irradiated with a 532-nm green light diode laser at 1.29 J/cm² for 60 s). The 3 controls were grouped as follows: biofilms exposed to the photosensitizers alone, biofilms exposed to the laser alone, and biofilms exposed to 0.12% chlorhexidine. All sample groups were cultured at 1, 3 and 6 h after PDT and incubated in an anaerobic chamber at 37 °C for 4 days. The surviving fraction was calculated from the log₁₀ CFU/ml. The 330 and 440 μM erythrosine and the 440 μM erythrosine + 404 μM cyanidin-3-glucoside were mixed with spin traps (TEMPO, DMPO), and the electron spin resonance spectra were evaluated.ResultsThe log₁₀ CFU/ml measurements showed that the PDT groups with 330 μM or 440 μM erythrosine and 440 μM erythrosine + 404 μM cyanidin-3-glucoside had statistically significant differences from the other groups (one-way ANOVA and Bonferroni’s multiple comparison test, p- value ≤ 0.05).ConclusionsPDT using 330 μM erythrosine, 440 μM erythrosine or 440 μM erythrosine + 404 μM cyanidin-3-glucoside irradiated with the laser more effectively inhibited P. gingivalis in biofilms.     

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.     

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

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

232Th(d,4n)230Pa cross-section measurements at ARRONAX facility for the production of 230U

Introduction²²⁶Th (T_1/2 = 31 min) is a promising therapeutic radionuclide since results, published in 2009, showed that it induces leukemia cells death and activates apoptosis pathways with higher efficiencies than ²¹³Bi. ²²⁶Th can be obtained via the ²³⁰U α decay. This study focuses on the ²³⁰U production using the ²³²Th(d,4n)²³⁰Pa(β−)²³⁰U reaction.MethodsExperimental cross sections for deuteron-induced reactions on ²³²Th were measured from 30 down to 19 MeV using the stacked-foil technique with beams provided by the ARRONAX cyclotron. After irradiation, all foils (targets as well as monitors) were measured using a high-purity germanium detector.ResultsOur new ²³⁰Pa cross-section values, as well as those of ²³²Pa and ²³³Pa contaminants created during the irradiation, were compared with previous measurements and with results given by the TALYS code. Experimentally, same trends were observed with slight differences in orders of magnitude mainly due to the nuclear data change. Improvements are ongoing about the TALYS code to better reproduce the data for deuteron-induced reactions on ²³²Th.ConclusionsUsing our cross-section data points from the ²³²Th(d,4n)²³⁰Pa reaction, we have calculated the thick-target yield of ²³⁰U, in Bq/μA·h. This value allows now to a full comparison between the different production routes, showing that the proton routes must be preferred.     

The reasons why stroke patients expend so much energy to walk slowly

BackgroundThe energy consumed per covered distance (C) is increased in hemiparetic stroke adults during walking.ObjectiveTo ascertain if increased C in stroke patients is a result of increased mechanical work, of decreased efficiency of work production by muscles or of slow walking speed.MethodsC and mechanical work were computed in 20 patients walking on a force measuring treadmill at speeds ranging from 1 km h^?1 to their own maximum speed (WS_MAX). Works done by healthy and pathological limbs were computed separately.ResultsFor hemiparetic patients, C was around 1.7 times greater than normal. When these patients had a slower WS_MAX, they had greater C and mechanical work (r = ?0.44 and ?0.57, respectively). The increased C was related to the external work performed to lift the center of body mass when the healthy limb was supporting the body weight (r = 0.77).ConclusionsThe increase of C in stroke patients is more pronounced when WS_MAX is slow. Moreover, this increase is related to increased mechanical work done by muscles and is not related to slow walking speed or decreased efficiency. As in healthy subjects, C and external work presented optimum speeds, indicating a preserved pendular mechanism of walking.     

Synthesis and biological evaluation of copper-64 radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a novel bivalent targeting vector having affinity for two distinct biomarkers (GRPr/PSMA) of prostate cancer

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH₂ = bombesin, a GRPr-specific peptide targeting probe.MethodsThe PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH₂], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with ⁶⁴CuCl₂ and ^natCuCl₂. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.).ResultsCompetitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-(^natCu-NODAGA)-5-Ava-BBN(7-14)NH₂] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18 h p.i. with collateral, background radiation also being observed in non-target tissue.ConclusionsDUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂ in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues.     

Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113

ObjectivesWith the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with ¹¹C and evaluated using small-animal PET.Methods[¹¹C]MC113 was synthesized by reaction of O-desmethyl MC113 with [¹¹C]methyl triflate. Small-animal PET was performed with [¹¹C]MC113 in FVB wild-type and Mdr1a/b^(-/-) mice (n = 3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n = 5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15 mg/kg).ResultsBrain uptake of [¹¹C]MC113, expressed as area under the time-activity curve from time 0 to 60 min (AUC_0-60), was moderately but not significantly increased in Mdr1a/b^(-/-) compared with wild-type mice (mean ± SD AUC_0-60, Mdr1a/b^(-/-): 88 ± 7 min, wild-type: 62 ± 6 min, P = 0.100, Mann Whitney test). In the tumor model, AUC_0-60 values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC₅₀) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375 ± 60 nM for MC113 versus 8.5 ± 2.5 nM for tariquidar.Conclusion[¹¹C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [¹¹C]MC113 was only to a low extent effluxed by Pgp. However, [¹¹C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.