Biological evaluation of an ornithine-modified 99mTc-labeled RGD peptide as an angiogenesis imaging agent

IntroductionRadiolabeled RGD peptides that specifically target integrin α_νβ₃ have great potential in early tumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous findings of our group on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfK derivative, c(RGDfK)-(Orn)₃-CGG. This new peptide availing the polar linker (Orn)₃ and the ^99mTc-chelating moiety CGG (Cys-Gly-Gly) is appropriately designed for ^99mTc-labeling, as well as consequent conjugation onto nanoparticles.MethodsA tumor imaging agent, c(RGDfK)-(Orn)₃-[CGG-^99mTc], is evaluated with regard to its radiochemical, radiobiological and imaging characteristics.ResultsThe complex c(RGDfK)-(Orn)₃-[CGG-^99mTc] was obtained in high radiochemical yield (> 98%) and was stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized ^185/187Re complex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the new radiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake (3.87 ± 0.48% ID/g at 60 min p.i.) was observed and was maintained relatively high even at 24 h p.i. (1.83 ± 0.05 % ID/g), thus providing well-defined scintigraphic imaging. Accumulation in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells.ConclusionDue to its relatively high tumor uptake, renal elimination and negligible abdominal localization, the new ^99mTc-RGD peptide is considered promising in the field of imaging α_νβ₃-positive tumors. However, the preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dual modality imaging of integrin expressing tumors should be further investigated.     

Specific uptake of 99mTc-NC100692, an αvβ3-targeted imaging probe,in subcutaneous and orthotopic tumors

IntroductionThe α_vβ₃ integrin, which is expressed by angiogenic epithelium and some tumor cells, is an attractive target for the development of both imaging agents and therapeutics. While optimal implementation of α_vβ₃-targeted therapeutics will require a priori identification of the presence of the target, the clinical evaluation of these compounds has typically not included parallel studies with α_vβ₃-targeted diagnostics. This is at least partly due to the relatively limited availability of PET radiopharmaceuticals in comparison to those labeled with ^99mTc. In an effort to begin to address this limitation, we evaluated the tumor uptake of ^99mTc-NC100692, a cyclic RGD peptide that binds to α_vβ₃ with ~ 1-nM affinity, in an α_vβ₃-positive tumor model as well as its in vivo specificity.MethodsMicroSPECT imaging was used to assess the ability of cilengitide, a therapeutic with high affinity for α_vβ₃, to block and displace ^99mTc-NC100692 in an orthotopic U87 glioma tumor. The specificity of ^99mTc-NC100692 was quantitatively evaluated in mice bearing subcutaneous U87MG tumors, by comparison of the biodistribution of ^99mTc-NC100692 with that of the non-specific structural analogue ^99mTc-AH-111744 and by blocking uptake of ^99mTc-NC100692 with excess unlabeled NC100692.ResultsMicroSPECT imaging studies demonstrated that uptake of ^99mTc-NC100692 in the intracranial tumor model was both blocked and displaced by the α_vβ₃-targeted therapeutic cilengitide. Biodistribution studies provided quantitative confirmation of these imaging results. Tumor uptake of ^99mTc-NC100692 at 1 h post-injection was 2.8 ± 0.7% ID/g compared to 0.38 ± 0.1% ID/g for ^99mTc-AH-111744 (p < 0.001). Blocking ^99mTc-NC100692 uptake by pre-injecting the mice with excess unlabeled NC100692 reduced tumor uptake by approximately five-fold, to 0.68 ± 0.3% ID/g (p = 0.01).ConclusionThese results confirm that ^99mTc-NC100692 does, in fact, target the α_vβ₃ integrin and may, therefore, be useful in identifying patients prior to anti-α_vβ₃ therapy as well as monitoring the response of these patients to therapy.     

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.     

Be spoilt for choice with radiolabelled RGD peptides: Preclinical evaluation of 68 Ga-TRAP(RGD)3

Gallium-68 is rapidly gaining importance, as this generator-produced PET isotope is available independent of on-site cyclotrons, enabling radiopharmaceutical production with comparably simple techniques at low cost. The recently introduced TRAP chelator combines the advantage of straightforward design of multimeric ⁶⁸ Ga-radiopharmaceuticals with very fast and efficient ⁶⁸ Ga-labeling. We synthesized a series of five cyclo(RGDfK) peptide trimers and determined their α_vβ₃ integrin affinities in competition assays on α_vβ₃-expressing M21 human melanoma cells against ¹²⁵I-echistatin. The compound with highest IC₅₀, Ga-TRAP(RGD)₃, showed more than 7-fold higher affinity compared to the monomers F-Galacto-RGD and Ga-NODAGA-c(RGDyK). TRAP(RGD)₃ was radiolabeled with ⁶⁸ Ga in a fully automated GMP compliant manner. CD-1 athymic nude mice bearing M21/M21L human melanoma xenografts were used for biodistribution studies, blockade experiments, metabolite studies and PET imaging. ⁶⁸ Ga-TRAP(RGD)₃ exhibited high M21 tumor uptake (6.08 ± 0.63% ID/g, 60 min p.i.), was found to be fully stable in vivo, and showed a fast renal clearance. Blockade studies showed that uptake in the tumor, as well as in all other tissues, is highly integrin specific. A comparison of biodistribution and PET data of ⁶⁸ Ga-TRAP(RGD)₃ with those of ⁶⁸ Ga-NODAGA-c(RGDyK) and ¹⁸ F-Galacto-RGD showed that the higher affinity of the trimer effects a larger dynamic response of tracer uptake to integrin expression, i.e., enhanced integrin-specific uptake in all tissues. We conclude that ⁶⁸ Ga-TRAP(RGD)₃ could allow for imaging of low-level integrin expression in tissues which are not visible with the two competitors. Overall, the study constitutes proof of concept for the favourable in vivo properties of TRAP-based ⁶⁸ Ga radiopharmaceuticals.     

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.     

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.     

Kit-like 18F-labeling of RGD-19F-Arytrifluroborate in high yield and at extraordinarily high specific activity with preliminary in vivo tumor imaging

IntroductionPositron Emission Tomography (PET) is a rapidly expanding, cutting edge technology for preclinical evaluation, cancer diagnosis and staging, and patient management. A one-step aqueous ¹⁸F-labeling method, which can be applied to peptides to provide functional in vivo images, has been a long-standing challenge in PET imaging. Over the past few years, we have sought a rapid and mild radiolabeling method based on the aqueous radiosynthesis of in vivo stable aryltrifluoroborate (ArBF₃^?) conjugates. Recent access to production levels of ¹⁸F-Fluoride led to a fluorescent-¹⁸F-ArBF₃^? at unprecedentedly high specific activities of 15 Ci/μmol. However, extending this method to labeling peptides as imaging agents has not been explored.MethodsIn order to extend these results to a peptide of clinical interest in the context of production-level radiosynthesis, we applied this new technology for labeling RGD, measured its specific activity by standard curve analysis, and carried out a preliminary evaluation of its imaging properties.ResultsRGD was labeled in excellent radiochemical yields at exceptionally high specific activity (~ 14 Ci/μmol) (n = 3). Preliminary tumor-specific images corroborated by ex vivo biodistribution data with blocking controls show statistically significant albeit relatively low tumor uptake along with reasonably high tumor:blood ratios (n = 3).ConclusionsIsotope exchange on a clinically useful ¹⁸F-ArBF₃^? radiotracer leads to excellent radiochemical yields and exceptionally high specific activities while the anionic nature of the aryltrifluoroborate prosthetic results in very rapid clearance. Since rapid clearance of the radioactive tracer is generally desirable for tracer development, these results suggest new directions for varying linker arm composition to slightly retard clearance rather than enhancing it.Advances in Knowledge and Implications for patient CareThis work is the first to use production levels of ¹⁸F-activity to directly label RGD at specific activities that are an order of magnitude higher than most reports and thereby increases the distribution window for radiotracer production and delivery.     

Initial characterization of a dually radiolabeled peptide for simultaneous monitoring of protein targets and enzymatic activity

ObjectiveThe goal of this study was to develop dually radiolabeled peptides for simultaneous imaging of cancer cell localization by targeting the α_vβ₃ integrin and their pathophysiology by targeting the activity of the proteolytic enzyme MMP2, involved in the metastatic process.MethodsA hybrid peptide c(RGDfE)K(DOTA)PLGVRY containing an RGD motif for binding to the α_vβ₃integrin, a metal chelator (DOTA) for radiolabeling with [⁶⁴Cu], and the MMP2 substrate cleavage sequence PLGVRY with terminal tyrosine for labeling with [¹²³I] was synthesized, labeled with [⁶⁴Cu] and [¹²³I], and evaluated in vitro as a potential imaging agent.ResultsThe peptide was synthesized and labeled with [⁶⁴Cu] and [¹²³I] with 300 and 40 μCi/μg (542 and 72.2 mCi/μmol) specific activities, respectively, and radiochemical purity of > 98%. c(RGDfE)K(DOTA)PLGVRY demonstrated high affinity for α_vβ₃ integrins (Kd = 83.4 + 13.2 nM) in both substrate competition and cell binding assays. c(RGDfE)K(DOTA)PLGVRY peptide, but not the scrambled version, c(RGDfE)K(DOTA)GRPLVY was specifically cleaved by MMP2.ConclusionsThese results demonstrate the feasibility of developing dually radiolabeled peptides for the simultaneous imaging of cancer cells and their pathophysiologic activity.     

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.     

64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

IntroductionWe synthesized and evaluated ⁶⁴Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin α_Vβ₃.MethodsTetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with ⁶⁴Cu at 40 °C for 1 h and purified using a PD-10 column. ⁶⁴Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/⁶⁴Cu-DOTA-liposomes or ⁶⁴Cu-DOTA-liposomes.ResultsHAEC binding studies exhibited that tetrac/⁶⁴Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while ⁶⁴Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/⁶⁴Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of ⁶⁴Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/⁶⁴Cu-DOTA-liposomes and ⁶⁴Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively.ConclusionThese results demonstrate that tetrac/⁶⁴Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to ⁶⁴Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/⁶⁴Cu-DOTA-liposomes.     

Design and biological evaluation of 99mTc-N2S2-Tat(49–57)-c(RGDyK): A hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins

The α(ν)β(3) integrin is over-expressed in the tumor neovasculature and the tumor cells of glioblastomas. The HIV Tat-derived peptide has been used to deliver various cargos into cells. The aim of this research was to synthesize and assess the in vitro and in vivo uptake of ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) (^99mTc-Tat-RGD) in α(ν)β(3) integrin positive cancer cells and compare it to that of a conventional ^99mTc-RGD peptide (^99mTc-EDDA/HYNIC-E-[c(RGDfK)]₂). Methods: The c(RGDyK) peptide was conjugated to a maleimidopropionyl (MP) moiety through Lys, and the MP group was used as the branch position to form a thioether with the Cys¹² side chain of the Tat(49–57)-spacer-N₂S₂ peptide. ^99mTc-Tat-RGD was prepared, and stability studies were carried out by size exclusion HPLC analyses in human serum. The in vitro affinity for α(v)β(3) integrin was determined by a competitive binding assay. In vitro internalization was determined using glioblastoma C6 cells. Biodistribution studies were accomplished in athymic mice with C6 induced tumors that had blocked and unblocked receptors. Images were obtained using a micro-SPECT/CT. Results: ^99mTc-Tat-RGD was obtained with a radiochemical purity higher than 95%, as determined by radio-HPLC and ITLC-SG analyses. Protein binding was 15.7% for ^99mTc-Tat-RGD and 5.6% for ^99mTc-RGD. The IC₅₀ values were 6.7 nM (^99mTc-Tat-RGD) and 4.6 nM (^99mTc-RGD). Internalization in C6 cells was higher in ^99mTc-Tat-RGD (37.5%) than in ^99mTc-RGD (10%). Biodistribution studies and in vivo micro-SPECT/CT images in mice showed higher tumor uptake for ^99mTc-Tat-RGD (6.98% ± 1.34% ID/g at 3 h) than that of ^99mTc-RGD (3.72% ± 0.52% ID/g at 3 h) with specific recognition for α(v)β(3) integrins. Conclusions: Because of the significant cell internalization (Auger and internal conversion electrons) and specific recognition for α(v)β(3) integrins, the hybrid ^99mTc-N₂S₂-Tat(49–57)-c(RGDyK) radiopharmaceutical is potentially useful for the imaging and possible therapy of tumors expressing α(v)β(3) integrins.     

Sodium bicarbonate improves sprint performance in endurance cycling

ObjectivesOral sodium bicarbonate intake (NaHCO₃) may improve performance in short maximal exercise by inducing metabolic alkalosis. However, it remains unknown whether NaHCO₃ also enhances all-out performance at the end of an endurance competition. Therefore, the present study investigated the effect of stacked NaHCO₃ loading on sprint performance following a 3-h simulated cycling race.DesignDouble-blind randomized placebo-controlled cross-over study.MethodsEleven trained male cyclists (22.3 (18.3–25.3) year; 73.0 (61.5–88) kg; VO₂max: 63.7 (57–72) ml kg^?1 min^?1) ingested either 300 mg kg^?1 body weight NaHCO₃ (BIC) or NaCl (PL). NaHCO₃ or NaCl was supplemented prior to (150 mg kg^?1) and during (150 mg kg^?1) a 3-h simulated cycling race with a 90-s all-out sprint (90S) at the end. Capillary blood samples were collected for determination of blood pH, lactate and HCO₃^? concentrations. Analysis of variance (lactate, pH, HCO₃^?) and paired t-test (power) were applied to compare variables across condition (and time).ResultsNaHCO₃ intake improved mean power during 90S by ～3% (541 ± 59 W vs. 524 ± 57 W in PL, p = 0.047, Cohen’s D = 0.28, medium). Peak blood lactate concentration and heart rate at the end of 90S were higher (p < 0.05) in BIC (16.2 ± 4.1 mmol l ¹, 184 ± 7 bpm) than in PL (12.4 ± 4.2 mmol l^?1, 181 ± 5 bpm). NaHCO₃ ingestion increased blood [HCO₃^?] (31.5 ± 1.3 vs. 24.4 ± 1.5 mmol l^?1 in PL, p < 0.001) and blood pH (7.50 ± 0.01 vs. 7.41 ± 0.03 in PL, p < 0.05) prior to 90S.ConclusionsNaHCO₃ supplementation prior and during endurance exercise improves short all-out exercise performance at the end of the event. Therefore, sodium bicarbonate intake can be applied as a strategy to increase success rate in endurance competitions.     

Comparison of two cross-bridged macrocyclic chelators for the evaluation of 64Cu-labeled-LLP2A,a peptidomimetic ligand targeting VLA-4-positive tumors

Integrin α₄β₁ (also called very late antigen-4 or VLA-4) plays an important role in tumor growth, angiogenesis and metastasis, and there has been increasing interest in targeting this receptor for cancer imaging and therapy. In this study, we conjugated a peptidomimetic ligand known to have good binding affinity for α₄β₁ integrin to a cross-bridged macrocyclic chelator with a methane phosphonic acid pendant arm, CB-TE1A1P. CB-TE1A1P-LLP2A was labeled with ⁶⁴Cu under mild conditions in high specific activity, in contrast to conjugates based on the “gold standard” di-acid cross-bridged chelator, CB-TE2A, which require high temperatures for efficient radiolabeling. Saturation binding assays demonstrated that ⁶⁴Cu-CB-TE1A1P-LLP2A had comparable binding affinity (1.2 nM vs 1.6 nM) but more binding sites (B_max = 471 fmol/mg) in B16F10 melanoma tumor cells than ⁶⁴Cu-CB-TE2A-LLP2A (B_max = 304 fmol/mg, p < 0.03). In biodistribution studies, ⁶⁴Cu-CB-TE1A1P-LLP2A had less renal retention but higher uptake in tumor (11.4 ± 2.3 %ID/g versus 3.1 ± 0.6 %ID/g, p < 0.001) and other receptor-rich tissues compared to⁶⁴Cu-CB-TE2A-LLP2A. At 2 h post-injection, ⁶⁴Cu-CB-TE1A1P-LLP2A also had significantly higher tumor:blood and tumor:muscle ratios than ⁶⁴Cu-CB-TE2A-LLP2A (CB-TE1A1P = 19.5 ± 3.0 and 13.0 ± 1.4, respectively, CB-TE2A = 4.2 ± 1.4 and 5.5 ± 0.9, respectively, p < 0.001). These data demonstrate that ⁶⁴Cu-CB-TE1A1P-LLP2A is an excellent PET radiopharmaceutical for the imaging of α₄β₁ positive tumors and also has potential for imaging other α₄β₁ positive cells such as those of the pre-metastatic niche.     

Prolonged local persistence of cisplatin-loaded gelatin microspheres and their chemoembolic anti-cancer effect in rabbits

PurposeTo confirm prolonged cisplatin release from drug-loaded gelatin microspheres (GMSs) and their improved chemoembolic anti-cancer effect against VX2 liver tumors in rabbits.Materials and methodsTwo groups of twelve rabbits each were treated intraarterially either with 2 mg/kg cisplatin-loaded GMSs (=0.04 mg/kg cisplatin) or 0.04 mg/kg cisplatin solution by administering them into the right renal artery. Platinum concentrations within the renal parenchyma were analyzed immediately following infusion (day 0) and on days 1, 3, and 7 using the atomic absorption method. In a second experiment four groups of five rabbits each with implanted VX2 liver tumors were treated intraarterially through the hepatic artery with the following drugs: 2 mg/kg cisplatin-loaded GMSs (=0.04 mg/kg cisplatin) (group I), 2 mg/kg GMSs without any drug (group II), 1.5 mg/kg cisplatin solution (group III) and saline (group IV). Tumor volumes were analyzed pre-injection and 7 days after with MRI allowing calculating the relative tumor growth rate (%). Degree of liver cell necrosis was assessed on the histopathological specimens.ResultsThe renal parenchymal platinum concentrations (μg/ml) with 4.51 ± 2.25 (day 0), 1.59 ± 0.70 (day 1), 0.72 ± 0.10 (day 3) and 0.20 ± 0.06 (day 7) were significantly more pronounced after cisplatin-loaded GMS on days one and three compared to cisplatin with 1.99 ± 0.55, 0.08 ± 0.03, 0.18 ± 0.01 and 0.10 ± 0.07, respectively. Relative tumor growth rates resulted in 84.5% ± 26.4 (group I); 241.4% ± 145.1 (II); 331.9% ± 72.2 (III), and 413.6% ± 103.6 (IV) with statistical significant differences between groups I and III, and groups I and IV. Similar degrees of necrosis were observed in both GMSs treated groups, while ballooning of hepatocytes was highest in cisplatin-loaded GMSs.ConclusionsWith cisplatin-loaded GMSs more pronounced and prolonged local parenchymal cisplatin concentrations may be achieved offering the advantage of an increased and prolonged anti-cancer effect compared to cisplatin alone or controls. Moreover this proves indirectly the breakdown and release of cisplatin from the GMSs which is of primary importance for drug delivery systems.     

Uptake studies with chondrotropic 99mTc-chondroitin sulfate in articular cartilage. Implications for imaging osteoarthritis in the knee

Chondroitin sulfate (CS) is an endogenous component of extracellular matrix in the cartilage and can be valuable for imaging of cartilage degeneration after radiolabeling. Data monitoring the uptake of ^99mTcCS by human cartilage are rare. Radiolabeling was performed by ^99mTcO₄^?/tin method at pH 5.0 in 0.5 M sodium acetate. For uptake studies human articular cartilage (n = 4, 65–79a) derived from individuals undergoing knee replacement (pieces of 3–5 mg wet weight), or frozen tissue sections (5 μ) for autoradiography (10 μCi) were used. The uptake was monitored from 10 min up to 96 h to achieve saturation. As the commercially available drug Condrosulf (IBSA, Lugano) contains Mg-stearate (0.25%) as additive (to improve its gastrointestinal resorption), we investigated the uptake ± additive. The washout of the tracer was examined by tissue incubation after uptake experiments (3 h and 24 h) with PBS-buffer for 10 min to 3 h. Using human articular cartilage the maximal uptake of ^99mTcCS (specific activity of 4.1–6.1 Ci/mmol) was continuously increasing with time amounting to a maximum of 53.2% ± 3.2% with additive, versus 39.4% ± 2.3%, without additive, at saturation. Additive increased the resorption of the drug and consecutively its uptake. The washout of the tracer from cartilage after 3 h uptake amounted to 1.5% ± 0.2% with additive, versus 2.6% ± 0.5%, without. After 24 h washout was lower amounting to 1.1% ± 0.1% versus 1.75% ± 0.15%, respectively. Autoradiography revealed also a continuous increase in uptake of ^99mTcCS with time. After 10 min of incubation the uptake increase was proportional to the incubation time, reaching the maximum at 48–72 h. Enhanced uptake at the surface (superficial zone) as compared to the subchondral part (deep zone) of slices, was observed. The non-specific uptake in the presence of 50-fold excess of cold CS was time-dependent up to a maximum of 15% (tissue) and 10% (autoradiography), at saturation. The uptake studies indicate, that ^99mTcCS accumulates in articular cartilage and prove its chondrotropic effects.     

[18F]Fallypride: Metabolism studies and quantification of the radiotracer and its radiometabolites in plasma using a simple and rapid solid-phase extraction method

Introduction[¹⁸F]Fallypride, a fluorinated and substituted benzamide with high affinity for D₂/D₃ receptors, is a useful PET radioligand for the study of striatal/extrastriatal areas. Since [¹⁸F]fallypride is extensively metabolized in vivo and since PET examinations are long lasting in humans, the rapid measurement of the unchanged radiotracer in plasma is essential for the quantification of images. The present study aims: i) to evaluate if the radiometabolites of [¹⁸F]fallypride cross the blood–brain barrier in rodents, ii) to identify these radiometabolites in baboon plasma and iii) to develop a rapid solid phase extraction method (SPE) suitable for human applications to quantify both [¹⁸F]fallypride and its radiometabolites in plasma.MethodsThe metabolites P450-dependant in rat and human liver microsomes were characterized by LC–MS–MS and compared to those detected in vivo. Sequential solvent elution on Oasis®-MCX-SPE cartridges was used to quantify [¹⁸F]fallypride and its radiometabolites.ResultIn rat microsomal incubations, five metabolites generated upon N/O-dealkylation or hydroxylation at the pyrrolidine and/or at the benzamide moiety were identified. No radiometabolite was detected in the rat brain. N-dealkylated and hydroxylated derivatives were detected in human microsomal incubations as well as in baboon plasma. The use of SPE (total recovery 100.2% ± 2.8%, extraction yield 95.5% ± 0.3%) allowed a complete separation of [¹⁸F]fallypride from its radiometabolites in plasma and evaluate [¹⁸F]fallypride at 150 min pi to be 22% ± 5% of plasma radioactivity.ConclusionsThe major in vivo radiometabolites of [¹⁸F]fallypride were produced by N-dealkylation and hydroxylation. Allowing the rapid analysis of multiple plasma samples, SPE is a method of choice for the determination of [¹⁸F]fallypride until late images required for quantitative PET imaging in humans.     

Demonstration of dose dependent cytotoxic activity in SW480 colon cancer cells by 177Lu-labeled siRNA targeting IGF-1R

PurposeThe radiolabeling of targeting biomolecules with gamma emitter radionuclides for tracing and beta emitters for therapy involves the conjugation of such biomolecules to the chelating agents to form complexes with the radionuclide of interest. In this study, radioconjugate of IGF-1R siRNA with lutetium-177 (¹⁷⁷Lu) was produced, and the anti-proliferation and apoptosis effects elicited by this ¹⁷⁷Lu-siRNA complex in the SW480 colon cancer cells were evaluated.MethodsIGF-1R and Luciferase siRNAs were conjugated with p-SCN-Bn-DTPA, and then radiolabeled with ¹⁷⁷Lu. The effects of labeled and non-labeled IGF-1R siRNAs on IGF-1R expression were assessed with RT-PCR analysis and ELISA assay. IGF-1R siRNAs induced cell death and apoptosis were investigated using MTT assay and Annexin-V/propidium iodide (PI) double staining, respectively.ResultsCombined purification using Vivaspin and PD-10 columns resulted in a radiochemical purity of 97.32% ± 1.97%. Knockdown effect of the labeled IGF-1R siRNA was not significantly different from the non-labeled duplex of the same sequence (P < 0.05), but it was significant compared to the Luciferase siRNAs (P < 0.001). Proliferation decreased significantly, but apoptosis increased in the cells treated with the ¹⁷⁷Lu-IGF-1R siRNA in comparison with either ¹⁷⁷Lu or IGF-1R siRNA (P < 0.001).ConclusionRadioconjugate of IGF-1R siRNA, p-SCN-Bn-DTPA and ¹⁷⁷Lu was successfully produced and characterized as radiopharmaceutical. The present study demonstrates the involvement of ¹⁷⁷Lu-labeled IGF-1R siRNA in the inhibition of cell growth and induction of apoptosis in colon cancer cells.     

Effect of co-ligands on chemical and biological properties of 99mTc(III) complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl,F, SCN and N3; CDOH2 = cyclohexanedione dioxime)

Introduction^99mTc-Teboroxime ([^99mTcCl(CDO)(CDOH)₂BMe]) is a member of the BATO (boronic acid adducts of technetium dioximes) class of ^99mTc(III) complexes. This study sought to explore the impact of co-ligands on solution stability, heart uptake and myocardial retention of [^99mTc(L)(CDO)(CDOH)₂BMe] (^99mTc-Teboroxime: L = Cl; ^99mTc-Teboroxime(F): L = F; ^99mTc-Teboroxime(SCN): L = SCN; and ^99mTc-Teboroxime(N₃): L = N₃).MethodsRadiotracers ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared by reacting ^99mTc-Teboroxime with NaF, NaSCN and NaN₃, respectively. Biodistribution and imaging studies were carried out in Sprague–Dawley rats. Image quantification was performed to compare their heart retention and liver clearance kinetics.ResultsComplexes ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared in high yield with high radiochemical purity. All new radiotracers were stable for > 6 h in the kit matrix. In its HPLC chromatogram, ^99mTc-Teboroxime showed one peak at ~ 15.5 min, which was shorter than that of ^99mTc-Teboroxime(F) (~ 16.4 min). There were two peaks for ^99mTc-Teboroxime(SCN) at 16.5 and 18.3 min. ^99mTc-Teboroxime(N₃) appeared as a single peak at 18.4 min. Their heart retention and liver clearance curves were best fitted to the bi-exponential decay function. The half-times of fast/slow components were 1.6 ± 0.4/60.7 ± 8.9 min for ^99mTc-Teboroxime, 0.8 ± 0.2/101.7 ± 20.7 min for ^99mTc-Teboroxime(F), 1.2 ± 0.3/84.8 ± 16.6 min for ^99mTc-Teboroxime(SCN), and 2.9 ± 0.9/51.6 ± 5.0 min for ^99mTc-Teboroxime(N₃). The 2-min heart uptake followed the order of ^99mTc-Teboroxime (3.00 ± 0.37%ID/g) > ^99mTc-Teboroxime(N₃) (2.66 ± 0.01 %ID/g) ≈ ^99mTc-Sestamibi (2.55 ± 0.46 %ID/g) > ^99mTcN-MPO (2.38 ± 0.15 %ID/g). ^99mTc-Teboroxime remains the best in first-pass extraction. The best image acquisition window is 0–5 min for ^99mTc-Teboroximine and 0–15 min for ^99mTc-Teboroximine(N₃).ConclusionCo-ligands had significant impact on the heart uptake and myocardial retention of complexes [^99mTc(L)(CDO)(CDOH)₂BMe] (L = Cl, F, SCN and N₃). Future studies should be directed towards minimizing the liver uptake and radioactivity accumulation in the blood vessels while maintaining their high heart uptake.     

3T diffusion-weighted MRI in the response assessment of colorectal liver metastases after chemotherapy: Correlation between ADC value and histological tumour regression grading

PurposeThe purpose of the study was to correlate the apparent diffusion coefficient (ADC) values of diffusion-weighted MR imaging (DW-MRI) by 3T device with the histological tumour regression grading (TRG) analysis of colorectal liver metastases after preoperative chemotherapy.Materials and methodsOur study included thirty-five patients with colorectal liver metastases who had undergone MRI by 3T device (GE DISCOVERY MR750; GE Healthcare) after preoperative chemotherapy. DW-MRI was performed using a single-shot spin-echo echo-planar sequence with multiple b-values (0, 150, 500, 1000, 1500 s/mm²), thus obtaining an ADC map. For each liver lesion (more than 1 cm in diameter) the fitted ADC values were calculated by two radiologists in conference and three ROIs were drawn: around the entire tumour (ADC_e), at the tumour periphery (ADC_p) and at the tumour center (ADC_c). All ADC values were correlated with histopathological findings after surgery. Hepatic metastases were pathologically classified into five groups on the basis of TRG. Statistical analysis was performed on a per-lesion basis utilizing the one-way analysis of variance (ANOVA). This retrospective study was approved by our institutional review board; written informed consent was obtained from all patients.ResultsA total of 106 colorectal liver metastases were included for image analysis. TRG1, TRG2, TRG3, TRG4 and TRG5 were observed in 4, 14, 36, 35 and 17 lesions, respectively. ADC_e and ADC_p values were significantly higher in lesions classified as TRG1 (2.40 ± 0.12 × 10⁻⁹ m²/s and 2.28 ± 0.26 × 10⁻⁹ m²/s, respectively) and as TRG2 (1.40 ± 0.31 × 10⁻⁹ m²/s and 1.44 ± 0.35 × 10⁻⁹ m²/s), compared to TRG3 (1.16 ± 0.13 × 10⁻⁹ m²/s and 1.01 ± 0.18 × 10⁻⁹ m²/s), TRG4 (1.10 ± 0.26 × 10⁻⁹ m²/s and 0.97 ± 0.24 × 10⁻⁹ m²/s), and TRG5 (0.93 ± 0.17 × 10⁻⁹ m²/s and 0.82 ± 0.28 × 10⁻⁹ m²/s). ADC_e, ADC_p and ADC_c values were significantly different in TRG classes (p < 0.0001). Statistical correlations were found between the ADC_e, ADC_p, ADC_c values and the TRG classes (Spearman correlation coefficient were −0.568, −0.542 and −0.554, respectively).ConclusionOur study showed a significant correlation between ADC values of 3T DW-MRI and histological TRG of colorectal liver metastases after preoperative chemotherapy.     

Aluminium salt-based antiperspirant coated prosthesis liners do not suppress local sweating during moderate intensity exercise in hot and temperate conditions

ObjectiveTo determine whether coating prosthesis liners with a 5% aluminium zirconium tetrachlorohydrate antiperspirant solution (AZCH) reduces local sweating on the thigh.DesignDouble-blinded counter-balanced crossover designMethodsFourteen able-bodied participants (age: 28 ± 5 y; body mass: 73.9 ± 7.9 kg, height: 1.73 ± 0.09 m; peak oxygen consumption [VO_2peak]: 50.7 ± 9.1 mlO₂⋅ kg⁻¹⋅ min⁻¹) simultaneously wore a prosthesis liner on each leg, one treated with AZCH and one untreated, for four days prior to running at 50% of VO_2peak for 60 min in a temperate (23.7 ± 0.7 °C and 42.2 ± 2.6% relative humidity) or hot (34.0 ± 1.6 °C and 40.8 ± 6.1% relative humidity) environment. Rectal temperature (T_re) and whole-body sweat rates (WBSR) were measured to characterize thermal strain. Local sweat rate (LSR) was measured bilaterally underneath the liners, continuously, and heat-activated-sweat gland density (HASGD) was measured bilaterally every 15 min.ResultsIn temperate condition, the mean change in T_re was 1.2±0.4 °C and WBSR was 723 ± 129 g⋅ h⁻¹, whereas in the hot condition, change in T_re was 1.2±0.5 °C and WBSR was 911 ± 231 g⋅ h⁻¹. In the temperate condition, AZCH treatment did not alter LSR (treated: 0.50±0.17 mg·cm^–2 min^–1, untreated: 0.50±0.17 mg·cm^–2 min^–1; P = 0.87) or HASGD (treated: 54±14 glands·cm^–2, untreated 55±14 glands·cm^–2; P = 0.38). In the hot condition, AZCH treatment paradoxically increased LSR (treated: 0.88 ± 0.38 mg·cm^–2 min^–1, untreated: 0.74 ± 0.28 mg·cm^–2 min^–1; P = 0.04) but not HASGD (treated: 52 ± 17 glands·cm^–2, untreated: 48 ± 19 glands·cm^–2; P = 0.77).ConclusionThese results indicate coating prosthesis liners with 5% AZCH is ineffective at reducing local sweating.