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.     

Evaluation of an anti-p185 (scFv-CH2-CH3)2 fragment following radioiodination using two different residualizing labels: SGMIB and IB-Mal-d-GEEEK

IntroductionA 105-kDa double mutant single-chain Fv-Fc fragment (scFv-Fc DM) derived from the anti-p185^HER2 hu4D5v8 antibody (trastuzumab; Herceptin) has been described recently. The goal of this study was to investigate whether improved tumor targeting could be achieved with this fragment through the use of residualizing radioiodination methods.MethodsThe scFv-Fc DM fragment was radioiodinated using N-succinimidyl 4-guanidinomethyl 3-[¹³¹I]iodobenzoate ([¹³¹I]SGMIB) and N^?-(3-[¹³¹I]iodobenzoyl)-Lys⁵-N^α- maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-d-GEEEK), two residualizing radioiodination agents that have been used successfully with intact antibodies. Paired-label internalization assays of the labeled fragments were performed in vitro using MCF7 human breast cancer cells transfected to express HER2 (MCF7-HER2); comparisons were made to scFv-Fc DM directly radioiodinated using Iodogen. The tissue distribution of the scFv-Fc DM labeled with [¹²⁵I]IB-Mal-d-GEEEK and [¹³¹I]SGMIB was compared in athymic mice bearing MCF7-HER2 xenografts.ResultsThe scFv-Fc DM fragment was labeled with [¹³¹I]SGMIB and [¹³¹I]IB-Mal-d-GEEEK in conjugation yields of 53% and 25%, respectively, with preservation of immunoreactivity for HER2. Internalization assays indicated that labeling via SGMIB resulted in a 1.6- to 3.5-fold higher (P<.05) retention of radioactivity, compared to that from the directly labeled fragment, in HER2-expressing cells during a 24-h observation period. Likewise, the amount of radioactivity retained in cells from the IB-Mal-d-GEEEK-labeled fragment was 1.4- to 3.3-fold higher (P<.05). Tumor uptake of radioiodine activity in athymic mice bearing MCF7-HER2 xenografts in vivo was significantly higher for the [¹²⁵I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment compared with that of the [¹³¹I]SGMIB-labeled fragment, particularly at later time points. The uptake of ¹²⁵I was threefold (3.6±1.1 %ID/g vs. 1.2±0.4 %ID/g) and fourfold (3.1±1.7 %ID/g vs. 0.8±0.4 %ID/g) higher than that for ¹³¹I at 24 and 48 h, respectively. However, the [¹²⁵I]IB-Mal-d-GEEEK-labeled scFv-Fc DM fragment also exhibited considerably higher levels of radioiodine activity in liver, spleen and kidney.ConclusionsThe overall results further demonstrate the potential utility of these two prosthetic groups for the radiohalogenation of internalizing monoclonal antibodies and their fragments. Specifically, the trastuzumab-derived double mutant fragment in combination with these residualizing agents warrants further evaluation for imaging and possibly treatment of HER2 expressing malignancies.     

Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

IntroductionAffibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL).MethodsThe HER2-targeting NCL-cyclized Affibody molecule Z_HER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. The binding affinity of DOTA-Z_HER2:342min was evaluated in vitro. The targeting properties of ¹¹¹In- and ⁶⁸ Ga-DOTA-Z_HER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of ¹¹¹In- and ⁶⁸ Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge.ResultsThe dissociation constant (K_D) for DOTA-Z_HER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K_D1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 ± 1.3 for ¹¹¹In- DOTA-Z_HER2:342min and 4.6 ± 0.7 for ⁶⁸ Ga-DOTA-Z_HER2:342min. However, the uptake of DOTA-Z_HER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.ConclusionsNative chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z_HER2:342min) with low nanomolar target affinity and specific tumor uptake.     

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.     

Single-step radiosynthesis and in vivo evaluation of a novel fluorine-18 labeled hippurate for use as a PET renal agent

ObjectiveThe objective of this study was to investigate a new fluorine-18 labeled hippurate, m-cyano-p-[¹⁸ F]fluorohippurate ([¹⁸ F]CNPFH), as a potential radiopharmaceutical for evaluating renal function by PET.Methods[¹⁸ F]CNPFH was synthesized by a direct one-step nucleophilic aromatic substitution using an ¹⁸ F-for-[N(CH₃)₃]⁺-reaction. In vivo stability was determined by HPLC analysis of urine collected from a healthy rat at 30 min p.i. of [¹⁸ F]CNPFH. The plasma protein binding (PPB) and erythrocyte uptake of [¹⁸ F]CNPFH were determined using blood collected from healthy rats at 5 min p.i. Biodistribution studies were conducted in healthy rats at 10 min and 1 h p.i. of [¹⁸ F]CNPFH. Dynamic PET/CT imaging data were acquired in normal rats. For comparison, the same rats underwent an identical imaging study using the previously reported p-[¹⁸ F]fluorohippurate ([¹⁸ F]PFH) renal agent.Results[¹⁸ F]CNPFH demonstrated high in vivo stability with no metabolic degradation. The in vivo PPB and erythrocyte uptake of [¹⁸ F]CNPFH were found to be comparable to those of [¹⁸ F]PFH. Biodistribution and dynamic PET/CT imaging studies revealed a rapid clearance of [¹⁸ F]CNPFH primarily through the renal–urinary pathway. However, unlike [¹⁸ F]PFH, a minor (about 12%) fraction was eliminated via the hepatobiliary route. The PET-derived [¹⁸ F]CNPFH renograms revealed an average time-to-peak (T_max) of 3.2 ± 0.4 min which was similar to [¹⁸ F]PFH, but the average time-to-half-maximal activity (11.4 ± 2.8 min) was found to be higher than that of [¹⁸ F]PFH (7.1 ± 1.3 min).ConclusionsOur in vivo results indicate that [¹⁸ F]CNPFH has renogram characteristics similar to those of [¹⁸ F]PFH, however, the unexpected hepatobiliary elimination is adding undesirable background signal in the PET images.     

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.     

Kinetic analyses of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid transport in Xenopus laevis oocytes expressing human ASCT2 and SNAT2

IntroductionTrans-1-amino-3-[¹⁸F]fluorocyclobutanecarboxylic acid (anti-[¹⁸F]FACBC) is a promising amino acid positron emission tomography (PET) radiotracer for visualizing prostate cancer. We previously showed that anti-FACBC is transported by amino acid transporters, especially by alanine-serine-cysteine transporter 2 (ASCT2), which is associated with tumor growth. We studied this affinity to assess the mechanism of anti-FACBC transport in prostate cancer cells.MethodsKinetic assays for trans-1-amino-3-fluoro-[1-¹⁴C]cyclobutanecarboxylic acid ([¹⁴C]FACBC) were performed in Xenopus laevis oocytes over-expressing either ASCT2 or sodium-coupled neutral amino acid transporter 2 (SNAT2), both of which are highly expressed in prostate cancer cells. We also examined the kinetics of [¹⁴C]FACBC uptake using mammalian cell lines over-expressing system L amino acid transporter 1 or 2 (LAT1 or LAT2).ResultsASCT2 and SNAT2 transported [¹⁴C]FACBC with Michaelis–Menten kinetics K_m values of 92.0 ± 32.3 μM and 222.0 ± 29.3 μM, respectively. LAT1 and LAT2 transported [¹⁴C]FACBC with Michaelis–Menten K_m values of 230.4 ± 184.5 μM and 738.5 ± 87.6 μM, respectively.ConclusionsBoth ASCT2 and SNAT2 recognize anti-FACBC as a substrate. Anti-FACBC has higher affinity for ASCT2 than for SNAT2, LAT1, or LAT2. The ASCT2-preferential transport of anti-[¹⁸F]FACBC in cancer cells could be used for more effective prostate cancer imaging.     

Radiosynthesis,biodistribution and imaging of [11C]YM155, a novel survivin suppressant,in a human prostate tumor-xenograft mouse model

IntroductionSepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [¹¹C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts.MethodsMethods utilizing [¹¹C]acetyl chloride and [¹¹C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [¹¹C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [¹¹C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS).ResultsSufficient quantities of radiopharmaceutical grade [¹¹C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16–22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29–60 GBq/μmol (EOS). High uptake levels of radioactivity (%ID/g, mean ± SE) were observed in tumor (0.0613 ± 0.0056), kidneys (0.0513 ± 0.0092), liver (0.0368 ± 0.0043) and cecum (0.0623 ± 0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [¹¹C]YM155, at 40 min after injection, were 26.5 (± 2.9) and 25.6 (± 3.6), respectively.ConclusionA rapid method for producing a radiopharmaceutical grade [¹¹C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [¹¹C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.     

(R)-[11C]verapamil is selectively transported by murine and human P-glycoprotein at the blood–brain barrier,and not by MRP1 and BCRP

IntroductionPositron emission tomography (PET) with [¹¹C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood–brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP.MethodsConcentration equilibrium transport assays were performed with [³H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[¹¹C]verapamil in female FVB/N (wild-type), Mrp1^(?/?), Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice, before and after Pgp inhibition with 15 mg/kg tariquidar.ResultsIn vitro transport experiments exclusively showed directed transport of [³H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5 μM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (K_b,brain) of (R)-[¹¹C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1^(?/?) (1.4 ± 0.1) and Bcrp1^(?/?) mice (1.8 ± 0.1) and high in Mdr1a/b^(?/?) (6.9 ± 0.8) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice (7.9 ± 0.5). In PET scans after tariquidar administration, K_b,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1^(?/?): 3.2 ± 0.6-fold, Bcrp1^(?/?): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?)).ConclusionOur combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[¹¹C]verapamil or racemic [¹¹C]verapamil as PET tracers of cerebral Pgp function.     

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

IntroductionFatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [¹¹C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor.MethodsPF-04457845 was radiolabeled at the carbonyl position via automated [¹¹C]CO₂-fixation. Ex vivo brain biodistribution of [¹¹C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [¹¹C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma.ResultsThe title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [¹¹C]CO₂ (decay uncorrected) within 25 min from end-of-bombardment in > 98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [¹¹C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2–4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71–81%, demonstrating specificity for FAAH. The binding of [¹¹C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted.Conclusions[¹¹C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents demonstrate that [11C PF-04457845 is a promising candidate radiotracer for imaging FAAH in the brain with PET. These results coupled with the known pharmacology and toxicology of PF-04457845 should facilitate clinical translation of this radiotracer.     

Desferrioxamine as an appropriate chelator for 90Nb: Comparison of its complexation properties for M-Df-Octreotide (M = Nb,Fe, Ga,Zr)

The niobium-90 radioisotope (90Nb) holds considerable promise for use in immuno-PET, due to its decay parameters (t½ = 14.6 h, positron yield = 53%, Eß+mean = 0.35 MeV and Eß+max = 1.5 MeV). In particular, 90Nb appears well suited to detect in vivo the pharmacokinetics of large targeting vectors (50–150 kDa). In order to be useful for immuno-PET chelators are required to both stabilize the radionuclide in terms of coordination chemistry and to facilitate the covalent attachment to the targeting vector. Different chelators were evaluated for this purpose in terms of radiolabelling efficiency and stability of the radiolabelled Nb(V) complex and in order to determine the most suitable candidate for conjugation to a biologically relevant targeting vector. For the purpose of studying the complexation properties the niobium radioisotope 95Nb was used as an analogue of 90Nb, by virtue of its longer half-life (35 days) and lower cost (reactor-based production). Acyclic and cyclic chelators were investigated, with desferroxamine [Df: (N'-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl) (hydroxy)amino]-4-oxobutanoyl} amino)pentyl]-N-hydroxysuccinamide)] emerging as the best candidate. Greater than 99% radiolabelling was achieved at room temperature over a wide pH range. The 95Nb-Df complex is sufficiently stable for immuno-PET (< 7% degradation over 7 days in vitro). As a proof-of-principle, a Df conjugate featuring a well-established targeting vector, (D)-Phe1-octreotide, was evaluated. The fast labelling kinetics of the unconjugated chelator (Df) were retained for Df-succinyl-(D)Phe1-octreotide (Df-OC), with > 90% labelling after 1 h at room temperature over the pH range 5–7. Stability studies, performed in vitro in serum at physiological temperature (37 °C), revealed that 87 ± 2% of the radiolabelled molecule remained intact after 7 days. Competition studies with relevant metal ions (zirconium(IV), gallium(III) and iron(III)) have been performed with Df-OC to gain insight to the relative stability [Nb-Df]-OC complex to transmetallation. At equimolar metal ion concentrations the [Nb-Df]-OC complex showed the greatest overall stability. The favourable radiolabelling characteristics of Df-OC and its stability indicate that Df is a potentially very useful chelator for the development of radiopharmaceuticals for 90Nb-PET.     

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.     

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.     

Evaluation of metabolism,plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

Introduction(−)-[¹⁸ F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α₄β₂ receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood.MethodsPlasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min.ResultsA fraction of 15% ± 2% of (−)-[¹⁸ F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[¹⁸ F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[¹⁸ F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts.Discussion(−)-[¹⁸ F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.     

Tattoos do not affect exercise-induced localised sweat rate or sodium concentration

ObjectivesSkin tattoos have been shown to reduce localised sweat rate and increase sweat sodium concentration ([Na⁺]) when sweating is artificially stimulated. This study investigated whether similar responses are observed with exercise-induced sweating.DesignUnblinded, within-participant control, single trial.MethodsTwenty-two healthy individuals (25.1 ± 4.8 y (Mean ± SD), 14 males) with a unilateral tattoo ≥11.4 cm² in size, ≥2 months in age, and shaded ≥50% participated in this investigation. Participants undertook 20 min of intermittent cycling (4 × 5 min intervals) on a stationary ergometer in a controlled environment (24.6 ± 1.1 °C; 64 ± 6% RH). Resultant sweat was collected into absorbent patches applied at two pairs of contralateral skin sites (pair 1: Tattoo vs. Non-Tattoo; pair 2: Control 1 vs. Control 2 (both non-tattooed)), for determination of sweat rate and sweat [Na⁺]. Paired samples t-tests were used to determine differences between contralateral sites.ResultsTattoo vs. Non-Tattoo: Neither sweat rate (Mean ± SD: 0.92 ± 0.37 vs. 0.94 ± 0.43 mg·cm⁻²·min⁻¹, respectively; p = 0.693) nor sweat [Na⁺] (Median(IQR): 37(32–52) vs. 37(31–45) mM·L⁻¹, respectively; p = 0.827) differed. Control 1 vs. Control 2: Neither sweat rate (Mean±SD: 1.19 ± 0.53 vs. 1.19 ± 0.53 mg·cm⁻²·min⁻¹, respectively; p = 0.917) nor sweat [Na⁺] (Median(IQR): 29(26–41) vs. 31(25–43) mM·L⁻¹, respectively; p = 0.147) differed. The non-significant differences for sweat rate and [Na⁺] between Tattoo vs. Non-Tattoo were inside the range of the within participant variability (sweat rate CVi = 5.4%; sweat [Na⁺] CVi = 4.4%).ConclusionsSkin tattoos do not appear to alter the rate or [Na⁺] of exercise-induced sweating. The influence of skin tattoos on localised sweat responses may have previously been over-estimated.     

Synthesis and evaluation of 2-amino-5-(4-[18F]fluorophenyl)pent-4-ynoic acid ([18F]FPhPA): A novel 18F-labeled amino acid for oncologic PET imaging

Introduction¹⁸ F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[¹⁸ F]fluorophenyl)pent-4-ynoic acid ([¹⁸ F]FPhPA) as a novel amino acid radiotracer for oncologic imaging.Methods¹⁸ F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[¹⁸ F]fluoroiodobenzene ([¹⁸ F]FIB) and propargylglycine. The radiopharmacological profile of [¹⁸ F]FPhPA was evaluated in comparison with O-(2-[¹⁸ F]fluoroethyl)-L-tyrosine ([¹⁸ F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging.Results¹⁸ F]FPhPA was prepared in 42 ± 10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[¹⁸ F]FPhPA reached a maximum of 58 ± 14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[¹⁸ F]FPhPA.Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na⁺-dependent system ASC, especially ASCT2, and Na⁺-independent system L are important amino acid transporters for [¹⁸ F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [¹⁸ F]FPhPA in EMT6 tumor-bearing mice compared to [¹⁸ F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [¹⁸ F]FPhPA was higher (SUV_30min = 0.65) compared to [¹⁸ F]FET (SUV_30min = 0.40), whereas [¹⁸ F]FPhPA showed a more rapid uptake and clearance from the brain compared to [¹⁸ F]FET.ConclusionL-[¹⁸ F]FPhPA is the first ¹⁸ F-labeled amino acid prepared through Pd-mediated cross-coupling reaction.Advances in Knowledge and Implications for patient CareL-[¹⁸ F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.     

Adrenergic pathway activation enhances brown adipose tissue metabolism: A [18 F]FDG PET/CT study in mice

ObjectivePharmacologic approaches to study brown adipocyte activation in vivo with a potential of being translational to humans are desired. The aim of this study was to examine pre- and postsynaptic targeting of adrenergic system for enhancing brown adipose tissue (BAT) metabolism quantifiable by [¹⁸ F]fluoro-2-deoxyglucose ([¹⁸ F]FDG) positron emission tomography (PET)/computed tomography (CT) in mice.MethodsA β₃-adrenoreceptor selective agonist (CL 316243), an adenylyl cyclase enzyme activator (forskolin) and a potent blocker of presynaptic norepinephrine transporter (atomoxetine), were injected through the tail vein of Swiss Webster mice 30 minutes before intravenous (iv) administration of [¹⁸ F]FDG. The mice were placed on the PET/CT bed for 30 min PET acquisition followed by 10 min CT acquisition for attenuation correction and anatomical delineation of PET images.ResultsActivated interscapular (IBAT), cervical, periaortic and intercostal BAT were observed in 3-dimentional analysis of [¹⁸ F]FDG PET images. CL 316243 increased the total [¹⁸ F]FDG standard uptake value (SUV) of IBAT 5-fold greater compared to that in placebo-treated mice. It also increased the [¹⁸ F]FDG SUV of white adipose tissue (2.4-fold), and muscle (2.7-fold), as compared to the control. There was no significant difference in heart, brain, spleen and liver uptakes between groups. Forskolin increased [¹⁸ F]FDG SUV of IBAT 1.9-fold greater than that in placebo-treated mice. It also increased the [¹⁸ F]FDG SUV of white adipose tissue (2.2-fold) and heart (5.4-fold) compared to control. There was no significant difference in muscle, brain, spleen, and liver uptakes between groups. Atomoxetine increased [¹⁸ F]FDG SUV of IBAT 1.7-fold greater than that in placebo-treated mice. There were no significant differences in all other organs compared to placebo-treated mice except liver (1.6 fold increase). A positive correlation between SUV levels of IBAT and CT Hounsfield unit (HU) (R² = 0.55, p < 0.001) and between CT HU levels of IBAT and liver (R² = 0.69, p < 0.006) was observed.ConclusionsThe three pharmacologic approaches reported here enhanced BAT metabolism by targeting different sites in adrenergic system as measured by [¹⁸ F]FDG PET/CT.     

[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.     

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.     

Dosimetry for 131Cs and 125I seeds in solid water phantom using radiochromic EBT film

PurposeTo measure the 2D dose distributions with submillimeter resolution for ¹³¹Cs (model CS-1 Rev2) and ¹²⁵I (model 6711) seeds in a Solid Water phantom using radiochromic EBT film for radial distances from 0.06 cm to 5 cm. To determine the TG-43 dosimetry parameters in water by applying Solid Water to liquid water correction factors generated from Monte Carlo simulations.MethodsEach film piece was positioned horizontally above and in close contact with a ¹³¹Cs or ¹²⁵I seed oriented horizontally in a machined groove at the center of a Solid Water phantom, one film at a time. A total of 74 and 50 films were exposed to the ¹³¹Cs and ¹²⁵I seeds, respectively. Different film sizes were utilized to gather data in different distance ranges. The exposure time varied according to the seed air-kerma strength and film size in order to deliver doses in the range covered by the film calibration curve. Small films were exposed for shorter times to assess the near field, while larger films were exposed for longer times in order to assess the far field. For calibration, films were exposed to either 40 kV (M40) or 50 kV (M50) x-rays in air at 100.0 cm SSD with doses ranging from 0.2 Gy to 40 Gy. All experimental, calibration and background films were scanned at a 0.02 cm pixel resolution using a CCD camera-based microdensitometer with a green light source. Data acquisition and scanner uniformity correction were achieved with Microd3 software. Data analysis was performed using ImageJ, FV, IDL and Excel software packages. 2D dose distributions were based on the calibration curve established for 50 kV x-rays. The Solid Water to liquid water medium correction was calculated using the MCNP5 Monte Carlo code. Subsequently, the TG-43 dosimetry parameters in liquid water medium were determined.ResultsValues for the dose-rate constants using EBT film were 1.069±0.036 and 0.923±0.031 cGy U⁻¹ h⁻¹ for ¹³¹Cs and ¹²⁵I seed, respectively. The corresponding values determined using the Monte Carlo method were 1.053±0.014 and 0.924±0.016 cGy U⁻¹ h⁻¹ for ¹³¹Cs and ¹²⁵I seed, respectively. The radial dose functions obtained with EBT film measurements and Monte Carlo simulations were plotted for radial distances up to 5 cm, and agreed within the uncertainty of the two methods. The 2D anisotropy functions obtained with both methods also agreed within their uncertainties.ConclusionEBT film dosimetry in a Solid Water phantom is a viable method for measuring ¹³¹Cs (model CS-1 Rev2) and ¹²⁵I (model 6711) brachytherapy seed dose distributions with submillimeter resolution. With the Solid Water to liquid water correction factors generated from Monte Carlo simulations, the measured TG-43 dosimetry parameters in liquid water for these two seed models were found to be in good agreement with those in the literature.