Design, synthesis and biological evaluation of choline based SPECT imaging agent: Ga(III)-DO3A-EA-Choline

The enhanced choline uptake and phosphorylation in tumor cells has motivated the development of radiolabeled choline derivatives as diagnostic markers for imaging cell membrane proliferation and noninvasive detection of prostate, brain and breast tumors. In the present work, we report a facile strategy for the synthesis of choline functionalized macrocyclic chelating agent (DO3A-EA-choline) and its radiocomplexation with (67)Ga for potential tumor imaging applications. The synthesis of the desired compound featured quaternization of N,N-dimethylaminoethanol with 1,2-dibromoethane followed by subsequent alkylation with trisubstituted cyclen (DO3A). All intermediates and final compounds have been fully characterized by spectroscopic techniques, namely, (1)H, (13)C NMR and mass spectroscopy. The compound has been successively labeled with (67)Ga-citrate in ammonium acetate buffer (pH 6.5) at 80 °C. MTT assays have been performed on the HEK cell line to determine the cytotoxicity of the compound. Cell uptake studies carried out on the U-87 MG cell line exhibited saturable binding of the radioconjugate in picomolar range with a K(d) value of 0.528 pM. The in vivo biodistribution and blood kinetics studies exhibited rapid clearance of the radiolabeled complex and excretion through the renal and hepatobiliary route. The present studies demonstrate the potential applications of (67)Ga-DO3A-EA-choline as a radiopharmaceutical for molecular imaging using ((67/68)Ga) SPECT and PET modalities.     

Linkage effects on binding affinity and activation of GPR30 and estrogen receptors ERalpha/beta with tridentate pyridin-2-yl hydrazine tricarbonyl-Re/(99m)Tc(I) chelates

We describe a new structural class of neutral tridentate pyridin-2-yl hydrazine chelates for labeling with tricarbonyl Re/99mTc(I) under aqueous conditions and investigate the receptor binding of synthetic estradiol derivatives with the novel G-protein-coupled receptor GPR30 and estrogen receptors ERalpha/beta. The steroid linkage affected the affinity and selectivity of estrogen binding with these receptors. Fluorescence assays based on calcium signaling demonstrate that membrane-permeable chelates 2 and 3 interact with the receptors in whole cells. These results suggest that in vitro assays will facilitate the development of targeted imaging agents for intracellular receptors and the feasibility of targeting GPR30 and ERalpha/beta for diagnostic tumor imaging.     

The first synthesis and cytostatic activity of novel 6-(fluoromethyl)purine bases and nucleosides

Two alternative approaches to the synthesis of novel 6-(fluoromethyl)purine bases and nucleosides are described either by direct deoxyfluorination or by multistep functional group transformations starting from 6-(hydroxymethyl)purines. 6-(fluoromethyl)purine ribonucleoside displayed significant cytostatic effects.     

Interbenzylic 19F,19F spin-spin coupling constants in bis(fluoromethyl) arenes

Five- to eleven-bond F,F spin coupling is observed in bis(fluoromethyl)arenes. According to INDO MO calculations, these couplings are transmitted mainly via the aromatic π-system. The low preference of 1,4-(CH₂F)₂-benzene for the conformation in which the C? F bonds are perpendicular to the aromatic ring is shown by the strong increase of⁷J(FF) upon double ortho-substitution.     

Single‐Molecule Force Measurement Guides the Design of Multivalent Ligands with Picomolar Affinity

Interaction of multiple entities and receptors, or multivalency is widely applied to achieve high affinity ligands for diagnostic and therapeutic purposes. However, lack of knowledge on receptor distribution in living subjects remains a challenge for rational structure design. Herein, we develop a force measurement platform to probe the distribution and separation of the cell surface vascular endothelial growth factor receptors (VEGFR) in live cells, and use this to assess the geometry of appropriate linkers for distinct multivalent binding modes. A tetravalent lead ZD‐4, which was developed from an antitumor drug ZD6474 (Vandetanib) with combined hybrid binding effects, yielded a 2000‐fold improvement in the binding affinity to VEGFR with IC₅₀ value of 25 pm . We confirmed the improved affinity by the associated increase of tumor uptake in the VEGFR‐targeting positron emission tomography (PET) imaging using U87 tumor xenograft mouse model.     

Stereoselective nucleophilic monofluoromethylation of N-(tert-butanesulfinyl)imines with fluoromethyl phenyl sulfone

[reaction: see text] Highly stereoselective nucleophilic monofluoromethylation of (R)-(tert-butanesulfinyl)imines with fluoromethyl phenyl sulfone was achieved to afford alpha-monofluoromethylamines with a nonchelation-controlled stereoselectivity mode. By using the same chemistry, (R)-(tert-butanesulfinyl)imines bearing a terminal tosylate (OTs) group can be converted to alpha-monofluoromethylated cyclic secondary amines with high stereoselectivity.     

Metal-ion-dependent biological properties of a chelator-derived somatostatin analogue for tumour targeting

Somatostatin-based radioligands have been shown to have sensitive imaging properties for neuroendocrine tumours and their metastases. The potential of [(55)Co(dotatoc)] (dotatoc =4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-ylacetyl-D-Phe-(Cys-Tyr-D-Trp-Lys-Thr-Cys)-threoninol (disulfide bond)) as a new radiopharmaceutical agent for PET has been evaluated. (57)Co was used as a surrogate of the positron emitter (55)Co and the pharmacokinetics of [(57)Co(dotatoc)] were investigated by using two nude mouse models. The somatostatin receptor subtype (sst1-sst5) affinity profile of [(nat)Co(dotatoc)] on membranes transfected with human somatostatin receptor subtypes was assessed by using autoradiographic methods. These studies revealed that [(57)Co(dotatoc)] is an sst2-specific radiopeptide which presents the highest affinity ever found for the sst2 receptor subtype. The rate of internalisation into the AR4-2J cell line also was the highest found for any somatostatin-based radiopeptide. Biodistribution studies, performed in nude mice bearing an AR4-2J tumour or a transfected HEK-sst2 cell-based tumour, showed high and specific uptake in the tumour and in other sst-receptor-expressing tissues, which reflects the high receptor binding affinity and the high rate of internalisation. The pharmacologic differences between [(57)Co(dotatoc)] and [(67)Ga(dotatoc)] are discussed in terms of the structural parameters found for the chelate models [Co(II)(dota)](2-) and [Ga(III)(dota)](-) whose X-ray structures have been determined. Both chelates show six-fold coordination in pseudo-octahedral arrangements.     

Design,Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. ⁶⁸Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the ^99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (^99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl₂ for labeling with ^99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, ¹H–NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The ^99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the ^99mTc-iFAP.     

Comparative molecular field analysis (CoMFA) model using a large diverse set of natural, synthetic and environmental chemicals for binding to the androgen receptor

A large number of natural, synthetic and environmental chemicals are capable of disrupting the endocrine systems of experimental animals, wildlife and humans. These so-called endocrine disrupting chemicals (EDCs), some mimic the functions of the endogenous androgens, have become a concern to the public health. Androgens play an important role in many physiological processes, including the development and maintenance of male sexual characteristics. A common mechanism for androgen to produce both normal and adverse effects is binding to the androgen receptor (AR). In this study, we used Comparative Molecular Field Analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (3D-QSAR) technique, to examine AR-ligand binding affinities. A CoMFA model with r2 = 0.902 and q2 = 0.571 was developed using a large training data set containing 146 structurally diverse natural, synthetic, and environmental chemicals with a 10(6)-fold range of relative binding affinity (RBA). By comparing the binding characteristics derived from the CoMFA contour map with these observed in a human AR crystal structure, we found that the steric and electrostatic properties encoded in this training data set are necessary and sufficient to describe the RBA of AR ligands. Finally, the CoMFA model was challenged with an external test data set; the predicted results were close to the actual values with average difference of 0.637 logRBA. This study demonstrates the utility of this CoMFA model for real-world use in predicting the AR binding affinities of structurally diverse chemicals over a wide RBA range.     

新型核素~(64)Cu标记硝基咪唑类肿瘤乏氧显像剂及正电子发射断层扫描显像研究

采用新型核素64Cu标记了含丙烯胺肟[Pn AO(3,3,9,9-Tetramethyl-4,8-diazaundecane-2,10-dione Dioxime)]结构的硝基咪唑类乏氧显像剂Pn AO-1-(2-nitroimidazole)[BMS181321],通过优化反应条件,于室温下反应10 min后即得到高放化纯度和高比活度的标记化合物64Cu-BMS181321.目标产物经放射性高效液相色谱检测验证和体外稳定性实验确认后,通过尾静脉注射到人源胰腺癌(PANC-1细胞系)裸鼠体内,分别于注射显像剂4和8 h后进行小动物正电子发射断层扫描显像(Micro-PET).结果表明,4 h左右肿瘤乏氧区域有良好的放射性浓聚.64Cu-BMS181321的合成及其分子显像研究开创了64Cu标记硝基咪唑类乏氧显像剂进行乏氧显像的先例,经进一步药物临床实验评价后,64Cu-BMS181321有望成为具有良好前景的PET乏氧显像药物.     

Streamlined in vivo selection and screening of human prostate carcinoma avid phage particles for development of peptide based in vivo tumor imaging agents

Bacteriophage (phage) display has been exploited for the purpose of discovering new cancer specific targeting peptides. However, this approach has resulted in only a small number of tumor targeting peptides useful as in vivo imaging agents. We hypothesize that in vivo screening for tumor uptake of fluorescently tagged phage particles displaying multiple copies of an in vivo selected tumor targeting peptide will expedite the development of peptide based imaging agents. In this study, both in vivo selection and in vivo screening of phage displaying foreign peptides were utilized to best predict peptides with the pharmacokinetic properties necessary for translation into efficacious in vivo imaging agents. An in vivo selection of phage display libraries was performed in SCID mice bearing human PC-3 prostate carcinoma tumors. Eight randomly selected phage clones and four control phage clones were fluorescently labeled with AlexaFluor 680 for subsequent in vivo screening and analyses. The corresponding peptides of six of these phage clones were tested as 111In-labeled peptide conjugates for single photon emission computed tomography (SPECT) imaging of PC-3 prostate carcinomas. Two peptide sequences, G1 and H5, were successful as in vivo imaging agents. The affinities of G1 and H5 peptides for cultured PC-3 cells were then analyzed via cell flow cytometry resulting in Kd values of 1.8 μM and 2.2 μM, respectively. The peptides bound preferentially to prostate tumor cell lines compared to that of other carcinoma and normal cell lines, and H5 appeared to possess cytotoxic properties. This study demonstrates the value of in vivo screening of fluorescently labeled phage for the prediction of the efficacy of the corresponding 111In-labeled synthetic peptide as an in vivo SPECT tumor imaging agent.     

Simultaneous determination of 15 steroids in rat blood via gas chromatography-mass spectrometry to evaluate the impact of emasculation on adrenal

Adrenal was believed to affect the prostate tumor tissue growth by its secretion of adrenal androgens. However, the mechanisms regulating these effects were not fully understood. In this work, a sensitive and specific method for the determination of 15 steroids in blood via gas chromatography-mass spectrometry in selective ion storage detection mode was established to evaluate the impact of emasculation on adrenal steroids metabolism. Steroids were isolated by solid-phase extraction using Oasis HLB cartridge, and then derivated with heptafluorobutyric anhydride before analysis. The limits of detection were between 0.15 and 1.0 ng/mL and limits of quantification were between 0.62 and 2.6 ng/mL. The recoveries of steroids were above 83%, and both the intra-day and inter-day precisions (RSD%) were lower than 8%. Pregnenolone, progesterone, 17α-hydroxyprogesterone (17αP), 17α-hydroxypregnenolone (17αH), dehydroepiandrosterone (DHEA), estrone, 17β-estradiol, dihydrotestosterone (DHT), testosterone (T), 4-androstenedione (4-A), 1,4-androstadiene-3,17-dione, 11-deoxycortisol, 11-deoxycorticosterone, cortisol and aldosterone were quantified in 156 major male SD rats at 0, 1, 2, 4, 7 days, 2, 4, 6, 8, 10, 12, 14, and 16 weeks following emasculation. T and DHT decreased by 86.2% and 73.4%, respectively in the first 7 days following emasculation, but adrenal androgens (DHEA, 4-A) stabled at the normal level accordingly. Adrenal androgens and their precursors (17αH, 17αP) increased from the 2nd week along with the increase of androgens and the decrease of mineralocorticoids. These facts revealed that adrenal possibly enhanced its function of producing adrenal androgens from the 2nd week responding to the low androgens level induced by emasculation.     

Effect of fluoromethyl substituents on the spectral properties of the benzene ring

The electronic spectra of benzyl fluoride, benzal fluoride, and benzotrifluoride have been studied in polarized light at 4 K. An attempt is made to interpret the special features observed in the spectra by associating them with the effect of the different fluoromethyl substituents.     

Improving Tumor-to-Background Contrast through Hydrophilic Tetrazines: The Construction of 18F-Labeled PET Agents Targeting Nonsmall Cell Lung Carcinoma

Bioorthogonal reactions have been widely used in the biomedical field. ¹⁸F-TCO/Tetrazine ligation is the most reactive radiolabelled inverse electron demand Diels–Alder reaction, but its application had been limited due to modest contrast ratios of the resulting conjugates. Herein, we describe the use of hydrophilic tetrazines to improve tumor-to-background contrast of neurotensin receptor targeted PET agents. PET agents were constructed using a rapid Diels–Alder reaction of the radiolabeled trans-cyclooctene (¹⁸F-sTCO) with neurotensin (NT) conjugates of a 3,6-diaryltetrazine, 3-methyl-6-aryltetrazine, and a derivative of 3,6-di(2-hydroxyethyl)tetrazine. Although cell binding assays demonstrated all agents have comparable binding affinity, the conjugate derived from 3,6-di(2-hydroxyethyl)tetrazine demonstrated the highest tumor to muscle contrast, followed by conjugates of the 3-methyl-6-aryltetrazine and 3,6-diaryltetrazine.     

Preparation and Biological Evaluation of [99mTc]Tc-CNGU as a PSMA-Targeted Radiotracer for the Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a well-established biological target that is overexpressed on the surface of prostate cancer lesions. Radionuclide-labeled small-molecule PSMA inhibitors have been shown to be promising PSMA-specific agents for the diagnosis and therapy of prostate cancer. In this study, a glutamate-urea-based PSMA-targeted ligand containing an isonitrile (CNGU) was synthesized and labeled with ^99mTc to prepare [^99mTc]Tc-CNGU with a high radiochemical purity (RCP). The CNGU ligand showed a high affinity toward PSMA (K_i value is 8.79 nM) in LNCaP cells. The [^99mTc]Tc-CNGU exhibited a good stability in vitro and hydrophilicity (log P = −1.97 ± 0.03). In biodistribution studies, BALB/c nude mice bearing LNCaP xenografts showed that the complex had a high tumor uptake with 4.86 ± 1.19% ID/g, which decreased to 1.74 ± 0.90% ID/g after a pre-injection of the selective PSMA inhibitor ZJ-43, suggesting that it was a PSMA-specific agent. Micro-SPECT imaging demonstrated that the [^99mTc]Tc-CNGU had a tumor uptake and that the uptake was reduced in the image after blocking with ZJ-43, further confirming its PSMA specificity. All of the results in this work indicated that [^99mTc]Tc-CNGU is a promising PSMA-specific tracer for the imaging of prostate cancer.     

Novel 17 substituted pregnadiene derivatives as 5 alpha-reductase inhibitors and their binding affinity for the androgen receptor

The in vitro antiandrogenic activity of four new progesterone derivatives: 4, 5, 6 and 7 (8 is a known compound) was determined. These compounds were evaluated as 5alpha-reductase inhibitors as well as by their capacity to bind to the androgen receptor in gonadectomized hamster prostate. The IC(50) value was determined using increasing concentrations of 4, 5, 6, 7 and 8 in the presence of [(3)H]T and the microsomal fraction of the hamster prostate containing the 5alpha-reductase enzyme. In this paper we also demonstrated the effect of increasing concentrations of the novel steroids upon [(3)H]DHT binding to the androgen receptors from hamster prostate which produces competition for the androgen receptor sites. The in vitro studies showed that steroids 4, 5, 6, 7 and 8 had an inhibitory activity for the 5alpha-reductase with IC(50) of: 4 (0.17 microM), 5 (0.19 microM), 6 (1 microM), 7 (4.2 microM), and 8 (2.7 microM). On the other hand, the IC(50) value for compounds 4, 5, 6, 7, 8 and DHT showed the following order of affinity for the androgen receptor: 6>7>5>DHT. Surprisingly compounds 4 and 8 did not bind to the androgen receptor. The overall data indicate that all synthesized compounds are inhibitors for the enzyme 5alpha-reductase present in the hamster prostate. In contrast, compounds 5, 6 and 7, which have a cyclohexyl group in the side chain showed a high affinity for the androgen receptor.     

Efficient Uptake of 177Lu‐Porphyrin‐PEG Nanocomplexes by Tumor Mitochondria for Multimodal‐Imaging‐Guided Combination Therapy

The benefits to intracellular drug delivery from nanomedicine have been limited by biological barriers and to some extent by targeting capability. We investigated a size‐controlled, dual tumor‐mitochondria‐targeted theranostic nanoplatform (Porphyrin‐PEG Nanocomplexes, PPNs). The maximum tumor accumulation (15.6 %ID g^?1, 72 h p.i.) and ideal tumor‐to‐muscle ratio (16.6, 72 h p.i.) was achieved using an optimized PPN particle size of approximately 10 nm, as measured by using PET imaging tracing. The stable coordination of PPNs with ¹⁷⁷Lu enables the integration of fluorescence imaging (FL) and photodynamic therapy (PDT) with positron emission tomography (PET) imaging and internal radiotherapy (RT). Furthermore, the efficient tumor and mitochondrial uptake of ¹⁷⁷Lu‐PPNs greatly enhanced the efficacies of RT and/or PDT. This work developed a facile approach for the fabrication of tumor‐targeted multi‐modal nanotheranostic agents, which enables precision and radionuclide‐based combination tumor therapy.     

Relation between location of elements in periodic table and affinity for the malignant tumor (author's transl)

Affinity of many inorganic compounds for the malignant tumor was examined, using the rats which were subcutaneously transplanted with Yoshida sarcoma. And the relations between the uptake rate into the malignant tumor and in vitro binding power to the protein were investigated in these compounds. In these experiments, the bipositive ions and anions had not affinity for the tumor tissue with a few exceptions. On the other hand, Hg, Au and Bi, which have strong binding power to the protein, showed high uptake rate into the malignant tumor. As Hg++, Au+ and Bi+++ are soft acids according to classification of Lewis acids, it was thought that these elements would bind strongly to soft base (R-SH, R-S-) present in the tumor tissue. In many hard acids (according to classification of Lewis acids), the uptake rate into the tumor was shown as a function of ionic potentials (valency/ionic radii) of the metal ions. It is presumed that the chemical bond of these hard acids in the tumor tissue is ionic bond to hard base (R-COO-, R-PO3(2-), R-SO3-, R-NH2).     

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.