SARS-CoV-2 Nucleocapsid Protein Is Not Responsible for Over-Activation of Complement Lectin Pathway.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.

Beta-Secretase 1 Recruits Amyloid-Beta Precursor Protein to ROCK2 Kinase, Resulting in Erroneous Phosphorylation and Beta-Amyloid Plaque Formation.

The amyloidogenic processing of APP depends on two events: its phosphorylation by ROCK2 (at Thr654) and the phosphorylation of the APP-cleaving enzyme BACE1 (at Ser498). However, the mechanisms and structural details of APP-ROCK2 and BACE1-ROCK2 binding are unknown. Using direct physical methods in combination with an in silico approach, we found that BACE1 binds into the substrate-binding groove of ROCK2 with a low affinity (Kd = 18 µM), while no binding of APP to ROCK2 alone could be detected. On the other hand, a strong association (Kd = 3.5 nM) of APP to the weak ROCK2-BACE1 complex was observed, although no stable ternary complex was detected, i.e., BACE1 was displaced by APP. We constructed a sequential functional model: (1) BACE1 weakly binds to ROCK2 and induces an allosteric conformational change in ROCK2; (2) APP strongly binds to the ROCK2-BACE1 complex, and BACE1 is released; and (3) ROCK2 phosphorylates APP at Thr654 (leading to a longer stay in the early endosome during APP processing). Direct fluorescence titration experiments showed that the APP646-664 or APP665-695 fragments did not bind separately to the ROCK2-BACE1 complex. Based on these observations, we conclude that two binding sites are involved in the ROCK2-APP interaction: (1) the substrate-binding groove, where the APP646-664 sequence containing Thr654 sits and (2) the allosteric binding site, where the APP665-695 sequence binds. These results open the way to attack the allosteric site to prevent APP phosphorylation at Thr654 by ROCK2 without inhibiting the activity of ROCK2 towards its other substrates.

Overview of Prostaglandin E2 (PGE2)-Targeting Radiolabelled Imaging Probes from Preclinical Perspective: Lessons Learned and Road Ahead.

As malignancies still represent one of the major health concerns worldwide, early tumor identification is among the priorities of today's science. Given the strong association between cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2), PGE2 receptors (EPs), and carcinogenesis, target-specific molecules directed towards the components of the COX2/PGE2/EP axis seem to be promising imaging probes in the diagnostics of PGE2pos. neoplasms and in the design of anti-cancer drugs. Featured with outstanding inclusion forming capability, ß-cyclodextrins (CDs) including randomly methylated ß-CD (RAMEB) were reported to complex with PGE2. Therefore, radiolabelled ß-CDs could be valuable vectors in the molecular imaging of PGE2-related tumorigenesis. In vivo preclinical small animal model systems applying positron emission tomography (PET) ensure a well-suited scenario for the assessment of PGE2-affine labelled CD derivatives. Previous translational studies dealt with the evaluation of the tumor-homing capability of Gallium-68 (68Ga) and Bismuth-205/206 (205/206Bi)-appended ß-CD compounds conjugated with chelator NODAGA or DOTAGA: [68Ga]Ga-NODAGA-2-hydroxypropyl-ß-cyclodextrin/HPBCD, [68Ga]Ga-NODAGA-RAMEB, [68Ga]Ga-DOTAGA-RAMEB, and [205/206Bi]Bi-DOTAGA-RAMEB in experimental tumors with different PGE2 expression. These imaging probes project the establishment of tailor-made PET diagnostics of PGE2pos. malignancies. In the present review, we provide a detailed overview of the in vivo investigations of radiolabelled PGE2-directed CDs, highlighting the importance of the integration of translational discoveries into routine clinical usage.

In Vivo Preclinical Assessment of ß-Amyloid-Affine [11C]C-PIB Accumulation in Aluminium-Induced Alzheimer's Disease-Resembling Hypercholesterinaemic Rat Model.

Aluminum (Al) excess and hypercholesterinaemia are established risks of Alzheimer's disease (AD). The aim of this study was to establish an AD-resembling hypercholesterinaemic animal model-with the involvement of 8 week and 48 week-old Fischer-344 rats-by Al administration for the safe and rapid verification of ß-amyloid-targeted positron emission tomography (PET) radiopharmaceuticals. Measurement of lipid parameters and ß-amyloid-affine [11C]C-Pittsburgh Compound B ([11C]C-PIB) PET examinations were performed. Compared with the control, the significantly elevated cholesterol and LDL levels of the rats receiving the cholesterol-rich diet support the development of hypercholesterinaemia (p ≤ 0.01). In the older cohort, a notably increased age-related radiopharmaceutical accumulation was registered compared to in the young (p ≤ 0.05; p ≤ 0.01). A monotherapy-induced slight elevation of mean standardised uptake values (SUVmean) was statistically not significant; however, adult rats administered a combined diet expressed remarkable SUVmean increment compared to the adult control (SUVmean: from 0.78 ± 0.16 to 1.99 ± 0.28). One and two months after restoration to normal diet, the cerebral [11C]C-PIB accumulation of AD-mimicking animals decreased by half and a third, respectively, to the baseline value. The proposed in vivo Al-induced AD-resembling animal system seems to be adequate for the understanding of AD neuropathology and future drug testing and radiopharmaceutical development.

PET Probes for Preclinical Imaging of GRPR-Positive Prostate Cancer: Comparative Preclinical Study of [68Ga]Ga-NODAGA-AMBA and [44Sc]Sc-NODAGA-AMBA.

Gastrin-releasing peptide receptors (GRPR) are overexpressed in prostate cancer (PCa). Since bombesin analogue aminobenzoic-acid (AMBA) binds to GRPR with high affinity, scandium-44 conjugated AMBA is a promising radiotracer in the PET diagnostics of GRPR positive tumors. Herein, the GRPR specificity of the newly synthetized [44Sc]Sc-NODAGA-AMBA was investigated in vitro and in vivo applying PCa PC-3 xenograft. After the in-vitro assessment of receptor binding, PC-3 tumor-bearing mice were injected with [44Sc]Sc/[68Ga]Ga-NODAGA-AMBA (in blocking studies with bombesin) and in-vivo PET examinations were performed to determine the radiotracer uptake in standardized uptake values (SUV). 44Sc/68Ga-labelled NODAGA-AMBA was produced with high molar activity (approx. 20 GBq/µmoL) and excellent radiochemical purity. The in-vitro accumulation of [44Sc]Sc-NODAGA-AMBA in PC-3 cells was approximately 25-fold higher than that of the control HaCaT cells. Relatively higher uptake was found in vitro, ex vivo, and in vivo in the same tumor with the 44Sc-labelled probe compared to [68Ga]Ga-NODAGA-AMBA. The GRPR specificity of [44Sc]Sc-NODAGA-AMBA was confirmed by significantly (p ≤ 0.01) decreased %ID and SUV values in PC-3 tumors after bombesin pretreatment. The outstanding binding properties of the novel [44Sc]Sc-NODAGA-AMBA to GRPR outlines its potential to be a valuable radiotracer in the imaging of GRPR-positive PCa.

In Vivo Assessments of Mesoblastic Nephroma (Ne/De) and Myelomonoblastic Leukaemia (My1/De) Tumour Development in Hypercholesterolemia Rat Models.

Given the rising prevalence of lipid metabolic disorders and malignant diseases, we aimed to establish an in vivo hypercholesterinaemic tumour-bearing rat model for the induction and assessment of these conditions. A normal standard CRLT/N, 2 (baseline),- or 4 (2 + 2, pretreated)-week-long butter and cholesterol rich (BCR) diet was applied to mesoblastic nephroma (Ne/De) and myelomonoblastic leukaemia (My1/De) tumour-bearing and healthy control Long­Evans and Fischer 344 rats. The beginning of chow administration started in parallel with tumour induction and the 2 weeks of pre-transplantation in the baseline and pretreated groups, respectively. Fourteen days post-inoculation, the measurement of lipid parameters and [18F]F-FDG PET/MRI examinations was executed. The comparable lipid status of baseline healthy and tumorous rats proves that regardless of tumour presence, BCR-based hypercholesterolemia was achieved. A higher tumour mass among pretreated tumorous animals was found when compared to the control groups (p < 0.05, p < 0.01). Further, a visually greater [18F]F-FDG accumulation was observed in pretreated BCR tumorous animals; however, the quantitative data (SUVmean: 9.86 ± 0.98, 9.68 ± 1.24; SUVmax: 19.63 ± 1.20; 17.56 ± 3.21 for Ne/De and My1/De, respectively) were not statistically significantly different from those of the CRLT/N tumorous rats (SUVmean: 8.40 ± 1.42, 7.22 ± 1.06 and SUVmax: 15.99 ± 2.22, 12.46 ± 1.96 for control Ne/De and My1/De, respectively). Our model seems to be appropriate for simultaneously investigating hypercholesterolemia and cancer in the same rat.

Dual Inhibitors of AChE and BACE-1 for Reducing Aß in Alzheimer's Disease: From In Silico to In Vivo.

Alzheimer's disease (AD) is a complex and widespread condition, still not fully understood and with no cure yet. Amyloid beta (Aß) peptide is suspected to be a major cause of AD, and therefore, simultaneously blocking its formation and aggregation by inhibition of the enzymes BACE-1 (ß-secretase) and AChE (acetylcholinesterase) by a single inhibitor may be an effective therapeutic approach, as compared to blocking one of these targets or by combining two drugs, one for each of these targets. We used our ISE algorithm to model each of the AChE peripheral site inhibitors and BACE-1 inhibitors, on the basis of published data, and constructed classification models for each. Subsequently, we screened large molecular databases with both models. Top scored molecules were docked into AChE and BACE-1 crystal structures, and 36 Molecules with the best weighted scores (based on ISE indexes and docking results) were sent for inhibition studies on the two enzymes. Two of them inhibited both AChE (IC50 between 4-7 µM) and BACE-1 (IC50 between 50-65 µM). Two additional molecules inhibited only AChE, and another two molecules inhibited only BACE-1. Preliminary testing of inhibition by F681-0222 (molecule 2) on APPswe/PS1dE9 transgenic mice shows a reduction in brain tissue of soluble Aß42.

Rapid Identification of Potential Drug Candidates from Multi-Million Compounds' Repositories. Combination of 2D Similarity Search with 3D Ligand/Structure Based Methods and In Vitro Screening.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach in early drug discovery. If structures of active compounds are available, rapid 2D similarity search can be performed on multimillion compounds' databases. This approach can be combined with physico-chemical parameter and diversity filtering, bioisosteric replacements, and fragment-based approaches for performing a first round biological screening. Our objectives were to investigate the combination of 2D similarity search with various 3D ligand and structure-based methods for hit expansion and validation, in order to increase the hit rate and novelty. In the present account, six case studies are described and the efficiency of mixing is evaluated. While sequentially combined 2D/3D similarity approach increases the hit rate significantly, sequential combination of 2D similarity with pharmacophore model or 3D docking enriched the resulting focused library with novel chemotypes. Parallel integrated approaches allowed the comparison of the various 2D and 3D methods and revealed that 2D similarity-based and 3D ligand and structure-based techniques are often complementary, and their combinations represent a powerful synergy. Finally, the lessons we learnt including the advantages and pitfalls of the described approaches are discussed.

111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model.

Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.

Design and Selection of Novel C1s Inhibitors by In Silico and In Vitro Approaches.

The complement system is associated with various diseases such as inflammation or auto-immune diseases. Complement-targeted drugs could provide novel therapeutic intervention against the above diseases. C1s, a serine protease, plays an important role in the CS and could be an attractive target since it blocks the system at an early stage of the complement cascade. Designing C1 inhibitors is particularly challenging since known inhibitors are restricted to a narrow bioactive chemical space in addition selectivity over other serine proteases is an important requirement. The typical architecture of a small molecule inhibitor of C1s contains an amidine (or guanidine) residue, however, the discovery of non-amidine inhibitors might have high value, particularly if novel chemotypes and/or compounds displaying improved selectivity are identified. We applied various virtual screening approaches to identify C1s focused libraries that lack the amidine/guanidine functionalities, then the in silico generated libraries were evaluated by in vitro biological assays. While 3D structure-based methods were not suitable for virtual screening of C1s inhibitors, and a 2D similarity search did not lead to novel chemotypes, pharmacophore model generation allowed us to identify two novel chemotypes with submicromolar activities. In three screening rounds we tested altogether 89 compounds and identified 20 hit compounds (<10 µM activities; overall hit rate: 22.5%). The highest activity determined was 12 nM (1,2,4-triazole), while for the newly identified chemotypes (1,3-benzoxazin-4-one and thieno[2,3-d][1,3]oxazin-4-one) it was 241 nM and 549 nM, respectively.

Inhibition of the LOX enzyme family members with old and new ligands. Selectivity analysis revisited.

Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

Combination of 2D/3D ligand-based similarity search in rapid virtual screening from multimillion compound repositories. Selection and biological evaluation of potential PDE4 and PDE5 inhibitors.

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost effective approach. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compound databases but the generated library requires further focusing by various 2D/3D chemoinformatics tools. We report here a combination of the 2D approach with a ligand-based 3D method (Screen3D) which applies flexible matching to align reference and target compounds in a dynamic manner and thus to assess their structural and conformational similarity. In the first case study we compared the 2D and 3D similarity scores on an existing dataset derived from the biological evaluation of a PDE5 focused library. Based on the obtained similarity metrices a fusion score was proposed. The fusion score was applied to refine the 2D similarity search in a second case study where we aimed at selecting and evaluating a PDE4B focused library. The application of this fused 2D/3D similarity measure led to an increase of the hit rate from 8.5% (1st round, 47% inhibition at 10 µM) to 28.5% (2nd round at 50% inhibition at 10 µM) and the best two hits had 53 nM inhibitory activities.

Concomitant [18F]F-FAZA and [18F]F-FDG Imaging of Gynecological Cancer Xenografts: Insight into Tumor Hypoxia.

BACKGROUND/AIM: Herein we assessed the feasibility of imaging protocols using both hypoxia-specific [18F]F-FAZA and [18F]F-FDG in bypassing the limitations derived from the non-specific findings of [18F]F-FDG PET imaging of tumor-related hypoxia. MATERIALS AND METHODS: CoCl2-generated hypoxia was induced in multidrug resistant (Pgp+) or sensitive (Pgp-) human ovarian (Pgp- A2780, Pgp+ A2780AD), and cervix carcinoma (Pgp- KB-3-1, Pgp+ KB-V-1) cell lines to establish corresponding tumor-bearing mouse models. Prior to [18F]F-FDG/[18F]F-FAZA-based MiniPET imaging, in vitro [18F]F-FDG uptake measurements and western blotting were used to verify the presence of hypoxia. RESULTS: Elevated GLUT-1, and hexokinase enzyme-II expression driven by CoCl2-induced activation of hypoxia-inducible factor-1α explains enhanced cellular [18F]F-FDG accumulation. No difference was observed in the [18F]F-FAZA accretion of Pgp+ and Pgp- tumors. Tumor-to-muscle ratios for [18F]F-FAZA measured at 110-120 min postinjection (6.2±0.1) provided the best contrasted images for the delineation of PET-oxic and PET-hypoxic intratumor regions. Although all tumors exhibited heterogenous uptake of both radiopharmaceuticals, greater differences for [18F]F-FAZA between the tracer avid and non-accumulating regions indicate its superiority over [18F]F-FDG. Spatial correlation between [18F]F-FGD and [18F]F-FAZA scans confirms that hypoxia mostly occurs in regions with highly active glucose metabolism. CONCLUSION: The addition of [18F]F-FAZA PET to [18F]F-FGD imaging may add clinical value in determining hypoxic sub-regions.

Acute Myelomonoblastic Leukemia (My1/De): A Preclinical Rat Model.

BACKGROUND/AIM: Since acute myeloid leukemias still represent the most aggressive type of adult acute leukemias, the profound understanding of disease pathology is of paramount importance for diagnostic and therapeutic purposes. Hence, this study aimed to explore the real-time disease fate with the establishment of an experimental myelomonoblastic leukemia (My1/De) rat model using preclinical positron emission tomography (PET) and whole-body autoradiography. MATERIALS AND METHODS: In vitro [18F]F-FDG uptake studies were performed to compare the tracer accumulation in the newly cultured My1/De tumor cell line (blasts) with that in healthy control and My1/De bone marrow suspensions. Post transplantation of My1/De cells under the left renal capsule of Long-Evans rats, primary My1/De tumorigenesis, and metastatic propagation were investigated using [18F]F-FDG PET imaging, whole-body autoradiography and phosphorimage analyses. To assess the organ uptake profile of the tumor-carrying animals we accomplished ex vivo biodistribution studies. RESULTS: The tracer accumulation in the My1/De culture cells exceeded that of both the tumorous and the healthy bone marrow suspensions (p<0.01). Based on in vivo imaging, the subrenally transplanted My1/De cells resulted in the development of leukemia in the abdominal organs, and metastasized to the mesenterial and thoracic parathymic lymph nodes (PTLNs). The lymphatic spread of metastasis was further confirmed by the significantly higher %ID/g values of the metastatic PTLNs (4.25±0.28) compared to the control (0.94±0.34). Cytochemical staining of the peripheral blood, autopsy findings, and wright-Giemsa-stained post-mortem histological sections proved the leukemic involvement of the assessed tissues/organs. CONCLUSION: The currently established My1/De model appears to be well-suited for further leukemia-related therapeutic and diagnostic investigations.

Evolution of the concept of conformational dynamics of enzyme functions over half of a century: A personal view.

To most physicists, it was always evident that conformational fluctuation is an inherent property of all molecules. Its existence in proteins was mentioned first by Linderström-Lang and Schellman in 1959 based on their hydrogen-deuterium exchange experiments. The "induced fit" mechanism to explain ligand-induced conformational changes was suggested by Koshland in 1958. Straub combined these two concepts in his "fluctuation fit" theory in 1964. The era of protein X-ray crystallography imposed a static view of protein structures. With proteins becoming accessible to NMR analysis, conformational dynamics could be mapped, and a new wave of dynamic interpretation of enzymatic catalysis and molecular recognition appeared. Energy landscapes, energy funnels, conformational selection, conformational distribution shifts are now frequent terms in interpreting biomolecular recognition and enzymatic catalysis. All these interpretations are based on the concept that evolution uses the conformational fluctuations of enzymes to develop efficient and dynamic catalytic machines. In a resurrection of the original "fluctuation fit" concept, it is generally recognized now that spatial and temporal events of catalysis are equally important to describe its mechanism. This special issue, dedicated to the memory of Henryk Eisenberg, prompted us to look back at the last 50 years of development of a concept that-like other important concepts-appeared, evolved and became accepted during the period covered by the scientific lifespan of Henryk.

[Selection of a melanine concentrating hormone receptor-1 (MCHR1) antagonists' focused library and its biological screening with AequoScreen]. / Melanin koncentráló hormon receptor-1 (MCHR1) antagonista fókuszált könyvtár kiválasztása és in vitro biológiai szurése AequosCreen esszével.

Target focused libraries can be rapidly selected by 2D virtual screening methods from multimillion compounds' repositories if structures of active compounds are available. In the present study a multi-step virtual and in vitro screening cascade is reported to select Melanin Concentrating Hormone Receptor-1 (MCHR1) antagonists. The 2D similarity search combined with physicochemical parameter filtering is suitable for selecting candidates from multimillion compounds' repository. The seeds of the first round virtual screening were collected from the literature and commercial databases, while the seeds of the second round were the hits of the first round. In vitro screening underlined the efficiency of our approach, as in the second screening round the hit rate (8.6 %) significantly improved compared to the first round (1.9%), reaching the antagonist activity even below 10 nM.

Insights into recent preclinical studies on labelled cyclodextrin-based imaging probes: Towards a novel oncological era.

As malignancies remain one of the major health concerns worldwide, increasing focus has been centered around the application of cyclodextrins (CDs) in cancer imaging and therapy due to their outstanding inclusion forming capability. Albeit the physicochemical properties of CDs were intensively elucidated, the spread of their clinical application is limited by the relative paucity of knowledge about their pharmacokinetic profile, especially biodistribution. Studies applying fluorescently- CDs, or CD-based MRI contrast agents revealed much about pharmacokinetics and diagnostic applications; however, derivatives labelled with positron emitters seem superior molecular probes in the investigation of the route of CDs in biological niche. In vivo imaging based on preclinical tumor-bearing model systems are well-suited to evaluate the whole-body distribution of the two most frequently assessed CDs: randomly methylated ß-cyclodextrin (RAMEB), and hydroxypropyl-ß-cyclodextrin (HPBCD). Exploiting the firm signaling interaction between cancer-related cyclooxygenase-2, prostaglandin E2 (PGE2) and RAS oncoprotein, radioconjugated, PGE2-affine CDs project the establishment of novel imaging probes and therapeutic agents. Currently, we provide an overview of the preclinical studies on CD pharmacokinetics highlighting the significance of the integration of translational discoveries into human patient care.

Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology.

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 µL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.

In vivo assessment of tumor targeting potential of 68Ga-labelled randomly methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl-ß-cyclodextrin (HPßCD) using positron emission tomography.

Cyclodextrin derivates (CyDs) can form complexes with cyclooxygenase-2 induced tumor promoting prostaglandin E2 (PGE2). Based on our previous observations, 68Ga-labelled CyDs may represent promising radiopharmaceuticals in the positron emission tomography (PET) diagnostics of PGE2 positive tumors. We aimed at evaluating the tumor-targeting potential of 68Ga-NODAGA conjugated randomly methylated beta-cyclodextrin (68Ga-NODAGA-RAMEB) and 2-hydroxypropyl-ß-cyclodextrin (68Ga-NODAGA-HPßCD) using in vivo PET imaging with experimental tumor models. Tumor radiopharmaceutical uptake was assessed applying PET and gamma counter in vivo and ex vivo respectively, following the administration of 18FDG, 68Ga-NODAGA-RAMEB or 68Ga-NODAGA-HPßCD via the lateral tail vein to the subsequent tumor-bearing animals: HT1080, A20, PancTu-1, BxPC3, B16-F10, Ne/De and He/De. All investigated tumors were identifiable with both 68Ga-labelled CyDs; however, in vivo results, in correlation with the ex vivo data, revealed that the PGE2 positive BxPC3, A20, Ne/De and He/De tumors presented the highest accumulation. In case of HT1080, A20, B16-F10 tumors significant differences were encountered between the accumulations of both 68Ga-labelled radiopharmaceuticals of the same tumor. Subcutaneously and the orthotopically transplanted Ne/De tumors differed significantly (p ≤ 0.01) regarding tracer uptake. 68Ga-labelled CyDs may open a novel field in the PET diagnostics of PGE2 positive primary tumors and metastases.