Introduction of a SiFA moiety into the D-glutamate chain of DOTA-PP-F11N results in radiohybrid-based CCK-2R-targeted compounds with improved pharmacokinetics in vivo

In order to enable 18F- and 177Lu-labelling within the same molecule, we introduced a silicon-based fluoride acceptor (SiFA) into the hexa-D-glutamate chain of DOTA-PP-F11N. In addition, minigastrin analogues with a prolonged as well as γ-linked D-glutamate chain were synthesised and evaluated. CCK-2R affinity (IC50, AR42J cells) and lipophilicity (logD7.4) were determined. Biodistribution studies at 24 h post-injection (p.i.) and µSPECT/CT imaging at 1, 4 and 24 h p.i. were carried out in AR42J tumour-bearing CB17-SCID mice. CCK-2R affinity of (R)-DOTAGA-rhCCK-1 to 18 was enhanced with increasing distance between the SiFA building block and the binding motif. Lipophilicity of [177Lu]Lu-(R)-DOTAGA-rhCCK-1 to 18 was higher compared to that of [177Lu]Lu-DOTA-PP-F11N and [177Lu]Lu-CP04. The respective α- and γ-linked rhCCK derivatives revealing the highest CCK-2R affinity were further evaluated in vivo. In comparison with [177Lu]Lu-DOTA-PP-F11N, [177Lu-]Lu-(R)-DOTAGA-rhCCK-9 and -16 exhibited three- to eight-fold increased activity levels in the tumour at 24 h p.i. However, activity levels in the kidneys were elevated as well. We could show that the introduction of a lipophilic SiFA moiety into the hydrophilic backbone of [177Lu]Lu-DOTA-PP-F11N led to a decelerated blood clearance and thus improved tumour retention. However, elevated kidney retention has to be addressed in future studies

Differential gene expression in pristane-induced arthritis susceptible DA versus resistant E3 rats

Arthritis susceptibility genes were sought by analysis of differential gene expression between pristane-induced arthritis (PIA)-susceptible DA rats and PIA-resistant E3 rats. Inguinal lymph nodes of naïve animals and animals 8 days after pristane injection were analyzed for differential gene expression. mRNA expression was investigated by microarray and real-time PCR, and protein expression was analyzed by flow cytometry or ELISA. Twelve genes were significantly differentially expressed when analyzed by at least two independent methods, and an additional five genes showed a strong a tendency toward differential expression. In naïve DA rats IgE, the bone marrow stromal cell antigen 1 (Bst1) and the MHC class II β-chain (MhcII) were expressed at a higher level, and the immunoglobulin kappa chain (Igκ) was expressed at a lower level. In pristane-treated DA rats the MHC class II β-chain, gelatinase B (Mmp9) and the protein tyrosine phosphatase CL100 (Ptpn16) were expressed at a higher level, whereas immunoglobulins, the CD28 molecule (Cd28), the mast cell specific protease 1 (Mcpt1), the carboxylesterase precursor (Ces2), K-cadherin (Cdh6), cyclin G1 (Ccng1), DNA polymerase IV (Primase) and the tumour associated glycoprotein E4 (Tage) were expressed at a lower level. Finally, the differentially expressed mRNA was confirmed with protein expression for some of the genes. In conclusion, the results show that animal models are well suited for reproducible microarray analysis of candidate genes for arthritis. All genes have functions that are potentially important for arthritis, and nine of the genes are located within genomic regions previously associated with autoimmune disease

Development of the first 18F-labeled radiohybrid-based minigastrin derivative with high target affinity and tumor accumulation by substitution of the chelating moiety

In order to optimize elevated kidney retention of previously reported minigastrin derivatives, we substituted (R)-DOTAGA by DOTA in (R)-DOTAGA-rhCCK-16/-18. CCK-2R-mediated internalization and affinity of the new compounds were determined using AR42J cells. Biodistribution and µSPECT/CT imaging studies at 1 and 24 h p.i. were carried out in AR42J tumor-bearing CB17-SCID mice. Both DOTA-containing minigastrin analogs exhibited 3- to 5-fold better IC50 values than their (R)-DOTAGA-counterparts. natLu-labeled peptides revealed higher CCK-2R affinity than their natGa-labeled analogs. In vivo, tumor uptake at 24 h p.i. of the most affine compound, [19F]F-[177Lu]Lu-DOTA-rhCCK-18, was 1.5- and 13-fold higher compared to its (R)-DOTAGA derivative and the reference compound, [177Lu]Lu-DOTA-PP-F11N, respectively. However, activity levels in the kidneys were elevated as well. At 1 h p.i., tumor and kidney accumulation of [19F]F-[177Lu]Lu-DOTA-rhCCK-18 and [18F]F-[natLu]Lu-DOTA-rhCCK-18 was high. We could demonstrate that the choice of chelators and radiometals has a significant impact on CCK-2R affinity and thus tumor uptake of minigastrin analogs. While elevated kidney retention of [19F]F-[177Lu]Lu-DOTA-rhCCK-18 has to be further addressed with regard to radioligand therapy, its radiohybrid analog, [18F]F-[natLu]Lu-DOTA-rhCCK-18, might be ideal for positron emission tomography (PET) imaging due to its high tumor accumulation at 1 h p.i. and the attractive physical properties of fluorine-18

Characterization and modeling of intermetallic phase formation during the joining of aluminum and steel in analogy to co-extrusion

The reinforcement of light metal components with steel allows to increase the strength of the part while keeping the weight comparatively low. Lateral angular co-extrusion (LACE) offers the possibility to produce hybrid coaxial profiles consisting of steel and aluminum. In the present study, the effect of the process parameters temperature, contact pressure and time on the metallurgical bonding process and the development of intermetallic phases was investigated. Therefore, an analogy experiment was developed to reproduce the process conditions during co-extrusion using a forming dilatometer. Based on scanning electron microscopy analysis of the specimens, the intermetallic phase seam thickness was measured to calculate the resulting diffusion coefficients. Nanoindentation and energy dispersive X-ray spectroscopy measurements were carried out to determine the element distribution and estimate properties within the joining zone. The proposed numerical model for the calculation of the resulting intermetallic phase seam width was implemented into a finite element (FE) software using a user-subroutine and validated by experimental results. Using the subroutine, a numerical prediction of the resulting intermetallic phase thicknesses is possible during the tool design, which can be exploited to avoid the weakening of the component strength due to formation of wide intermetallic phase seams. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Dimer formation of GdDO3A-arylsulfonamide complexes causes loss of pH-dependency of relaxivity

[Abstract] Gadolinium(III) complexes with pH-dependent relaxivities have been proposed as responsive magnetic resonance imaging (MRI) contrast agents (CA) for mapping of pH value in living subjects. The latter is clinically relevant because hypoxia-induced reduction of interstitial pH (acidosis) is a hallmark of tumor progression and resistance against chemotherapy. In order to obtain versatile building blocks for integration of a pH-responsive MRI-CA functionality into larger molecular assemblies, such as bioconjugates, micelles or nanoparticles, we equipped the structural motif GdDO3A-ethylene(arylsulfonic acid) with additional carboxylic acid moieties in the aromatic para-position. Two novel compounds were characterized concerning their pH-dependent relaxivity as well as by 17O NMR and 1H NMRD, augmented by determination of luminescence lifetimes of the respective Eu(III) complexes and structural modelling by density functional theory (TPSSh/LCRECP/6-31G(d)). Unexpected involvement of the peripheral carboxylates into metal ion complexation effected self-assembly of the lanthanide(III) complexes, resulting in dimeric species comprising two lanthanide ions, two symmetrically bridging ligands, and one coordinated water molecule per Gd(III) (q = 1). These structures are stable even at low concentrations and in presence of competing anions like phosphate. The pH-sensitive sulfonamide moieties are not involved into Gd(III) coordination, resulting in virtually constant relaxivities of r1 = 6.7 mM−1 s−1 (298 K, 20 MHz) over the biologically relevant pH range (4 to 9). Since further functionalisation on the peripheral carboxylates would effectively inhibit dimer formation, the compounds are nonetheless suited for the initially envisaged field of application.Lorenzo Tei sincerely acknowledges the Alexander von Humboldt Foundation (AvH) for a Research fellowship. The authors also thank Dr. Claudio Cassino for the help in acquisition of 1H NMR DOSY spectra, and the Deutsche Forschungsgemeinschaft (CRC 824) for suppor

A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version

[99mTc]Tc-PentixaTec: development, extensive pre-clinical evaluation, and first human experience

Purpose The clinical success non-invasive imaging of CXCR4 expression using [(68) Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of Tc-99m-labeled cyclic pentapeptides based on the PentixaFor scaffold.Methods Six mas(3)-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa(3) linkers (L1-L6) as well as the corresponding HYNIC- and N-4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC(50)inv) were carried out using Jurkat T cell lymphoma cells and [I-125]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [Tc-99m]Tc-N-4-L6-CPCR4 ([Tc-99m]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [Tc-99m]Tc-N-4-L6-CPCR4 SPECT/planar imaging with individual dosimetry.Results Of the six mas(3)-conjugated peptides, mas(3)-L6-CPCR4 (mas(3)-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 & PLUSMN; 1.3 nM). Conjugation with N-4 (N-4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 & PLUSMN; 0.1 nM. [Tc-99m]Tc-N-4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 & PLUSMN; 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [Tc-99m]Tc-N-4-L6-CPCR4 (termed [Tc-99m]Tc-PentixaTec) was selected for first-in-human application. [Tc-99m]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1-3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging.Conclusion The successive optimization of the amino acid composition of the linker structure and the N-terminal Tc-99m-labeling strategies (mas(3) vs HYNIC vs N-4) has provided [Tc-99m]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use