Radiolabelling and preliminary evaluation of 68Ga-tetrapyrrole derivatives as potential tracers for PET.

Tetrapyrroles are multisided natural products which are of relevance in clinical medicine. Owing to their specific accumulation in tumour tissue, porphyrins, metalloporphyrins and chlorins have been used as in photodynamic therapy and optical imaging. Moreover, their specific uptake into inflammatory atheromatous plaques via LDL endocytosis has been reported. The present study is concerned with the synthesis of (68)Ga labelled porphyrin derivatives and an in vitro assessment of the utility of radiotracers in positron emission tomography. A set of five porphyrin derivatives were labelled using (68)Ga from a commercially obtained radionuclide generator. Dedicated post-processing of the generator eluate was conducted to allow for labelling in aqueous media and also under anhydrous conditions. Challenge studies and incubation in human serum confirmed the stability of the tracers. Plasma protein binding was investigated in order to confirm the presence of freely diffusible radioligand in plasma. A preliminary microPET study in a tumour-bearing rat resulted in a clear visualisation of the tumour.

Combination of phage display and molecular grafting generates highly specific tumor-targeting miniproteins.

Frankenstein's peptide: the grafting of the binding domain from miniprotein Min-23 into the sunflower trypsin inhibitor (SFTI-I) peptide scaffold preserved its in vitro and in vivo binding specificity and proteolytic stability. The combination of these peptides was shown to be tumor-specific with a good binding affinity for delta-like ligand 4 (Dll4) protein. The use of SFTI-I as a peptide scaffold is ideal for hit-to-lead development.

A dimerized urea-based inhibitor of the prostate-specific membrane antigen for 68Ga-PET imaging of prostate cancer.

BACKGROUND: Alternative positron-emission tomography (PET) probes like labeled inhibitors of the prostate-specific membrane antigen (PSMA) are of emerging clinical impact as they show the ability to image small lesions of recurrent prostate cancer. Here, the dimerization of the pharmacophore Glu-ureido-Lys via the 68Ga chelator N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was investigated to further improve the binding characteristics and pharmacokinetics. METHODS: The peptidomimetic structures were synthesized by solid-phase chemistry, and the resulting products were coupled with the respective 2,3,5,6-tetrafluorophenol esters of HBED-CC to form the monomeric reference and the dimeric Glu-ureido-Lys derivative. The binding properties were analyzed in competitive binding, internalization, and cell surface retention experiments. PET images and biodistribution data were obtained 1 h after injection in BALB/c nu/nu mice bearing LNCaP tumor xenografts. RESULTS: Cell binding data revealed significant better binding properties of the dimer (IC50 = 3.9 ± 1.8 nM; IC50 (monomer) = 12.1 ± 2.1 nM). The inhibition potency investigated by the enzyme-based NAALADase assay confirmed these results. Specific internalization in LNCaP cells was demonstrated for both, the monomer and dimer. As shown by efflux measurements, the dimeric compound was more effectively retained on the cell surface, resulting in advanced in vivo properties (T/BMonomer = 9.2; T/BDimer = 26.5). CONCLUSIONS: The dimeric [68Ga]7 is a promising imaging agent for PSMA-expressing tumors as it shows higher tumor uptake while observing more favorable background clearance. As compared to the respective monomer, the higher affinity and prolonged tumor retention additionally represent promising features and warrant further evaluation regarding 68Ga-PET imaging of PSMA expression.

Engineering and functionalization of the disulfide-constrained miniprotein min-23 as a scaffold for diagnostic application.

Miniproteins are scaffolds for the development of alternative non-immunoglobin binding agents for medical applications. This peptide format features high tolerance to sequence mutagenesis, excellent proteolytic stability, and fast blood pool clearance. Herein we present the total chemical synthesis of the disulfide-constrained scaffold Min-23 and its functionalization for in vitro and in vivo application. Optimized solid-phase peptide chemistry and oxidative folding strategies were developed to engineer this miniprotein with native-like disulfide configuration. High levels of serum stability and proteolytic resistance, as well as a beneficial pharmacokinetic profile for diagnostic imaging, were determined by using radiolabeling techniques such as positron emission tomography. The reported achievements highlight Min-23 as a promising scaffold for the development of novel recognition molecules for medical application.

Miniproteins as phage display-scaffolds for clinical applications.

Miniproteins are currently developed as alternative, non-immunoglobin proteins for the generation of novel binding motifs. Miniproteins are rigid scaffolds that are stabilised by alpha-helices, beta-sheets and disulfide-constrained secondary structural elements. They are tolerant to multiple amino acid substitutions, which allow for the integration of a randomised affinity function into the stably folded framework. These properties classify miniprotein scaffolds as promising tools for lead structure generation using phage display technologies. Owing to their high enzymatic resistance and structural stability, miniproteins are ideal templates to display binding epitopes for medical applications in vivo. This review summarises the characteristics and the engineering of miniproteins as a novel class of scaffolds to generate of alternative binding agents using phage display screening. Moreover, recent developments for therapeutic and especially diagnostic applications of miniproteins are reviewed.

Challenges in optimizing a prostate carcinoma binding peptide, identified through the phage display technology.

The transfer of peptides identified through the phage display technology to clinical applications is difficult. Major drawbacks are the metabolic degradation and label instability. The aim of our work is the optimization of DUP-1, a peptide which was identified by phage display to specifically target human prostate carcinoma. To investigate the influence of chelate conjugation, DOTA was coupled to DUP-1 and labeling was performed with ¹¹¹In. To improve serum stability cyclization of DUP-1 and targeted D-amino acid substitution were carried out. Alanine scanning was performed for identification of the binding site and based on the results peptide fragments were chemically synthesized. The properties of modified ligands were investigated in in vitro binding and competition assays. In vivo biodistribution studies were carried out in mice, carrying human prostate tumors subcutaneously. DOTA conjugation resulted in different cellular binding kinetics, rapid in vivo renal clearance and increased tumor-to-organ ratios. Cyclization and D-amino acid substitution increased the metabolic stability but led to binding affinity decrease. Fragment investigation indicated that the sequence NRAQDY might be significant for target-binding. Our results demonstrate challenges in optimizing peptides, identified through phage display libraries, and show that careful investigation of modified derivatives is necessary in order to improve their characteristics.

Endoradiotherapy in cancer treatment--basic concepts and future trends.

Endoradiotherapy represents an alternative therapeutic method in cancer treatment with advantageous features compared to chemotherapy and radiation therapy. Intelligent dose delivery concepts using small drugs, peptides or antibodies as radionuclide carriers enable the verification of a selective accumulation in the tumour lesion and to reduce radiation toxicity for the peripheral organs. The development of endoradiotherapeutic agents, especially chelator-conjugated biomolecules, for example ibritumomab tiuxetan or DOTATOC, gains importance due to the stable complexation of versatile radiometals, such as (90)Y or (177)Lu. The rational design of novel target binding sides and their grafting into a drug scaffold is a highly promising strategy, which may promote further implication in endoradiotherapy. This review highlights the basic concepts of endoradiotherapy and discusses the potential of targeted therapy and the properties of energy-rich particles emitted by radionuclides for tumour therapy.

[(68)Ga]Ga-DO(2)A-(OBu-l-tyr)(2): synthesis, (68)Ga-radiolabeling and in vitro studies of a novel (68)Ga-DO(2)A-tyrosine conjugate as potential tumor tracer for PET.

The synthesis, (68)Ga-labeling and in vitro study of the novel tyrosine chelate derivative [(68)Ga]Ga-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid-4,10-di-(O-butyl)-l-tyrosine ([(68)Ga]Ga-DO(2)A-(OBu-l-tyr)(2)) as a potential tracer for imaging tumor metabolism by positron emission tomography (PET) is presented. This approach combines the biological amino acid transporter targeting properties of l-tyrosine with the outstanding availability of (68)Ga(III) via the (68)Ge/(68)Ga generator. In vitro studies utilizing the F98-glioblastoma cell line revealed specific uptake of [(68)Ga]Ga-DO2A-(OBu-l-tyr)(2) that was comparable to that of the reference O-(2-[(18)F]fluoroethyl)-l-tyrosine (FET). These promising results indicate a high potential of [(68)Ga]Ga-DO(2)A-(OBu-l-tyr)(2) for molecular imaging of tumor-driven amino acid uptake by PET.