[68Ga]Ga-NODAGA-TriGalactan, a low molecular weight tracer for the non-invasive imaging of the functional liver reserve.

BACKGROUND: Determination of the functional liver mass is important in a variety of clinical settings including liver surgery and transplantation. [99mTc]Tc-diethylenetriamine-pentaacetic acid galactosyl human serum albumin (99mTc-GSA) is a radiotracer targeting the asialoglycoprotein receptor (ASGR) and is routinely used in Japan for this purpose. Here we describe the development and evaluation of [68Ga]Ga-NODAGA-TriGalactan a low molecular weight PET-tracer targeting this structure. RESULTS: For synthesis TRIS as branching unit and NODAGA as chelator for labelling with [68Ga]Ga are included. Three galactose moieties are conjugated via a click chemistry approach resulting in the desired labelling precursor.68Ga-labelling could be accomplished in high radiochemical yield and purity. [68Ga]Ga-NODAGA-TriGalactan is very hydrophilic and revealed high plasma stability and low plasma protein binding. Fluorescence imaging showed binding on ASGR-positive organoids and the IC50-value was in the nanomolar range. Most importantly, both biodistribution as well as animal imaging studies using normal mice demonstrated high liver uptake with rapid elimination from all other organs leading to even higher liver-to-background ratios as found for 99mTc-GSA. CONCLUSION: [68Ga]Ga-NODAGA-TriGalactan shows high in vitro stability and selectively binds to the ASGR allowing imaging of the functional liver mass with high contrast. Thus, our first generation compound resulted already in an alternative to 99mTc-GSA for imaging the functional liver reserve and might allow the broader use of this imaging technique.

Development of Cytotoxic GW7604-Zeise's Salt Conjugates as Multitarget Compounds with Selectivity for Estrogen Receptor- Positive Tumor Cells.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a carrier was esterified with alkenols of various lengths and coordinated through the ethylene moiety to PtCl3, similar to Zeise's salt (K[PtCl3(C2H4)]). The resulting GW7604-Alk-PtCl3 complexes (Alk = Prop, But, Pent, Hex) degraded in aqueous solution only by exchange of the chlorido ligands. For example, GW7604-Pent-PtCl3 coordinated the amino acid alanine in the cell culture medium, bound the isolated nucleotide 5'-GMP, and interacted with the DNA (empty plasmid pSport1). It accumulated in estrogen receptor (ER)-positive MCF-7 cells primarily via cytosolic vesicles, while it was only marginally taken up in ER-negative SKBr3 cells. Accordingly, GW7604-Pent-PtCl3 and related complexes were inactive in SKBr3 cells. GW7604-Pent-PtCl3 showed high affinity to ERα and ERß without mediating agonistic or ER downregulating properties. GW7604-Alk ligands also increased the cyclooxygenase (COX)-2 inhibitory potency of the complexes. In contrast to Zeise's salt, the GW7604-Alk-PtCl3 complexes inhibited COX-1 and COX-2 to the same extent.

Development of heterodimeric estrogen receptor alpha antagonists to target simultaneously the ligand and coactivator binding site.

One-third of breast cancer patients will develop recurrent cancer within 15 years of endocrine treatment. Notably, tumor growth in a hormone-refractory state still relies on the interaction between estrogen receptor alpha (ERα) and upregulated coactivators. Herein, we suggest that simultaneous targeting of the primary ligand binding site (LBS) and the coactivator binding site (CABS) at ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. We synthesized two series of compounds that connect the LBS-binder (E)-3-{4-[8-fluoro-4-(4-hydroxyphenyl)-2,3-dihydrobenzo[b]oxepin-5-yl]phenyl}acrylic acid 8 with the coactivator binding site inhibitors (CBIs) 4,6-bis(isobutyl(methyl)amino)pyrimidine or 3-(5-methoxy-1H-benzo[d]imidazol-2-yl)propanoic acid via covalent linkage. The most active benzoxepine-pyrimidine conjugate 31 showed strong inhibition of estradiol-induced transactivation (IC50 = 18.2 nM (ERα) and 61.7 nM (ERß)) in a luciferase reporter gene assay as well as high antiproliferative effects in MCF-7 (IC50 = 65.9 nM) and tamoxifen-resistant MCF-7/TamR (IC50 = 88.9 nM) breast cancer cells. All heterodimers exhibited two- to sevenfold higher antagonism at ERα (compared with ERß) and were superior to the acrylic acid precursor 8 in terms of ER antagonism and antiproliferative activity. It was demonstrated on the example of 31 that the compounds did not influence the ERα content in MCF-7 cells and therefore act as pure antiestrogens without downregulating potency. Possible interactions of the CBI at the receptor surface, which enhanced the biological activities, were evaluated using molecular docking studies.

Synthesis, characterization and evaluation of hyaluronic acid-based polymers for nasal delivery.

Epidemiological research has found that between 5 and 12 percent of the population suffers from chronic rhinosinusitis. Patients are dealing with local side effects such as nasal dryness, sporadic sneezing, and nasal pain in addition to the inflammation. The aim of this study was to synthesize a polymer based on hyaluronic acid in order to provide lubrication combined with a ligand leading to a covalent binding on the nasal mucosa. Hyaluronic acid (HA) was modified with L-cysteine ethyl ester hydrochloride (CYS) via amid bond formation. Ellman's assay, together with spectroscopic techniques like IR and 1H NMR, confirmed that HACys had been successfully synthesized. It was demonstrated that HACys is safe for administration on the nasal mucosa. The mucoadhesive potential was determined by 3.26-fold with the rotating cylinder assay and 1.4-fold in terms of bioadhesive examination, respectively. Further, the stability of the modified polymer was improved by 7.6-fold compared to the unmodified polymer. Spraying the formulation on the nasal mucosa, the residence time of a model drug was 1.74-fold prolonged at the site of action compared to unmodified polymer. In light of these findings, modified hyaluronic acid (HACys) displayed compelling properties such as lubricity, targeted application, long-lasting effect, and safety and therefore could be an excellent candidate for nasal application.

Charge converting nanostructured lipid carriers containing a cell-penetrating peptide for enhanced cellular uptake.

HYPOTHESIS: The aim of this study was the development of nanostructured lipid carriers (NLCs) decorated with a polycationic cell-penetrating peptide (CPP). A coating with polyphosphates (PP) enables charge conversion at target cells being triggered by the membrane bound enzyme intestinal alkaline phosphatase (IAP). EXPERIMENTS: The CPP, stearyl-nona-L-arginine (R9SA) was obtained by solid phase synthesis. Formed nanocarriers were characterized regarding size, polydispersity index, zeta potential and charge conversion in the presence of IAP and on Caco-2 cells. The BCS class IV drug saquinavir (SQV) was loaded into NLCs in different concentrations. Mucus diffusion ability of the NLCs was evaluated by the rotating tube method. Furthermore, cellular uptake was evaluated on Caco-2 cells and endosomal escape properties were investigated using erythrocytes. FINDINGS: All NLCs were obtained in a size range between 146 nm and 152 nm and a polydispersity index of 0.2. Incubation of PP coated PP-R9SA-NLCs with IAP led to a charge conversion from -41.8 mV to 6.4 mV (Δ48.2 mV). After four hours of incubation with IAP, phosphate release reached a plateau, indicating a faster polyphosphate cleavage than on Caco-2. Drug load and encapsulation efficiency of SQV was obtained up to 80.6% and 46.5 µg/mg. Mucus diffusion was increasing in the following rank order: R9SA-NLCs < blank NLCs < PP-R9SA-NLCs. R9SA-NLCs and PP-R9SA-NLCs increased the cellular uptake 15.6- and 13.2-fold, respectively, compared to the control NLCs. Erythrocytes interaction study revealed enhanced endosomal escape properties for R9SA-NLCs and PP-R9SA-NLCs when incubated with IAP.

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177.

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo.