EGFR-targeting peptide-coupled platinum(IV) complexes

The high mortality rate of lung cancer patients and the frequent occurrence of side effects during cancer therapy demonstrate the need for more selective and targeted drugs. An important and well-established target for lung cancer treatment is the occasionally mutated epidermal growth factor receptor (EGFR). As platinum(II) drugs are still the most important therapeutics against lung cancer, we synthesized in this study the first platinum(IV) complexes coupled to the EGFR-targeting peptide LARLLT (and the shuffled RTALLL as reference). Notably, HPLC–MS measurements revealed two different peaks with the same molecular mass, which turned out to be a transcyclization reaction in the linker between maleimide and the coupled cysteine moiety. With regard to the EGFR specificity, subsequent biological investigations (3-day viability, 14-day clonogenic assays and platinum uptake) on four different cell lines with different verified EGFR expression levels were performed. Unexpectedly, the results showed neither an enhanced activity nor an EGFR expression-dependent uptake of our new compounds. Consequently, fluorophore-coupled peptides were synthesized to re-evaluate the targeting ability of LARLLT itself. However, also with these molecules, flow cytometry measurements showed no correlation of drug uptake with the EGFR expression levels. Taken together, we successfully synthesized the first platinum(IV) complexes coupled to an EGFR-targeting peptide; however, the biological investigations revealed that LARLLT is not an appropriate peptide for enhancing the specific uptake of small-molecule drugs into EGFR-overexpressing cancer cells.© The Author(s) 201

Comparison of metabolic pathways of different α-N-heterocyclic thiosemicarbazones

Clinical failure of novel drugs is often related to their rapid metabolism and excretion. This highlights the importance of elucidation of their pharmacokinetic profile already at the preclinical stage of drug development. Triapine, the most prominent representative of α-N-heterocyclic thiosemicarbazones, was investigated in more than 30 clinical phase I/II trials, but the results against solid tumors were disappointing. Recent investigations from our group suggested that this is, at least partially, based on the fast metabolism and excretion. In order to establish more detailed structure/activity/metabolism relationships, herein a panel of 10 different Triapine derivatives was investigated for their metabolic pathways. From the biological point of view, the panel consists of terminally dimethylated thiosemicarbazones with nanomolar IC50 values, derivatives with micromolar cytotoxicities comparable to Triapine and a completely inactive representative. To study the oxidative metabolism, a purely instrumental approach based on electrochemistry/mass spectrometry was applied and the results were compared to the data obtained from microsomal incubations. Overall, the investigated thiosemicarbazones underwent the phase I metabolic reactions dehydrogenation, hydroxylation, oxidative desulfuration (to semicarbazone and amidrazone) and demethylation. Notably, dehydrogenation resulted in a ring-closure reaction with formation of thiadiazoles. Although strong differences between the metabolic pathways of the different thiosemicarbazones were observed, they could not be directly correlated to their cytotoxicities. Finally, the metabolic pathways for the most cytotoxic compound were elucidated also in tissues collected from drug-treated mice, confirming the data obtained by electrochemical oxidation and microsomes. In addition, the in vivo experiments revealed a very fast metabolism and excretion of the compound.© The Author(s) 201

Understanding the metabolism of the anticancer drug Triapine: electrochemical oxidation, microsomal incubation and in vivo analysis using LC-HRMS

α-N-Heterocyclic thiosemicarbazones are among the most promising ribonucleotide reductase inhibitors identified so far. Triapine, the most prominent representative of this class of substances, has been investigated in multiple phase I and II clinical trials. With regard to clinical practice, Triapine showed activity against hematological diseases, but ineffectiveness against a variety of solid tumors. However, the reasons are still vague and the amount of ADME (absorption, distribution, metabolism and excretion) data for Triapine available in the literature is very limited. Therefore, different analytical tools were used to investigate the metabolism of Triapine including electrochemical oxidations, liver microsomes and in vivo samples from mice. The main metabolic reactions, observed by all three methods, were dehydrogenation and hydroxylations, confirming that electrochemistry, as a purely instrumental approach, can be applied for the simulation of metabolic pathways. The dehydrogenated metabolite M1 was identified as a thiadiazole ring-closed oxidation product of Triapine. From a biological point of view, M1, as a key metabolite, is of interest since the crucial chemical property of α-N-heterocyclic thiosemicarbazones to bind metal ions is lost and cytotoxicity studies showed no anticancer activity of M1. The in vivo data of the urine samples revealed very high levels of the metabolites and Triapine itself already 15 min after treatment. This clearly indicates that Triapine is rapidly metabolised and excreted, which represents an important step forward to understand the possible reason for the inefficiency of Triapine against solid tumors

Ruthenium–arene complexes bearing naphthyl-substituted 1,3-dioxoindan-2-carboxamides ligands for G-quadruplex DNA recognition

Quadruplex nucleic acids – DNA/RNA secondary structures formed in guanine rich sequences – proved to have key roles in the biology of cancers and, as such, in recent years they emerged as promising targets for small molecules. Many reports demonstrated that metal complexes can effectively stabilize quadruplex structures, promoting telomerase inhibition, downregulation of the expression of cancer-related genes and ultimately cancer cell death. Although extensively explored as anticancer agents, studies on the ability of ruthenium arene complexes to interact with quadruplex nucleic acids are surprisingly almost unknown. Herein, we report on the synthesis and characterization of four novel Ru(II) arene complexes with 1,3-dioxoindan-2-carboxamides ligands bearing pendant naphthyl-groups designed to bind quadruplexes by both stacking and coordinating interactions. We show how improvements on the hydrolytic stability of such complexes, by substituting the chlorido leaving ligand with pyridine, have a dramatic impact on their interaction with quadruplexes and on their cytotoxicity against ovarian cancer cells

Synthesis and in vivo anticancer evaluation of poly(organo)phosphazene-based metallodrug conjugates

Within this work we aimed to improve the pharmacodynamics and toxicity profile of organoruthenium and -rhodium complexes which had previously been found to be highly potent in vitro but showed unselective activity in vivo. Different organometallic complexes were attached to a degradable poly(organo)phosphazene macromolecule, prepared via controlled polymerization techniques. The conjugation to hydrophilic polymers was designed to increase the aqueous solubility of the typically poorly soluble metal-based half-sandwich compounds with the aim of a controlled, pH-triggered release of the active metallodrug. The synthesized conjugates and their characteristics have been thoroughly studied by means of 31P NMR and UV-Vis spectroscopy, ICP-MS analyses and SEC coupled to ICP-MS. In order to assess their potential as possible anticancer drug candidates, the complexes, as well as their respective macromolecular prodrug formulations were tested against three different cancer cell lines in cell culture. Subsequently, the anticancer activity and organ distribution of the poly(organo)phosphazene drug conjugates were explored in vivo in mice bearing CT-26 colon carcinoma. Our investigations revealed a beneficial influence of this macromolecular prodrug by a significant reduction of adverse effects compared to the free metallodrugs

An albumin-based tumor-targeted oxaliplatin prodrug with distinctly improved anticancer activity in vivo

The design of targeted platinum(IV) prodrugs is a very promising approach to enhance the low selectivity of platinum(II) drugs towards cancerous tissue in order to reduce the impact on healthy tissue and, consequently, the often severe side-effects. Herein, we report a set of mono-functionalized cis- and oxaliplatin-based platinum(IV) complexes bearing a maleimide moiety, which allows selective binding to serum albumin in the bloodstream. This leads not only to a prolonged plasma half-life by avoidance of fast renal clearance, but also to preferential accumulation of the drug in the tumor tissue due to the EPR-effect. Additionally, analogous succinimide-functionalized derivatives were prepared to verify the influence of the maleimide moiety. First experiments showed that all the maleimide compounds are stable and also possess good albumin-binding properties in whole serum. Further analytical studies on in vivo samples proved the highly increased plasma half-life, as well as tumor accumulation of the maleimide-functionalized substances. In vivo antitumor experiments with CT-26-bearing mice showed that, in contrast to the cisplatin derivatives, the oxaliplatin-based complexes had exceptionally better activity than the free drug resulting in the cure of the majority of treated mice. Subsequent analysis suggested that a distinctly faster reduction as well as reduced tumor accumulation of the cisplatin derivative might explain the worse performance compared to the oxaliplatin(IV) complexes. Taken together, a novel lead platinum(IV) complex with outstanding antitumor activity is presented, which will now be further developed towards clinical phase I trials

Another step toward DNA selective targeting: NiIIand CuIIcomplexes of a Schiff base ligand able to bind gene promoter G-quadruplexes

DNA G-rich sequences are able to form four-stranded structures organized in stacked guanine tetrads. These structures, called G-quadruplexes, were found to have an important role in the regulation of oncogenes expression and became, for such a reason, appealing targets for anticancer drugs. Aiming at finding selective G-quadruplex binders, we have designed, synthesized and characterized a new water soluble Salen-like Schiff base ligand and its NiII and CuII metal complexes. UV-Vis, circular dichroism and FRET measurements indicated that the nickel complex can stabilize oncogene promoter G-quadruplexes with high selectivity, presenting no interactions with duplex DNA at all. The same compound exhibited dose-dependent cytotoxic activity in MCF-7 breast cancer cells when combined with lipofectamine as lipophilic carrier

Lysosomal Sequestration Impairs the Activity of the Preclinical FGFR Inhibitor PD173074

Knowledge of intracellular pharmacokinetics of anticancer agents is imperative for understanding drug efficacy as well as intrinsic and acquired cellular resistance mechanisms. However, the factors driving subcellular drug distribution are complex and poorly understood. Here, we describe for the first time the intrinsic fluorescence properties of the fibroblast growth factor receptor inhibitor PD1703074 as well as utilization of this physicochemical feature to investigate intracellular accumulation and compartmentalization of this compound in human lung cancer cells. Cell-free PD173074 fluorescence, intracellular accumulation and distribution were investigated using analytical chemistry and molecular biology approaches. Analyses on a subcellular scale revealed selective drug accumulation in lysosomes. Coincubation with inhibitors of lysosomal acidification strongly enhanced PD173074-mediated fibroblast growth factor receptor (FGFR) inhibition and cytotoxicity. In conclusion, intrinsic fluorescence enables analysis of molecular factors influencing intracellular pharmacokinetics of PD173074. Lysosome-alkalinizing agents might represent candidates for rational combination treatment, preventing cancer cell-intrinsic PD173074 resistance based on lysosomal trapping.© 2018 by the author

Electronic State of Sodium trans-[Tetrachloridobis(1H-indazole)ruthenate(III)] (NKP-1339) in Tumor, Liver and Kidney Tissue of a SW480-bearing Mouse

Ruthenium complexes are promising candidates for anticancer agents, especially NKP-1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which is on the edge to clinical applications. The anticancer mechanism seems to be tightly linked to the redox chemistry but despite progress in human clinical trials the in vivo Ru oxidation state and the coordination of Ru remains unclear. The Ru-based anticancer drug NKP-1339 was studied applying XANES (Cl K- and Ru L2,3-edges) in tumor, kidney and liver tissue of a SW480 bearing mouse. Based on coordination charge and 3D XANES plots containing a series of model compounds as well as pre-edge analysis of the ligand Cl K-edge it is suggested that NKP-1339 remains in its +III oxidation state after 24 hours and at least one of the four chlorido ligands remain covalently bound to the Ru ion showing a biotransformation from RuIIIN2Cl4 to RuIIIClx(N/O)6−x (X = 1 or 2).© The Author(s) 201

Serum-binding properties of isosteric ruthenium and osmium anticancer agents elucidated by SEC–ICP–MS

Size-exclusion chromatography–inductively coupled plasma–mass spectrometry (SEC–ICP–MS) was used to study the serum-binding preferences of two metallodrugs with anticancer activities in vivo, namely the organoruthenium compound plecstatin-1 and its isosteric osmium analog. The complexes were administered intraperitoneally into mice bearing a CT-26 tumor. Comparing the total metal content of mouse whole blood and serum underlined that the metallodrugs are mainly located in serum and not in the cellular fraction of the blood samples. In mouse serum, both compounds were not only found to bind extensively to the serum albumin/transferrin fraction but also to immunoglobulins. Free drug was not observed in any of the samples indicating rapid protein binding of the metallodrugs. These findings were validated by spiking human serum with the respective compounds ex vivo. An NCI-60 screen is reported for the osmium analog, which revealed a relative selectivity for cancer cell lines of the ovary and the central nervous system with respect to plecstatin-1. Finally, a COMPARE 170 analysis revealed disruption of DNA synthesis as a possible treatment effect of the osmium-based drug candidate.© The Author(s) 201