A Comparative Study on the Complexation of the Anticancer Iron Chelator VLX600 with Essential Metal Ions.

As cancer cells exhibit an increased uptake of iron, targeting the interaction with iron has become a straightforward strategy in the fight against cancer. This work comprehensively characterizes the chemical properties of 6-methyl-3-{(2E)-2-[1-(2-pyridinyl)ethylidene]hydrazino}-5H-[1,2,4]triazino[5,6-b]indole (VLX600), a clinically investigated iron chelator, in solution. Its protonation processes, lipophilicity, and membrane permeability as well as its complexation with essential metal ions were investigated using UV-visible, electron paramagnetic resonance, and NMR spectroscopic and computational methods. Formation constants revealed the following order of metal binding affinity at pH 7.4: Cu(II) > Fe(II) > Zn(II). The structures of VLX600 (denoted as HL) and the coordination modes in its metal complexes [Cu(II)(LH)Cl2], [Cu(II)(L)(CH3OH)Cl], [Zn(II)(LH)Cl2], and [Fe(II)(LH)2](NO3)2 were elucidated by single-crystal X-ray diffraction. Redox properties of the iron complexes characterized by cyclic voltammetry showed strong preference of VLX600 toward Fe(II) over Fe(III). In vitro cytotoxicity of VLX600 was determined in six different human cancer cell lines, with IC50 values ranging from 0.039 to 0.51 µM. Premixing VLX600 with Fe(III), Zn(II), and Cu(II) salts in stoichiometric ratios had a rather little effect overall, thus neither potentiating nor abolishing cytotoxicity. Together, although clinically investigated as an iron chelator, this is the first comprehensive solution study of VLX600 and its interaction with physiologically essential metal ions.

Degradable Bottlebrush Polypeptides and the Impact of their Architecture on Cell Uptake, Pharmacokinetics, and Biodistribution In Vivo.

Bottlebrush polymers are highly promising as unimolecular nanomedicines due to their unique control over the critical parameters of size, shape and chemical function. However, since they are prepared from biopersistent carbon backbones, most known bottlebrush polymers are non-degradable and thus unsuitable for systemic therapeutic administration. Herein, we report the design and synthesis of novel poly(organo)phosphazene-g-poly(α-glutamate) (PPz-g-PGA) bottlebrush polymers with exceptional control over their structure and molecular dimensions (Dh ≈ 15-50 nm). These single macromolecules show outstanding aqueous solubility, ultra-high multivalency and biodegradability, making them ideal as nanomedicines. While well-established in polymer therapeutics, it has hitherto not been possible to prepare defined single macromolecules of PGA in these nanosized dimensions. A direct correlation was observed between the macromolecular dimensions of the bottlebrush polymers and their intracellular uptake in CT26 colon cancer cells. Furthermore, the bottlebrush macromolecular structure visibly enhanced the pharmacokinetics by reducing renal clearance and extending plasma half-lives. Real-time analysis of the biodistribution dynamics showed architecture-driven organ distribution and enhanced tumor accumulation. This work, therefore, introduces a robust, controlled synthesis route to bottlebrush polypeptides, overcoming limitations of current polymer-based nanomedicines and, in doing so, offers valuable insights into the influence of architecture on the in vivo performance of nanomedicines.

Insertion of (Bioactive) Equatorial Ligands into Platinum(IV) Complexes.

Platinum(IV) prodrugs are highly interesting alternatives to platinum(II) anticancer therapeutics due to their increased tumor selectivity and reduced side effects. In contrast to the established theory, we recently observed that the equatorial ligand(s) of e.g. oxaliplatin(IV) complexes can be hydrolyzed with formation of [(DACH)Pt(OHeq )2 (OAcax )2 ]. In the work presented here, we investigated the reactivity and synthetic usability of this complex to be exploited as a precursor for the development of novel platinum(IV) complexes, not able to be synthesized by conventional protocols. Indeed, we could substitute the equatorial hydroxido ligand(s) e.g. by one or two monodentate biotin ligands (which would be oxidized under standard methods). The formed complexes turned out to be very stable with slow ligand release after reduction, ideal for long-circulating tumor-targeting strategies. Therefore, two platinum(IV) complexes with equatorial maleimides, capable of exploiting serum albumin as a natural nanocarrier, were synthesized as well. The complexes showed massively prolonged plasma half-life and distinctly improved anticancer activity in vivo compared to oxaliplatin. Taken together, the newly developed synthetic platform allows the simple and specific insertion of equatorial ligands into platinum(IV) complexes. This will enable the attachment of three different (bioactive) moieties generating targeted triple-action platinum(IV) prodrugs within one single platinum complex.

Copper-Catalyzed Glutathione Oxidation is Accelerated by the Anticancer Thiosemicarbazone Dp44mT and Further Boosted at Lower pH.

Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, CuIn(GS)m clusters are formed, in which CuI is very slowly reoxidized by O2 at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by CuII was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that CuII-Dp44mT catalyzes GSH oxidation faster than CuII alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favors the rate-limiting reduction step without subsequent dissociation of the CuI intermediate. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of CuII-Dp44mT.

Oxoplatin-Based Pt(IV) Lipoate Complexes and Their Biological Activity.

α-Lipoic acid, known for its anti-inflammatory and antioxidant activity, represents a promising ligand for Pt(IV) prodrugs. Three new Pt(IV) lipoate complexes were synthesized and characterized by NMR spectroscopy (1 H, 13 C, 195 Pt), mass spectrometry and elemental analysis. Due to the low solubility of the complex containing two axial lipoate ligands, further experiments to examine the biological activity were performed with two Pt(IV) complexes containing just one axial lipoate ligand. Both complexes exhibit anticancer activity and produce reactive oxygen species (ROS) in the cell lines tested. Especially, the monosubstituted complex can be reduced by ascorbic acid and forms adducts with 9-methylguanine (9MeG), which is favorable for the formation of DNA-crosslinks in the cells.

Metal Drugs and the Anticancer Immune Response.

The immune system deploys a multitude of innate and adaptive mechanisms not only to ward off pathogens but also to prevent malignant transformation ("immune surveillance"). Hence, a clinically apparent tumor already reflects selection for those malignant cell clones capable of evading immune recognition ("immune evasion"). Metal drugs, besides their well-investigated cytotoxic anticancer effects, massively interact with the cancer-immune interface and can reverse important aspects of immune evasion. This topic has recently gained intense attention based on combination approaches with anticancer immunotherapy (e.g., immune checkpoint inhibitors), a strategy recently delivering first exciting results in clinical settings. This review summarizes the promising but still extremely fragmentary knowledge on the interplay of metal drugs with the fidelity of anticancer immune responses but also their role in adverse effects. It highlights that, at least in some cases, metal drugs can induce long-lasting anticancer immune responses. Important steps in this process comprise altered visibility and susceptibility of cancer cells toward innate and adaptive immunity, as well as direct impacts on immune cell populations and the tumor microenvironment. On the basis of the gathered information, we suggest initiating joint multidisciplinary programs to implement comprehensive immune analyses into strategies to develop novel and smart anticancer metal compounds.

Lipid droplet-mediated scavenging as novel intrinsic and adaptive resistance factor against the multikinase inhibitor ponatinib.

Ponatinib is a small molecule multi-tyrosine kinase inhibitor clinically approved for anticancer therapy. Molecular mechanisms by which cancer cells develop resistance against ponatinib are currently poorly understood. Likewise, intracellular drug dynamics, as well as potential microenvironmental factors affecting the activity of this compound are unknown. Cell/molecular biological and analytical chemistry methods were applied to investigate uptake kinetics/subcellular distribution, the role of lipid droplets (LDs) and lipoid microenvironment compartments in responsiveness of FGFR1-driven lung cancer cells toward ponatinib. Selection of lung cancer cells for acquired ponatinib resistance resulted in elevated intracellular lipid levels. Uncovering intrinsic ponatinib fluorescence enabled dissection of drug uptake/retention kinetics in vitro as well as in mouse tissue cryosections, and revealed selective drug accumulation in LDs of cancer cells. Pharmacological LD upmodulation or downmodulation indicated that the extent of LD formation and consequent ponatinib incorporation negatively correlated with anticancer drug efficacy. Co-culturing with adipocytes decreased ponatinib levels and fostered survival of cancer cells. Ponatinib-selected cancer cells exhibited increased LD levels and enhanced ponatinib deposition into this organelle. Our findings demonstrate intracellular deposition of the clinically approved anticancer compound ponatinib into LDs. Furthermore, increased LD biogenesis was identified as adaptive cancer cell-defense mechanism via direct drug scavenging. Together, this suggests that LDs represent an underestimated organelle influencing intracellular pharmacokinetics and activity of anticancer tyrosine kinase inhibitors. Targeting LD integrity might constitute a strategy to enhance the activity not only of ponatinib, but also other clinically approved, lipophilic anticancer therapeutics.

Improving the Stability of Maleimide-Thiol Conjugation for Drug Targeting.

Maleimides are essential compounds for drug conjugation reactions via thiols to antibodies, peptides and other targeting units. However, one main drawback is the occurrence of thiol exchange reactions with, for example, glutathione resulting in loss of the targeting ability. A new strategy to overcome such retro-Michael exchange processes of maleimide-thiol conjugates by stabilization of the thiosuccinimide via a transcyclization reaction is presented. This reaction enables the straightforward synthesis of stable maleimide-thiol adducts essential in drug-conjugation applications.

Improving the Stability of EGFR Inhibitor Cobalt(III) Prodrugs.

Although tyrosine kinase inhibitors (TKIs) have revolutionized cancer therapy in the past two decades, severe drawbacks such as strong adverse effects and drug resistance limit their clinical application. Prodrugs represent a valuable approach to overcoming these disadvantages by administration of an inactive drug with tumor-specific activation. We have recently shown that hypoxic prodrug activation is a promising strategy for a cobalt(III) complex bearing a TKI of the epidermal growth factor receptor (EGFR). The aim of this study was the optimization of the physicochemical properties and enhancement of the stability of this compound class. Therefore, we synthesized a series of novel derivatives to investigate the influence of the electron-donating properties of methyl substituents at the metal-chelating moiety of the EGFR inhibitor and/or the ancillary acetylacetonate (acac) ligand. To understand the effect of the different methylations on the redox properties, the newly synthesized complexes were analyzed by cyclic voltammetry and their behavior was studied in the presence of natural low-molecular weight reducing agents. Furthermore, it was proven that reduction to cobalt(II) resulted in a lower stability of the complexes and subsequent release of the coordinated TKI ligand. Moreover, the stability of the cobalt(III) prodrugs was investigated in blood serum as well as in cell culture by diverse cell and molecular biological methods. These analyses revealed that the complexes bearing the methylated acac ligand are characterized by distinctly enhanced stability. Finally, the cytotoxic activity of all new compounds was tested in cell culture under normoxic and various hypoxic conditions, and their prodrug nature could be correlated convincingly with the stability data. In summary, the performed chemical modifications resulted in new cobalt(III) prodrugs with strongly improved stabilities together with retained hypoxia-activatable properties.

Development and biological investigations of hypoxia-sensitive prodrugs of the tyrosine kinase inhibitor crizotinib.

Despite the huge success of tyrosine kinase inhibitors as anticancer agents, severe side effects are a major problem. In order to overcome this drawback, the first hypoxia-activatable 2-nitroimidazole-based prodrugs of the clinically approved ALK and c-MET inhibitor crizotinib were developed. The 2-aminopyridine functionality of crizotinib (essential for target kinase binding) was considered as ideal position for prodrug derivatization. Consequently, two different prodrugs were synthesized with the nitroimidazole unit attached to crizotinib either via carbamoylation (A) or alkylation (B) of the 2-aminopyridine moiety. The successful prodrug design could be proven by docking studies and a dramatically reduced ALK and c-MET kinase-inhibitory potential. Furthermore, the prodrugs showed high stability in serum and release of crizotinib in an enzymatic nitroreductase-based cleavage assay was observed for prodrug A. The in vitro activity of both prodrugs was investigated against ALK- and c-MET-dependent or -overexpressing cells, revealing a distinct hypoxia-dependent activation for prodrug A. Finally, inhibition of c-MET phosphorylation and cell proliferation could also be proven in vivo. In summary of the theoretical, chemical and biological studies, prodrug derivatization of the 2-aminopyridine position can be considered as a promising strategy to reduce the side effects and improve the anticancer activity of crizotinib.

Reactive Oxygen Species (ROS)-Sensitive Prodrugs of the Tyrosine Kinase Inhibitor Crizotinib.

Tyrosine kinase inhibitors revolutionized cancer therapy but still evoke strong adverse effects that can dramatically reduce patients' quality of life. One possibility to enhance drug safety is the exploitation of prodrug strategies to selectively activate a drug inside the tumor tissue. In this study, we designed a prodrug strategy for the approved c-MET, ALK, and ROS1 tyrosine kinase inhibitor crizotinib. Therefore, a boronic-acid trigger moiety was attached to the 2-aminopyridine group of crizotinib, which is a crucial position for target kinase binding. The influence of the modifications on the c-MET- and ALK-binding ability was investigated by docking studies, and the strongly reduced interactions could be confirmed by cell-free kinase inhibition assay. Furthermore, the newly synthesized compounds were tested for their activation behavior with H2O2 and their stability in cell culture medium and serum. Finally, the biological activity of the prodrugs was investigated in three cancer cell lines and revealed a good correlation between activity and intrinsic H2O2 levels of the cells for prodrug A. Furthermore, the activity of this prodrug was distinctly reduced in a non-malignant, c-MET expressing human lung fibroblast (HLF) cell line.

Synthesis and Cytotoxicity of Water-Soluble Dual- and Triple-Action Satraplatin Derivatives: Replacement of Equatorial Chlorides of Satraplatin by Acetates.

Pt(II) complexes, such as cisplatin and oxaliplatin, are in widespread use as anticancer drugs. Their use is limited by the toxic side effects and the ability of tumors to develop resistance to the drugs. A popular approach to overcome these drawbacks is to use their kinetically inert octahedral Pt(IV) derivatives that act as prodrugs. The most successful Pt(IV) complex in clinical trials to date is satraplatin, cct-[Pt(NH3)(c-hexylamine)Cl2(OAc)2], that upon cellular reduction releases the cytotoxic cis-[Pt(NH3)(c-hexylamine)Cl2]. In an attempt to obtain water-soluble and more effective cytotoxic Pt(IV) complexes, we prepared a series of dual- and triple-action satraplatin analogues, where the equatorial chlorido ligands were replaced with acetates and the axial ligands include innocent and bioactive ligands. Replacement of the chlorides with acetates enhanced the water solubility of the compounds and, with one exception, all of the compounds were very stable in buffer. In general, compounds with one or two axial hydroxido ligands were reduced by ascorbate significantly more quickly than compounds with two axial carboxylates. While replacement of the chlorides with acetates in satraplatin led to a reduction in cytotoxicity, the dual- and triple-action analogues with equatorial acetates had low- to sub-micromolar IC50 values in a panel of eight cancer cells. The triple-action compound cct-[Pt(NH3)(c-hexylamine)(OAc)2(PhB)(DCA)] was active in all cell lines, causing DNA damage that induced cell cycle inhibition and apoptosis. Its good activity against CT26 cells in vitro translated into good in vivo efficacy against the CT26 allograft, an in vivo model with intrinsic satraplatin resistance. This indicates that multiaction Pt(IV) derivatives of diamine dicarboxylates are interesting anticancer drug candidates.

Synthesis, Characterization and in†vitro Studies of a Cathepsin B-Cleavable Prodrug of the VEGFR Inhibitor Sunitinib.

Since several decades, the prodrug concept has raised considerable interest in cancer research due to its potential to overcome common problems associated with chemotherapy. However, for small-molecule tyrosine kinase inhibitors, which also cause severe side effects, hardly any strategies to generate prodrugs for therapeutic improvement have been reported so far. Here, we present the synthesis and biological investigation of a cathepsin B-cleavable prodrug of the VEGFR inhibitor sunitinib. Cell viability assays and Western blot analyses revealed, that, in contrast to the non-cathepsin B-cleavable reference compound, the prodrug shows activity comparable to the original drug sunitinib in the highly cathepsin B-expressing cell lines Caki-1 and RU-MH. Moreover, a cathepsin B cleavage assay confirmed the desired enzymatic activation of the prodrug. Together, the obtained data show that the concept of cathepsin B-cleavable prodrugs can be transferred to the class of targeted therapeutics, allowing the development of optimized tyrosine kinase inhibitors for the treatment of cancer.

A Dogma in Doubt: Hydrolysis of Equatorial Ligands of PtIV Complexes under Physiological Conditions.

Due to their high kinetic inertness and consequently reduced side reactions with biomolecules, PtIV complexes are considered to define the future of anticancer platinum drugs. The aqueous stability of a series of biscarboxylato PtIV complexes was studied under physiologically relevant conditions. Unexpectedly and in contrast to the current chemical understanding, especially oxaliplatin and satraplatin complexes underwent fast hydrolysis in equatorial position (even in cell culture medium and serum). Notably, the resulting hydrolysis products strongly differ in their reduction kinetics, a crucial parameter for the activation of PtIV drugs, which also changes the anticancer potential of the compounds in cell culture. The discovery that intact PtIV complexes can hydrolyze at equatorial position contradicts the dogma on the general kinetic inertness of PtIV compounds and needs to be considered in the screening and design for novel platinum-based anticancer drugs.

Critical assessment of different methods for quantitative measurement of metallodrug-protein associations.

Quantitative screening for potential drug-protein binding is an essential step in developing novel metal-based anticancer drugs. ICP-MS approaches are at the core of this task; however, many applications lack in the capability of large-scale high-throughput screenings and proper validation. In this work, we critically discuss the analytical figures of merit and the potential method-based quantitative differences applying four different ICP-MS strategies to ex vivo drug-serum incubations. Two candidate drugs, more specifically, two Pt(IV) complexes with known differences of binding affinity towards serum proteins were selected. The study integrated centrifugal ultrafiltration followed by flow injection analysis, turbulent flow chromatography (TFC), and size exclusion chromatography (SEC), all combined with inductively coupled plasma-mass spectrometry (ICP-MS). As a novelty, for the first time, UHPLC SEC-ICP-MS was implemented to enable rapid protein separation to be performed within a few minutes at > 90% column recovery for protein adducts and small molecules. Graphical abstract Quantitative screening for potential drug-protein binding is an essential step in developingnovel metal-based anticancer drugs.

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. Graphical abstract Structure/activity/metabolisation relationships for 10 anticancer thiosemicarbazones were established using electrochemical oxidation coupled to mass spectrometry (EC-MS) and human liver microsomes analyzed by LC-MS.

Nanoformulations of anticancer FGFR inhibitors with improved therapeutic index.

Fibroblast growth factor receptor (FGFR) inhibitors like ponatinib and nintedanib are clinically approved for defined cancer patient cohorts but often exert dose-limiting adverse effects. Hence, we encapsulated the FGFR inhibitors ponatinib, PD173074, and nintedanib into polylactic acid nanoparticles and liposomes to enable increased tumor accumulation/specificity and reduce side effects. Different methods of drug loading were tested and the resulting formulations compared regarding average size distribution as well as encapsulation efficiency. Appropriate encapsulation levels were achieved for liposomal preparations only. Nanoencapsulation resulted in significantly decelerated uptake kinetics in vitro with clearly decreased short-term (up to 72 h) cytotoxicity at higher concentrations. However, in long-term clonogenic assays liposomal formations were equally or even more active as compared to the free drugs. Accordingly, in an FGFR inhibitor-sensitive murine osteosarcoma transplantation model (K7M2), only liposomal but not free ponatinib resulted in significant tumor growth inhibition (by 60.4%) at markedly reduced side effects.

Multifunctional αvß6 Integrin-Specific Peptide-Pt(IV) Conjugates for Cancer Cell Targeting.

Increasing the specificity of cancer therapy, and thereby decreasing damage to normal cells, requires targeting to cancer-cell specific features. The αvß6 integrin is a receptor involved in cell adhesion and is frequently up-regulated in cancer cells compared to normal cells. We have selected a peptide ligand reported to bind specifically to the ß6 integrin and have synthesized a suite of multispecific molecules to explore the potential for targeting of cancer cells. A combination of solid-phase peptide synthesis and chemoselective ligations was used to synthesize multifunctional molecules composed of integrin-targeting peptides, cytotoxic platinum(IV) prodrugs, and fluorescent or affinity probes joined with flexible linkers. The modular synthesis approach facilitates the construction of peptide-drug conjugates with various valencies and properties in a convergent manner. The binding and specificity of the multifunctional peptide conjugates were investigated using a cell line transfected with the ß6 integrin and fluorescence microscopy. This versatile and highly controlled approach to synthesizing labeled peptide-drug conjugates has the potential to target potent cytotoxic drugs specifically to cancer cells, reducing the doses required for effective treatment.

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.

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.