Combination of Drug Delivery Properties of PAMAM Dendrimers and Cytotoxicity of Platinum(IV) Complexes-A More Selective Anticancer Treatment?

Based on their drug delivery properties and activity against tumors, we combined PAMAM dendrimers with various platinum(IV) complexes in order to provide an improved approach of anticancer treatment. Platinum(IV) complexes were linked to terminal NH2 moieties of PAMAM dendrimers of generation 2 (G2) and 4 (G4) via amide bonds. Conjugates were characterized by 1H and 195Pt NMR spectroscopy, ICP-MS and in representative cases by pseudo-2D diffusion-ordered NMR spectroscopy. Additionally, the reduction behavior of conjugates in comparison to corresponding platinum(IV) complexes was investigated, showing a faster reduction of conjugates. Cytotoxicity was evaluated via the MTT assay in human cell lines (A549, CH1/PA-1, SW480), revealing IC50 values in the low micromolar to high picomolar range. The synergistic combination of PAMAM dendrimers and platinum(IV) complexes resulted in up to 200 times increased cytotoxic activity of conjugates in consideration of the loaded platinum(IV) units compared to their platinum(IV) counterparts. The lowest IC50 value of 780 ± 260 pM in the CH1/PA-1 cancer cell line was detected for an oxaliplatin-based G4 PAMAM dendrimer conjugate. Finally, in vivo experiments of a cisplatin-based G4 PAMAM dendrimer conjugate were performed based on the best toxicological profile. A maximum tumor growth inhibition effect of 65.6% compared to 47.6% for cisplatin was observed as well as a trend of prolonged animal survival.

Not the usual suspect - an unexpected organometallic product during the synthesis of cytotoxic platinum(II) complexes.

The reaction of (1R,2R)-(cyclohexane-1,2-diamine)dichloridoplatinum(II) with maleic acid unexpectedly resulted in the formation of an organometallic platinum(II) complex featuring a C,O-coordinating ligand. Additionally, a small series of close derivatives with increasing lipophilicity was synthesized. All complexes were fully characterized by multinuclear one- and two-dimensional (1H, 13C, 15N, and 195Pt) NMR spectroscopy, high resolution mass spectrometry, and in one case by X-ray diffraction. The lipophilicity and the impact on the DNA secondary structure as well as the cytotoxic properties in three human cancer cell lines (A549, SW480, and CH1/PA-1) were investigated. Unexpectedly, no clear-cut trend in cytotoxicity was observed with increasing lipophilicity. Also unexpectedly, the complexes showed only a low potential to inhibit cancer cell growth and no sign of interaction with DNA, in sharp contrast to the parent drug oxaliplatin, which seems to be caused by the low reactivity of the investigated compounds.

Quaternary Ammonium Palmitoyl Glycol Chitosan (GCPQ) Loaded with Platinum-Based Anticancer Agents-A Novel Polymer Formulation for Anticancer Therapy.

Quaternary ammonium palmitoyl glycol chitosan (GCPQ) has already shown beneficial drug delivery properties and has been studied as a carrier for anticancer agents. Consequently, we synthesised cytotoxic platinum(IV) conjugates of cisplatin, carboplatin and oxaliplatin by coupling via amide bonds to five GCPQ polymers differing in their degree of palmitoylation and quaternisation. The conjugates were characterised by 1H and 195Pt NMR spectroscopy as well as inductively coupled plasma mass spectrometry (ICP-MS), the latter to determine the amount of platinum(IV) units per GCPQ polymer. Cytotoxicity was evaluated by the MTT assay in three human cancer cell lines (A549, non-small-cell lung carcinoma; CH1/PA-1, ovarian teratocarcinoma; SW480, colon adenocarcinoma). All conjugates displayed a high increase in their cytotoxic activity by factors of up to 286 times compared to their corresponding platinum(IV) complexes and mostly outperformed the respective platinum(II) counterparts by factors of up to 20 times, also taking into account the respective loading of platinum(IV) units per GCPQ polymer. Finally, a biodistribution experiment was performed with an oxaliplatin-based GCPQ conjugate in non-tumour-bearing BALB/c mice revealing an increased accumulation in lung tissue. These findings open promising opportunities for further tumouricidal activity studies especially focusing on lung tissue.

Platinum(IV)-Loaded Degraded Glycol Chitosan as Efficient Platinum(IV) Drug Delivery Platform.

A new class of anticancer prodrugs was designed by combining the cytotoxicity of platinum(IV) complexes and the drug carrier properties of glycol chitosan polymers: Unsymmetrically carboxylated platinum(IV) analogues of cisplatin, carboplatin and oxaliplatin, namely (OC-6-44)-acetatodiammine(3-carboxypropanoato)dichloridoplatinum(IV), (OC-6-44)-acetaodiammine(3-carboxypropanoato)(cyclobutane-1,1-dicarboxylato)platinum(IV) and (OC-6-44)-acetato(3-carboxypropanoato)(1R,2R-cyclohexane-1,2-diamine)oxalatoplatinum(IV) were synthesised and conjugated via amide bonding to degraded glycol chitosan (dGC) polymers with different chain lengths (5, 10, 18 kDa). The 15 conjugates were investigated with 1H and 195Pt NMR spectroscopy, and average amounts of platinum(IV) units per dGC polymer molecule with ICP-MS, revealing a range of 1.3-22.8 platinum(IV) units per dGC molecule. Cytotoxicity was tested with MTT assays in the cancer cell lines A549, CH1/PA-1, SW480 (human) and 4T1 (murine). IC50 values in the low micromolar to nanomolar range were obtained, and higher antiproliferative activity (up to 72 times) was detected with dGC-platinum(IV) conjugates in comparison to platinum(IV) counterparts. The highest cytotoxicity (IC50 of 0.036 ± 0.005 µM) was determined in CH1/PA-1 ovarian teratocarcinoma cells with a cisplatin(IV)-dGC conjugate, which is hence 33 times more potent than the corresponding platinum(IV) complex and twice more potent than cisplatin. Biodistribution studies of an oxaliplatin(IV)-dGC conjugate in non-tumour-bearing Balb/C mice showed an increased accumulation in the lung compared to the unloaded oxaliplatin(IV) analogue, arguing for further activity studies.

Multifunctional Pt(iv) prodrug candidates featuring the carboplatin core and deferoxamine.

The synergistic combination of the anticancer drug carboplatin and the iron chelator deferoxamine (DFO) served as a foundation for the development of novel multifunctional prodrugs. Hence, five platinum(iv) complexes, featuring the equatorial coordination sphere of carboplatin, and one or two DFO units incorporated at axial positions, were synthesized and characterized using ESI-HRMS, multinuclear (1H, 13C, 15N, 195Pt) NMR spectroscopy and elemental analysis. Analytical studies demonstrated that the chelating properties of the DFO moiety were not compromised after coupling to the platinum(iv) core. The cytotoxic activity of the compounds was evaluated in monolayer (2D) and spheroid (3D) cancer cell models, derived from ovarian teratocarcinoma (CH1/PA-1), colon carcinoma (SW480) and non-small cell lung cancer (A549). The platinum(iv)-DFO prodrugs demonstrated moderate in vitro cytotoxicity (a consequence of their slow activation kinetics) but with less pronounced differences between intrinsically chemoresistant and chemosensitive cell lines as well as between 2D and 3D models than the clinically used platinum(ii) drug carboplatin.

Nano-scale imaging of dual stable isotope labeled oxaliplatin in human colon cancer cells reveals the nucleolus as a putative node for therapeutic effect.

Oxaliplatin shows a superior clinical activity in colorectal cancer compared to cisplatin. Nevertheless, the knowledge about its cellular distribution and the mechanisms responsible for the different range of oxaliplatin-responsive tumors is far from complete. In this study, we combined highly sensitive element specific and isotope selective imaging by nanometer-scale secondary ion mass spectrometry (NanoSIMS) with transmission electron microscopy to investigate the subcellular accumulation of oxaliplatin in three human colon cancer cell lines (SW480, HCT116 wt, HCT116 OxR). Oxaliplatin bearing dual stable isotope labeled moieties, i.e. 2H-labeled diaminocyclohexane (DACH) and 13C-labeled oxalate, were applied for comparative analysis of the subcellular distribution patterns of the central metal and the ligands. In all the investigated cell lines, oxaliplatin was found to have a pronounced tendency for cytoplasmic aggregation in single membrane bound organelles, presumably related to various stages of the endocytic pathway. Moreover, nuclear structures, heterochromatin and in particular nucleoli, were affected by platinum-drug exposure. In order to explore the consequences of oxaliplatin resistance, subcellular drug distribution patterns were investigated in a pair of isogenic malignant cell lines with distinct levels of drug sensitivity (HCT116 wt and HCT116 OxR, the latter with acquired resistance to oxaliplatin). The subcellular platinum distribution was found to be similar in both cell lines, with only slightly higher accumulation in the sensitive HCT116 wt cells which is inconsistent with the resistance factor of more than 20-fold. Instead, the isotopic analysis revealed a disproportionally high accumulation of the oxalate ligand in the resistant cell line.

Resistance against novel anticancer metal compounds: differences and similarities.

The platinum antitumor drugs cisplatin, carboplatin and oxaliplatin are widely used components of modern cancer chemotherapy. However, their success is limited by severe adverse effects and because of the impact of intrinsic and acquired resistance mechanisms on tumor responses. Consequently, intense efforts have been made to develop new metal compounds that either display enhanced tumor specificity or are less prone to the development of resistance. Despite the synthesis of thousands of compounds during the last decades only very few novel metal drugs have successfully reached clinical development and/or approval so far. In this review we summarize the current knowledge on drug resistance mechanisms against novel metal compounds (including platinum, arsenic, ruthenium, gallium, titanium, copper, and lanthanum drugs), and address the question whether there might exist a general metal-drug resistance phenotype.

Unprecedented twofold intramolecular hydroamination in diam(m)ine-dicarboxylatodichloridoplatinum(IV) complexes - ethane-1,2-diamine vs. ammine ligands.

Reaction of (OC-6-13)-bis(2Z-3-carboxyacrylato)dichlorido(ethane-1,2-diamine)platinum(IV) and (OC-6-13)-diamminebis(2Z-3-carboxyacrylato)dichloridoplatinum(IV) with propylamine in the presence of 1,1'-carbonyl diimidazole afforded not the expected amides; instead, beside amide formation, a twofold intramolecular attack of the am(m)ine ligand at the C[double bond, length as m-dash]C bonds was observed involving either both (ethane-1,2-diamine) or only one (ammine) coordinated nitrogen atom(s).

Analysis of quaternary ammonium and phosphonium ionic liquids by reversed-phase high-performance liquid chromatography with charged aerosol detection and unified calibration.

Several hydrophobic ionic liquids (ILs) based on long-chain aliphatic ammonium- and phosphonium cations and selected aromatic anions were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) employing trifluoroacetic acid as ion-pairing additive to the acetonitrile-containing mobile phase and adopting a step-gradient elution mode. The coupling of charged aerosol detection (CAD) for the non-chromophoric aliphatic cations with diode array detection (DAD) for the aromatic anions allowed their simultaneous analysis in a set of new ILs derived from either tricaprylmethylammonium chloride (Aliquat 336) and trihexyltetradecylphosphonium chloride as precursors. Aliquat 336 is a mix of ammonium cations with distinct aliphatic chain lengths. In the course of the studies it turned out that CAD generates an identical detection response for all the distinct aliphatic cations. Due to lack of single component standards of the individual Aliquat 336 cation species, a unified calibration function was established for the quantitative analysis of the quaternary ammonium cations of the ILs. The developed method was validated according to ICH guidelines, which confirmed the validity of the unified calibration. The application of the method revealed molar ratios of cation to anion close to 1 indicating a quantitative exchange of the chloride ions of the precursors by the various aromatic anions in the course of the synthesis of new ILs. Anomalies of CAD observed for the detection of some aromatic anions (thiosalicylate and benzoate) are discussed.

The impact of whole human blood on the kinetic inertness of platinum(iv) prodrugs - an HPLC-ICP-MS study.

The potential advantage of platinum(iv) complexes as alternatives to classical platinum(ii)-based drugs relies on their kinetic stability in the body before reaching the tumor site and on their activation by reduction inside cancer cells. In this study, an analytical workflow has been developed to investigate the reductive biotransformation and kinetic inertness of platinum(iv) prodrugs comprising different ligand coordination spheres (respectively, lipophilicity and redox behavior) in whole human blood. The distribution of platinum(iv) complexes in blood pellets and plasma was determined by inductively coupled plasma-mass spectrometry (ICP-MS) after microwave digestion. An analytical approach based on reversed-phase (RP)-ICP-MS was used to monitor the parent compound and the formation of metabolites using two different extraction procedures. The ligand coordination sphere of the platinum(iv) complexes had a significant impact on their accumulation in red blood cells and on their degree of kinetic inertness in whole human blood. The most lipophilic platinum(iv) compound featuring equatorial chlorido ligands showed a pronounced penetration into blood cells and a rapid reductive biotransformation. In contrast, the more hydrophilic platinum(iv) complexes with a carboplatin- and oxaliplatin-core exerted kinetic inertness on a pharmacologically relevant time scale with notable amounts of the compound accumulated in the plasma fraction.

Effect of metal ion complexation and chalcogen donor identity on the antiproliferative activity of 2-acetylpyridine N,N-dimethyl(chalcogen)semicarbazones.

Three chalcogensemicarbazones, viz., 2-acetylpyridine N,N-dimethylsemicarbazone (HL(1)), 2-acetylpyridine N,N-dimethylthiosemicarbazone (HL(2)) and 2-acetylpyridine N,N-dimethylselenosemicarbazone (HL(3)), their corresponding gallium(III) complexes [Ga(L(1-3))(2)]PF(6) and the ruthenium(III) compound [Ru(L(2))(2)]PF(6) have been prepared and characterised by X-ray crystallography and spectroscopic techniques (IR, UV/vis, (1)H, (13)C, (15)N, (77)Se NMR) in order to elucidate the effect of metal ion complexation and chalcogen donor identity on the cytotoxicity of chalcogensemicarbazones in two human tumour cell lines 41M (ovarian carcinoma) and SK-BR-3 (mammary carcinoma).

Oxaliplatin reacts with DMSO only in the presence of water.

Herein we show that oxaliplatin reacts rapidly with DMSO in aqueous solutions, despite being stable in pure DMSO and pure water. Furthermore, the reactivity of the clinically applied Pt(ii) drugs in water/DMSO and PBS/DMSO mixtures, and the nature of the species formed were investigated by MS, NMR and RP-HPLC techniques.

Impact of the equatorial coordination sphere on the rate of reduction, lipophilicity and cytotoxic activity of platinum(IV) complexes.

The impact of the equatorial coordination sphere on the reduction behavior (i.e. rate of reduction) of platinum(IV) complexes with axial carboxylato ligands was studied. Moreover, the influence of equatorial ligands on the stability, lipophilicity and cytotoxicity of platinum(IV) compounds was evaluated. For this purpose, a series of platinum(IV) complexes featuring axial carboxylato ligands (succinic acid monoesters) was synthesized; anionic carboxylato (OAc-, oxalate) and halido (Cl-, Br-, I-) ligands served as leaving groups and am(m)ine carrier ligands were provided by monodentately (isopropylamine, ammine+cyclohexaneamine) or bidentately (ethane-1,2-diamine) coordinating am(m)ines. All platinum(IV) products were fully characterized based on elemental analysis, high resolution mass spectrometry and multinuclear (1H, 13C, 15N, 195Pt) NMR spectroscopy as well as by X-ray diffraction in some cases. The rate of reduction in the presence of ascorbic acid was determined by NMR spectroscopy and the lipophilicity of the complexes was investigated by analytical reversed phase HPLC measurements. Cytotoxic properties were studied by means of a colorimetric microculture assay in three human cancer cell lines derived from cisplatin sensitive ovarian teratocarcinoma (CH1/PA-1) as well as cisplatin insensitive colon carcinoma (SW480) and non-small cell lung cancer (A549).

Signal separation and determination of the enantiomeric purity of primary amines with (-)-myrtenal--a 13C NMR study.

The applicability of (-)-myrtenal as a chiral derivatizing agent in combination with (13)C NMR spectroscopy was investigated. (13)C NMR was found to be a valuable tool for the identification and enantiomer differentiation of primary amines including beta-amino alcohols and vicinal diamines. The enantiomeric excess could be determined via automated deconvolution and integration, and was found to be in good accordance with the expected values even in the cases, when enantiomer differentiation was not possible in (1)H NMR spectra.

The role of the equatorial ligands for the redox behavior, mode of cellular accumulation and cytotoxicity of platinum(IV) prodrugs.

The current study aims to elucidate the possible reasons for the significantly different pharmacological behavior of platinum(IV) complexes with cisplatin-, carboplatin- or nedaplatin-like cores and how this difference can be related to their main physicochemical properties. Chlorido-containing complexes are reduced fast (within hours) by ascorbate and are able to unwind plasmid DNA in the presence of ascorbate, while their tri- and tetracarboxylato analogs are generally inert under the same conditions. Comparison of the lipophilicity, cellular accumulation and cytotoxicity of the investigated platinum compounds revealed the necessity to define new structure-property/activity relationships (SPRs and SARs). The higher activity and improved accumulation of platinum(IV) complexes bearing Cl(-) in equatorial position cannot only be attributed to passive diffusion facilitated by their lipophilicity. Therefore, further platinum accumulation experiments under conditions where active/facilitated transport mechanisms are suppressed were performed. Under hypothermic conditions (4°C), accumulation of dichloridoplatinum(IV) complexes is reduced down to 10% of the amount determined at 37°C. These findings suggest the involvement of active and/or facilitated transport in cellular uptake of platinum(IV) complexes with a cisplatin-like core. Studies with ATP depletion mediated by oligomycin and low glucose partially confirmed these observations, but their feasibility was severely limited in the adherent cell culture setting.

Prediction of logP for Pt(II) and Pt(IV) complexes: Comparison of statistical and quantum-chemistry based approaches.

The octanol/water partition coefficient, logP, is one of the most important physico-chemical parameters for the development of new metal-based anticancer drugs with improved pharmacokinetic properties. This study addresses an issue with the absence of publicly available models to predict logP of Pt(IV) complexes. Following data collection and subsequent development of models based on 187 complexes from literature, we validate new and previously published models on a new set of 11 Pt(II) and 35 Pt(IV) complexes, which were kept blind during the model development step. The error of the consensus model, 0.65 for Pt(IV) and 0.37 for Pt(II) complexes, indicates its good accuracy of predictions. The lower accuracy for Pt(IV) complexes was attributed to experimental difficulties with logP measurements for some poorly-soluble compounds. This model was developed using general-purpose descriptors such as extended functional groups, molecular fragments and E-state indices. Surprisingly, models based on quantum-chemistry calculations provided lower prediction accuracy. We also found that all the developed models strongly overestimate logP values for the three complexes measured in the presence of DMSO. Considering that DMSO is frequently used as a solvent to store chemicals, its effect should not be overlooked when logP measurements by means of the shake flask method are performed. The final models are freely available at http://ochem.eu/article/76903.

Synthesis and structure-activity relationships of mono- and dialkyl-substituted oxaliplatin derivatives.

In order to improve the pharmacological profile of the anticancer drug oxaliplatin, (trans-R,R-cyclohexane-1,2-diamine)oxalatoplatinum(II), and to explore activity-structure relationships, new mono- and dialkyl substituted oxaliplatin analogues have been synthesized. Following a new synthetic strategy, racemates with a defined stereochemistry at carbon atoms 1, 2, 4, and 5 of the cyclohexane ring could be prepared, which are the bases for reliable structure-activity relationships and the following enantiomer resolution. The cytotoxicity was evaluated in nine tumor cell lines, indicating that bulky substituents have a negative influence on the cytotoxic potency of the oxaliplatin derivatives. With respect to the antiproliferative properties, the 4-methyl-, cis-4,5-dimethyl-, and especially the 4,4-dimethyl-trans-cyclohexane-1,2-diamine(oxalato)platinum(II) complexes are the most promising candidates to be further evaluated.

Tetracarboxylatoplatinum(IV) complexes featuring monodentate leaving groups - A rational approach toward exploiting the platinum(IV) prodrug strategy.

A series of novel symmetrically and unsymmetrically coordinated platinum(IV) complexes with monodentate carboxylato ligands was synthesized. The compounds exhibit a general coordination sphere of [Pt(en)(OCOR)2(OCOR')(OCOR″)], where the carboxylato ligands are represented by acetato and succinic acid monoester ligands. Dicarboxylatoplatinum(II) complexes were synthesized and oxidized symmetrically or unsymmetrically to obtain platinum(IV) complexes, which were subsequently carboxylated with noncyclic anhydrides. The compounds were investigated in detail by elemental analysis, mass spectrometry, infrared and multinuclear ((1)H, (13)C, (15)N, (195)Pt) NMR spectroscopy as well as by X-ray diffraction in some cases. The reduction behavior was followed by NMR spectroscopy, while stability and lipophilicity were examined by analytical reversed phase HPLC measurements. Cytotoxic properties were studied in three human cancer cell lines derived from cisplatin sensitive ovarian teratocarcinoma (CH1/PA-1), cisplatin insensitive colon carcinoma (SW480) and non-small cell lung cancer (A549). Thereby, the most lipophilic (yet water soluble) platinum(IV) complexes showed promising IC50 values in the low micromolar and even nanomolar range, demonstrating the significant advantage of using equatorially coordinated monodentate carboxylato ligands.

Capillary electrophoretic study of cisplatin interaction with nucleoside monophosphates, di- and trinucleotides.

cis-Diamminedichloroplatinum(II) (cisplatin) is applied against different kinds of cancer although toxic side effects are known. Screening systems for alternative compounds with higher effectiveness but minimizing toxic side effects are required. We investigated the adduct formation of cisplatin with nucleoside monophosphates, di- and trinucleotides. Capillary electrophoretic separations were performed in a sodium phosphate buffer using an instrument equipped with a diode array detector. Adduct formation results in a significant shift of lambda(max) to lower energy compared to free nucleotides. Therefore, UV spectra are an important tool for peak identification. We could separate and identify all four common nucleotides and their major platinum adducts in a single run demonstrating the suitability of CE for these kinds of investigations. Furthermore, kinetic studies of these reactions are performed.

Capillary electrophoretic study of carboplatin and analogues with nucleoside monophosphates, di- and trinucleotides.

Carboplatin (cis-diammine-1,1-cyclobutane-dicarboxylatoplatinum(II)) is the only cisplatin (cis-diammine-dichloroplatinum(II)) derivative currently available for the treatment of cancer worldwide. The higher stability of the carboxylate ligand compared to the coordinated chloride in cisplatin results in a reduced reactivity of the molecule. Capillary electrophoresis has been applied for investigating the adduct formation of carboplatin and analogues with nucleoside monophosphates, di- and trinucleotides. Adduct formation results in a significant shift of the absorption maximum to lower energy compared to free nucleotides. Therefore, characterization of the analytes was performed by UV additionally to NMR spectroscopy. A preference for GMP- and AMP-coordination was found. The ability of separating all four common nucleotides and their major platinum adducts in a single run demonstrates the suitability of CE for this kind of investigations.