Bart A. J. Jansen

Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs

The sulfur-containing tripeptide glutathione (GSH) is one of the most abundant molecules in cells. Elevated levels of GSH render some types of cancer cells resistant against well-known platinum anti-cancer drugs such as cisplatin and carboplatin. Platinum complexes are often very reactive towards the cysteine residue of GSH, which detoxifies these compounds by a rapid binding mechanism. Clearly, this resistance mechanism poses a severe obstacle to any new platinum drugs designed to overcome cisplatin resistance. In the present study the cytotoxicity of dinuclear platinum compounds of the 1,1/t,t type, as developed by Farrell, is determined in human ovarium A2780 cells and in the cisplatin-resistant cell line A2780cisR, which possesses elevated levels of GSH. Further, the effect of depletion of GSH levels by L-buthionine-S,R-sulfoximine (L-BSO) in A2780cisR was investigated. The experiments show that detoxification by GSH is an effective resistance mechanism against dinuclear platinum compounds. However, the dinuclear complexes are less sensitive towards detoxification compared to cisplatin. This is probably because of the rapid binding of dinuclear cationic complexes to DNA. Compared to cisplatin, the rapid binding to DNA reduces the time during which the drug molecules are exposed to GSH in the cytosol. The reaction of a representative dinuclear compound with glutathione (pH 7, 37 degrees C) was studied in detail by 195Pt NMR. The dinuclear complex BBR3005 ([trans-PtCl(2)(NH(3))(2)(mu-H(2)N(CH(2))(6)NH(2))](2+), abbreviated as 1,1/t,t n=6), follows different pathways in the reaction with GSH, depending on the molar ratio of the reactants. When reacted in stoichiometric amounts (1:1), first a chloride on each platinum is replaced by a sulfur, forming a PtN(3)S product at -2977 ppm. After 2-3 h, this intermediate reacts further to form a sulfur-bridged N(3)Pt-S-PtN(3) species as the main product at -2811 ppm. When BBR3005 is reacted with GSH in a ratio of 1:4, the sulfur-bridged species is not observed. Instead, the final product is trans-Pt(GS)(2)(NH(3))(2) (at -3215 ppm); the same product appears if GSH is reacted with trans-PtCl(2)(NH(3))(2). Apparently, GSH first replaces the chlorides and subsequently degrades the dinuclear compound by replacement of the diaminealkyl linker.

A TETRANUCLEAR PLATINUM COMPOUND DESIGNED TO OVERCOME CISPLATIN RESISTANCE

The synthesis and characterisation of the first generation of a poly(propyleneimine) dendrimer DAB(PA)4, substituted with four trans-diamminechloroplatinum moieties is reported. The compound DAB(PA-tPt-Cl)4 was designed to overcome two problems often associated with cisplatin resistance in cancer cells: (i) deactivation of the platinum species by intracellular thiolates and (ii) improved repair of crosslinks with DNA. The four-armed molecule can be expected to form crosslinks with DNA that are very different from the adducts formed by cisplatin. Also, the tetranuclear compound has four leaving groups, while cisplatin has only two. Therefore, DAB(PA-tPt-Cl)4 would be less susceptible towards inactivation by reaction with intracellular thiolates. A reaction with an excess of the model nucleobase guanosine 5′-monophosphate (GMP) confirmed that the tetranuclear compound is capable of binding a maximum of four nucleobases. Therefore, the inactivation of one or two arms would still leave the molecule with enough reactivity to form crosslinks with DNA. Cytotoxicity tests were performed on two mouse leukemia L1210 cell lines, both sensitive and resistant towards cisplatin, and in seven human tumor cell lines. In all cell lines, the tetranuclear compound showed a low cytotoxicity. It is suggested that the low activity is related to the structure of the compound. Probably the high charge (+6) at physiological pH and its branched structure hamper the molecule in crossing the cell membranes.

Dinuclear platinum complexes with N, N'-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780 cisplatin-resistant human ovarian carcinoma cells: new insights into the mechanism of resistance.

The cellular processing of three fluorescent N,N′-bis(aminoalkyl)-1,4-diaminoanthraquinones (aminoalkyl=2-aminoethyl, 3-aminoprop-1-yl or 4-aminobut-1-yl) and their dinuclear platinum complexes in A2780 human ovarian carcinoma cells with acquired resistance to cisplatin has been monitored over time by time-lapse fluorescence microscopy. The results were compared with the previously reported observations in the parent A2780 cell line. The cellular distribution pattern for the free ligands is similar in sensitive and resistant cells, whereas significant differences in cellular distribution were observed in the case of the platinum complexes. In the cisplatin-resistant cell line the platinum complexes were found to be sequestrated in acidic vesicles in the cytosol from the very beginning of the incubation. This sequestration was not observed in the case of sensitive cells. Platinum accumulation in vesicles possibly presents a mechanism of resistance to platinum complexes. This mechanism appears to be unrelated to the mechanism of deactivation of platinum compounds by glutathione. Encapsulation of the dinuclear platinum complexes in lysosomal vesicles provides a plausible explanation for the decreased activity of these compounds in the resistant cell line, as compared to the sensitive cell line.

Dinuclear platinum anticancer complexes with fluorescent N,N'-bis(aminoalkyl)-1,4-diaminoanthraquinones: cellular processing in two cisplatin-resistant cell lines reflects the differences in their resistance profiles.

The biological activity of N,N′-bis(aminoalkyl)-1,4-diaminoanthraquinones (aminoalkyl is 2-aminoethyl, 3-aminoprop-1-yl and 4-aminobut-1-yl) and their dinuclear platinum complexes has been evaluated in the U2-OS human osteosarcoma cell line and its cisplatin-resistant U2-OS/Pt subline. All the compounds have been found to exhibit high cytotoxicity in the sensitive cell line, and to overcome cisplatin resistance in U2-OS/Pt cells. Cellular processing of N,N′-bis(2-aminoethyl)-1,4-diaminoanthraquinone and the respective dinuclear platinum complex in the sensitive and resistant U2-OS cells has been studied over time using digital fluorescence microscopy. Cellular processing of the compounds has been found to be similar in sensitive and resistant U2-OS cells, which is in agreement with the lack of cross-resistance in the U2-OS/Pt cell line. Both the platinum complex and the free ligand quickly enter the cell and accumulate in the nucleus. The platinum complex is excreted from the cell via the Golgi apparatus, while the weakly basic anthraquinone ligand accumulates in the Golgi complex, where it is taken up by lysosomes and then transported to the cell surface. The cellular distribution of the fluorescent anthraquinones and their dinuclear platinum complexes in the sensitive/resistant pair of U2-OS osteosarcoma cell lines is compared with the earlier studied cellular processing in the sensitive/resistant pair of A2780 ovarian carcinoma cell lines. In the A2780cisR cell line, the platinum complexes (and not the free ligands) are sequestered in lysosomes, which is not the case in A2780 sensitive cells. The differences in cellular distribution of the compounds in these two sensitive/resistant pairs of cell lines most likely result from different resistance profiles in A2780cisR and U2-OS/Pt cells. Lysosomes of A2780cisR cells are less acidic than lysosomes of A2780 sensitive cells, which is likely to be the cause of a defect in endocytosis. The disruption of normal endocytosis might facilitate sequestration of the platinum complexes in lysosomes, which partly confers the cross-resistance of these complexes with cisplatin in the A2780cisR cell line. In contrast, sequestration in acidic vesicles does not occur in U2-OS/Pt cells that do not exhibit enhanced lysosomal pH and which are likely to have normal endocytosis.

Solid-phase synthesis of peptide-platinum complexes using platinum-chelating building blocks derived from amino acids

Amino acids have been employed as precursors in the synthesis of platinum-chelating solid-phase building blocks. These chelating molecules were subsequently successfully used in the solid-phase synthesis of peptide-platinum complexes. The newly introduced functionality in the chelating part, as well as the nature of the pendant peptide, was shown to have an important influence on the anticancer activity of the complexes.

Pharmaceutical Development of a Parenteral Lyophilized Formulation of the Investigational Polymer-Conjugated Platinum Anticancer Agent AP 5280

Abstract AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ® resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2–8°C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.

Oxa-aza Crown Ethers as Ligands for Mixed-Ligand Cisplatin Derivatives and Dinuclear Platinum Anticancer Drugs

In the search for novel platinum anticancer drugs, cisplatin derivatives and dinuclear platinum complexes containing oxa-aza crown ether and oxa-diaza crown ether ligands have been prepared. The cisplatin derivatives cis[PtCl2(NH3)(1,4,7,10,13-pentaoxa-16-azacyclooctadecaneN)] (AO18) and cis-[PtCl2(NH3)(1,4,7,10-tetraoxa-13-azacyclopentadecane-N)] (AO15), and the dinuclear cationic platinum complexes [{trans-PtCl(NH3)2}2(μ-1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-N,N′)](NO3)2 (DAO18) and [{trans-PtCl(NH3)2}2(μ-1,4,10-trioxa-7,13-diazacyclopentadecane-N,N′)](NO3)2 (DAO15) were investigated for their cytotoxic properties, cellular uptake and intracellular DNA binding in A2780 human ovarian cancer. The cisplatin derivative AO15 shows the highest cytotoxic activity, whereas the cationic dinuclear complexes DAO18 and DAO15 display a disappointing lack of biological activity at concentrations up to 100 μM. In the intracallular DNA platination experiments, the fifteen-membered rings were found to have a threefold higher intracellular DNA binding compared to their 1eight-membered analogues for the neutral cisplatin derivatives as well as for the cationic dinuclear complexes. The crown ether complex with the most effective binding to DNA, AO15, also shows the highest cytotoxicity against A2780 cancer cells. It is remarkable that, although AO15 binds more effectively to DNA than cisplatin, its cytotoxic effect is much lower. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Mononuclear Tetraamineplatinum(II) Complexes: Synthesis, Anticancer Activity, DNA Binding, and Cellular Uptake

The synthesis of three bis[(tert-butoxy)carbonyl]-protected (tetramine)dichloroplatinum complexes 2a – c of formula cis-[PtCl2(LL)] and of their cationic deprotected analogs 3a – c and their evaluation with respect to in vitro cytotoxicity, intramolecular stability, DNA binding, and cellular uptake is reported. The synthesis comprises the complexation of K2[PtCl4] with di-N-protected tetramines 1a – c to give 2a – c and subsequent acidolysis, yielding 3a – c. The cytotoxicity of the complexes is in direct relation to the length of the polyamine. Complexes 3a – c display a significant higher affinity for CT DNA as well as for cellular DNA in A2780 cells than cisplatin.