Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin

Clinical application of platinum-based anticancer drugs is largely limited by severe general toxicity and drug resistance. Drug delivery systems with tumor-targeting potential are highly desired for improving the efficacy and applicability of these drugs. This study describes an alternative strategy for the delivery of platinum drugs (cisplatin, carboplatin and oxaliplatin) by encapsulating each of them in the cavity of apoferritin (AFt). The encapsulation was achieved through manipulating the pH-dependent unfolding-refolding process of AFt at pH 2.0 and 7.4, respectively, in saturated drug solution. UV-vis spectrometry, circular dichroism spectrometry, dynamic light scattering, and inductively coupled plasma mass spectrometry were used to characterize the AFt-drug complexes. The loading capacity of AFt varies with respective drugs and the structural integrity of the protein shell remains intact after encapsulation. In vitro assays on the rat pheochromocytoma cell line (PC12) show that AFt-cisplatin inhibits the cells in a slow but sustaining mode and the cellular uptake of platinum is enhanced by AFt. AFt-carboplatin and AFt-oxaliplatin complexes only exhibit a marginal cytotoxicity towards this cell line under similar concentrations.     

Unique platinum-DNA interactions may lead to more effective platinum-based antitumor drugs

Platinum coordination compounds are among the most utilized anticancer agents, even though platinum has not been determined to be an essential trace element in any living organism. The success of platinum-based drugs has catalyzed research on other metal-containing agents that can be used to achieve therapeutic goals that cannot be achieved with organic compounds. The antitumor activities of recently reported platinum(ii) complexes indicate that further modification of platinum coordination compounds will lead to the development of anticancer agents with higher efficacies against chemotherapy-insensitive tumors.     

Cross-linking of glutathione to DNA by cancer chemotherapeutic platinum coordination complexes

Glutathione can modulate the toxicity of a variety of drugs, although its role in modulating toxicity by anticancer platinum drugs is ambivalent. At physiologically relevant concentrations, glutathione can inhibit the reaction between DNA and cis-dichloro(ethylenediamine)platinum(II) (cis-DEP). Glutathione can also react with monofunctional adducts in DNA to produce a glutathione-Pt-deoxyguanosine cross-link which would reduce the potential toxicity of the drug. The relative importance of these two mechanisms of detoxification is unknown, although both mechanisms probably contribute to glutathione modulation of platinum toxicity.     

Synthesis, crystal structure and pH dependent cytotoxicity of (SP-4-2)-bis(2-aminoethanolato-κN,O)platinum(II) - a representative of novel pH sensitive anticancer platinum complexes

Solid tumors are often hypoxic and consequently the pH in the tumoral tissue is decreasing with increasing tumor size (pH 5.5-7.4 in solid tumors versus pH 7.4 in normal tissues). This marked difference in pH value is a problem for weak base organic drugs and could advantageously be used for the introduction of pH sensitive anticancer platinum drugs. Synthesis and structure determination of (SP-4-2)-bis(2-aminoethanolato-κ²N,O)platinum(II), its binding behavior to 5^′-GMP and its cytotoxicity against cisplatin sensitive cell lines under standard pH screening conditions (pH 7.4) as well as in acidified cell culture medium (pH 6.0) mimicking the conditions in a number of solid tumors is presented. There is evidence that this concept in anticancer platinum therapy, namely administration of rather unreactive drugs and activation under acidic pH conditions, can be realized.     

Site selectivity of platinum anticancer therapeutics

X-ray crystallographic and biochemical investigation of the reaction of cisplatin and oxaliplatin with nucleosome core particle and naked DNA reveals that histone octamer association can modulate DNA platination. Adduct formation also occurs at specific histone methionine residues, which could serve as a nuclear platinum reservoir influencing adduct transfer to DNA. Our findings suggest that the nucleosome center may provide a favorable target for the design of improved platinum anticancer drugs.     

Improving platinum(II)-based anticancer drug delivery using cucurbit[n]urils

Despite the synthesis of hundreds of new platinum(II) and platinum(IV)-based complexes each year as potential anticancer drugs, only three have received world-wide approval: cisplatin, carboplatin and oxaliplatin. The next big advance in platinum-based chemotherapy is not likely to come from the development of new drugs, but from the controlled and targeted delivery of already approved drugs or those in late stage clinical trials. Encapsulation of platinum drugs inside macromolecules has already demonstrated promise, and encapsulation within cucurbit[n]urils has shown particular potential. Partial or full encapsulation within cucurbit[n]urils provides steric hindrance to drug degradation by peptides and proteins, and the use of different sized cucurbit[n]urils allows for the tuning of drug release rates, cytotoxicity and toxicity.     

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is ¹H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.     

Meeting report on 8th International Symposium on Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy

The platinum-based drugs, cisplatin and carboplatin, represent major agents in the chemotherapeutic treatment of a variety of types of cancer. Novel, "third-generation" agents aimed at broadening the clinical activity of this class of drug are currently undergoing clinical evaluation. These include oxaliplatin, ZD0473 and BBR3464. Clinical trials and preclinical studies are also being conducted with liposomal (SPI-077 and L-NDDP) and polymeric platinum complexes (linked to HPMA or albumin). Combination studies of cisplatin/carboplatin with other anticancer drugs such as gemcitabine and UCN-01 (7-hydroxystaurosporine) and agents designed to reduce platinum drug toxicities (e.g., BNP-7787, DIMESNA) are ongoing. Preclinically, there is interest in trans platinum complexes, terpyridine platinum(II) complexes and other metal-containing agents (ruthenium and gold).     

Metalloproteomics in conjunction with other omics for uncovering the mechanism of action of metallodrugs: Mechanism-driven new therapy development

Medicinal inorganic chemistry has been largely stimulated by the clinic success of platinum anticancer drugs. An array of metal-based drugs (e.g. platinum, gold, bismuth, and silver) are currently used clinically for the treatment of various diseases. Integrating multiomics approaches, particularly metalloproteomics, with other biochemical characterizations enables comprehensive understanding of cellular responses of metallodrugs, which in turn will guide the rational design of a new drug and modification of the presently used drugs. This review aims to summarize the recent progress in this area. We will describe the technology platforms and their applications for uncovering the mechanisms of action of metallodrugs, for which remarkable advances have been achieved recently. Moreover, we will also highlight the application of newly generated knowledge for the development of novel therapeutic strategies.     

Unusual DNA binding modes for metal anticancer complexes

DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophobic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes.     

Sequence specificity, conformation, and recognition by HMG1 protein of major DNA interstrand cross-links of antitumor dinuclear platinum complexes

Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [?trans-PtCl(NH(3))(2)?H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site-specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues.     

Modulation of platinum antitumor drug binding to DNA by linked and free intercalators

We report the DNA binding site preferences of the novel molecule AO-Pt, in which the anticancer drug dichloro(ethylenediamine)platinum(II) is linked by a hexamethylene chain to acridine orange. The sequence specificity of platinum binding was mapped by exonuclease III digestion of 165 and 335 base pair restriction fragments from pBR322 DNA. Parallel studies were carried out with the unmodified anticancer drugs cis-diamminedichloroplatinum(II) (cis-DDP) and dichloro(ethylenediamine)platinum(II), [Pt(en)Cl2]. Oligo(dG) sequences are the most prevalent binding sites for AO-Pt, with secondary binding occurring mainly at d(AG) sites. cis-DDP and [Pt(en)Cl2] bind less readily to the secondary sequences, with cis-DDP showing greater binding site selectivity than [Pt(en)Cl2]. The DNA intercalator ethidium bromide promotes binding of [Pt(en)Cl2] and cis-DDP to many sites containing d(CGG) and, to a lesser extent, d(AG) sequences. AO-Pt exhibits enhanced binding to these sequences without the need for an external intercalator. Unlinked acridine orange, however, does not promote binding of [Pt(en)Cl2] and cis-DDP to d(CGG) and d(AG) sequences. These results are discussed in terms of the sequence preferences, stereochemistry, and relative residence times of the intercalators at their DNA binding sites. By modulating local structure in a sequence-dependent manner, both linked and, in the case of ethidium, free intercalators can influence the regioselectivity of covalent modification of DNA by platinum antitumor drugs.     

A novel technique to assay adducts of DNA induced by anticancer agent cis-diamminedichloroplatinum(II).

The dideoxynucleotides d(pGpG) and d(pApG) and the tetradeoxynucleotide d(CpTpApG) were synthesized in solution phase by a modified phosphotriester technique and reacted with the anticancer agent cis-diamminedichloroplatinum(II) (cisplatin). The major products were isolated by HPLC and characterized by NMR and mass spectrometry as cross-link adducts of cisplatin with the neighboring purine bases. The cross-link adducts of d(pGpG) and d(pApG) were dansylated through a 5'-phosphoramidate linkage with ethylenediammine. The labeling efficiency of the adducts was quantitative as in the case of the normal dinucleotides. The modified tetramer was digested with nuclease P1. The excised adduct was enriched by HPLC and labeled with dansyl chloride. The analysis of the postlabeled adduct by HPCL, using a fluorescence detector, detected a peak with retention time corresponding to that of the dansylated cis-Pt(NH3)2d(pApG). Cochromatography with the authentic marker confirmed the identification. The same overall procedure was used to assay calf thymus DNA exposed to cisplatin. The major adducts were identified as cis-Pt(NH3)2d(pGpG) and cis-Pt(NH3)2d(pApG). The quantitative labeling efficiency of platinum adducts combined with highly sensitive fluorescence detection technique (subfemtomol) suggests that fluorescence postlabeling assay could be a novel approach for real-time analysis of DNA modification induced by platinated drugs in biological system.     

Direct determination of paracetamol and its metabolites in urine and serum by capillary electrophoresis with ultraviolet and mass spectrometric detection

The use of capillary electrophoresis (CE) for the determination of paracetamol and its main metabolites in urine and serum is described. Due to its high efficacy, CE enables the analysis of drugs directly in complex matrices. Thus, simple, rapid and reliable assays could be developed that made use of some of the main advantages of this analytical technique. In order to prevent the peaks from tailing, a water zone was injected behind the sample. Occasionally occurring peak splittings of paracetamol were investigated and methods to suppress these splittings were developed. Paracetamol, its main metabolites, paracetamol glucuronide, paracetamol sulfate as well as paracetamol cysteinate and paracetamol mercapturate, as metabolites of the oxidative pathway were identified in urine using diode-array detection and coupling of the CE instruments to electrospray–mass spectrometry. The assays were validated. Their usefulness was demonstrated by applying them to the analysis of urine and serum samples of healthy volunteers as well as to urine samples from children under anticancer therapy.     

Metal Complexes in Cancer Treatment: Journey So Far

Metal complexes in cancer therapy have attracted much interest mainly because metals exhibit unique characteristics, such as redox activity, metal-ligand interaction, structure and bonding, Lewis acid properties etc. In 1965, Barnett Rosenberg serendipitously discovered the metal-based compound cisplatin, an outstanding breakthrough in the history of metal-based anticancer complexes and led to a new area of anticancer drug discovery. Many metal-based compounds have been studied for their potential anticancer properties. Some of these compounds have FDA approval for clinical use, while others are now undergoing clinical trials for cancer therapy and detection. In the present study, we have highlighted the primary mode of action of metallic complexes and all FDA-approved/under clinical trial drugs with reference to cancer treatment. This review also focuses on recent progress on metal-based complexes such as platinum, ruthenium, iron, etc. with potential anticancer activities.     

Speciation of platinum compounds: a review of recent applications in studies of platinum anticancer drugs

This is a review of investigations involving speciation studies of five important platinum-containing drugs used in cancer treatments. The information presented here is drawn from recent reports published during the period 1995–1999. The work includes detection, separations and identifications of degradation and biotransformation products. In addition, important information is reported on the number and nature of products of reactions of platinum anticancer drugs with thiol compounds. HPLC is employed effectively for separations of reaction products in speciation investigations. Information derived from speciation is very helpful in studies of pharmacokinetics as well as side effects and toxicities of the drugs as they are administered to patients.     

Mechanism of tumor resistance to cisplatin mediated by the copper transporter ATP7B

The Wilson disease protein (ATP7B) is a copper-transporting ATPase that is responsible for regulating copper homeostasis in human tissues. ATP7B is associated with cancer resistance to cisplatin, one of the most widely used anticancer drugs. This minireview discusses the possible mechanisms of tumor resistance to cisplatin mediated by ATP7B. Cisplatin binds to the N-terminal cytosolic domain of ATP7B, which contains multiple copper-binding sites. Active platinum efflux catalyzed by ATP7B is unlikely to significantly contribute to cisplatin resistance in vivo. Transient platinum sequestration in the metal-binding domain followed by transfer to an acceptor protein or a low molecular weight compound is proposed as an alternative mechanism of cisplatin detoxification in the cell.     

Mini-review: discovery and development of platinum complexes designed to circumvent cisplatin resistance

The discovery and development of new platinum-containing anticancer drugs have represented an integral part of anticancer drug development at the Institute of Cancer Research, Sutton, over almost 20 years. As part of a collaboration with chemists at Johnson Matthey, later AnorMED, four major new classes of platinum drug have been discovered, three of which have entered clinical trial. Earlier studies led to the clinical development of the less toxic analogue carboplatin and JM216, the first orally administerable platinum drug. In recent years, the focus has been on two lead complexes designed to overcome the major mechanisms of tumour resistance to cisplatin: JM335 (trans-ammine (cyclohexylaminedichlorodihydroxo) platinum(IV)), an active trans platinum complex; and ZD0473 (cis-amminedichloro(2-methylpyridine) platinum(II)), a sterically hindered complex shown to be less reactive towards thiol-containing molecules than cisplatin. JM335 shows some circumvention of acquired cisplatin resistance in vitro and exhibits unique cellular pharmacological properties in comparison to cisplatin or its cis-isomer in terms gene-specific repair of adducts on DNA and the rate of induction of apoptosis. ZD0473 is now in phase I clinical trial. Myelosuppression is the dose-limiting toxicity at a dose of 130 mg/m2 given i.v. every 3 weeks and there has been evidence of antitumour activity. ZD0473-resistant human ovarian carcinoma cell lines have been established in vitro. Some mechanisms of resistance common to those described for cisplatin (decreased drug uptake, increased glutathione) have been observed plus, in one cell line, increased BCL2 levels and loss of the DNA mismatch repair protein MLH1.     

Fast and slow versus strong and weak metal-DNA binding: consequences for anti-cancer activity

The binding of transition metal compounds to nucleic acids is discussed in the perspectives of kinetics and their anticancer activity. Kinetics of ligand exchange is primarily determined by the intrinsic properties of the metal ions, and to a lesser degree by the ligands coordinated already to the metal ion. Metal compounds having ligand-exchange rates of the same order of magnitude as cell-division processes, e.g. many Pt(iIIi), Ru(II) and Ru(III) compounds, are in use as chemotherapeutic drugs. Detailed knowledge of ligand exchange in such compounds is important for design of derivative and entirely new compounds. Metal coordination compounds of metal ions with much faster ligand-exchange reactions interact with DNA in a quite different way, namely primarily by compensation of negative charge of the polyanionic chain and are usually not active as anticancer agents. Examples of our recent work are presented in relation with experiments performed by others on new generations of platinum anti-cancer drugs.