The interaction of DNA-targeted platinum phenanthridinium complexes with DNA

Cisplatin analogues were synthesised that consisted of platinum(II) diamine complexes tethered via a polymethylene chain ( n = 3, 5, 8 and 10) to a phenanthridinium cation. Both chloro and iodo leaving groups were examined. DNA adduct formation was quantitatively analysed using a linear amplification system with the plasmid pGEM-3Zf(+). This system utilised Taq DNA polymerase to extend from an oligonucleotide primer to the damage site. This damage site inhibited the extension of the DNA polymerase. The products were electrophoresed on a DNA sequencing gel enabling adduct formation to be determined at base pair resolution. The damage intensity at each site was determined by densitometry. The platinum phenanthridinium complexes were shown to damage DNA at shorter incubation times than cisplatin. To produce similar levels of damage, an 18 h incubation was required for cisplatin compared to 30 min for the n = 3 platinum phenanthridinium complexes; this indicates that the intercalating chromophore causes a large increase in the rate of platination. A reaction mechanism involving direct displacement of the chloride by the N-7 of guanine may account for the rate increase. These results indicate that further development of these compounds could lead to more effective cancer chemotherapeutic agents.     

Action of antitumoral platinum complexes on in vitro platelet functions

This report presents a comparison of the effects of cis- and trans-diamminedichloroplatinum complexes on in vitro platelet functions. Pretreatment of platelets with cis-platinum (cisplatin) induced a slow, dose-dependent (0.1-0.45 mM), increase in the cytosolic Ca2+ concentration, pleckstrin (47 kDa) phosphorylation and serotonin secretion, as well as a slight shape modification with emission of a few pseudopodia. All these effects were remarkably increased in platelets exposed to trans-platinum (transplatin). The rise in cytosolic Ca2+ concentration and serotonin secretion evoked by stimulation of platelets with thrombin were not significantly influenced by cellular exposure to cis-platinum, whereas they were enhanced and inhibited, respectively, by exposure to trans-platinum. Trans-platinum also inhibited thrombin-promoted platelet aggregation to a greater extent than the cis-isomer. While the viscosity of platelet rich-plasma tended to decrease in the presence of cis-platinum, it tended to increase in the presence of trans-platinum. Taken together, these results indicate that the effects on platelet functions of the efficacious antitumor complex cis-platinum is rather different from that of the inactive complex trans-platinum. Therefore, the in vitro tests of platelet functions employed in this study might provide an index of antitumor drug toxicity and serve as a preliminary indicator of therapeutic efficacy.     

Direct physical measurements on substituted agarose gels: evidence for intercalation of gel-bound ethidium into transfer RNA

The cation of the salt ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) has been covalently linked to an agarose matrix through an intermediate 3,3'-diaminodipropylaminosuccinyl spacer arm. Partition binding and visible absorption spectral measurements on the gel were used to monitor the binding of transfer RNA to the covalently bound ethidium group. Direct fluorescence measurements of the formation of the gel-bound complex indicate that this binding involves the intercalation of the ethidium groups into the tRNA molecule. Dissociation of the ethidium-tRNA complex was monitored as a function of sodium chloride concentration by both direct solution spectral measurement of the released tRNA and by fluorescence quenching measurements of the dissociation of the intercalation complex. The derivatized gel has been shown to be capable of the fractionation of tRNA species by elution with a positive salt gradient under column flow conditions.     

Synthesis and in vitro cytotoxicity of lipophilic platinum(II) complexes

A number of lipophilic platinum(II) complexes of the general structures cis-[Pt(LA)2Cl2] and [Pt(LD)Cl2] were synthesised. Long chain amines (LA) and diamines (LD), prepared from lipidic amino acids, were used as ligands. The in vitro cytotoxicity of the complexes was evaluated against four cell lines (P388, NSCLC-N6, E39, M96). cis-Dichloro-bis(2-aminohexadecanol)platinum(II) was the most active against P388, NSCLC-N6 and E39 (IC50: 11 micrograms/ml, 25 micrograms/ml, 31 micrograms/ml), while dichloro(1,3-heptadecanediamine)platinum(II) presented the highest activity against M96 (IC50: 13 micrograms/ml).     

Antitumor activity of five new platinum complexes having a glycolate leaving ligand

Ganglioside GM3, one of acidic components of membrane glycosphingolipids (GSL), has been known to change its content quantitatively during growth and differentiation of various cells in vitro. Detailed analysis of lipid portion of GM3 of rat renal glomerular SGE1 cells revealed that fatty acids with long carbon chains, especially that of C24:0 and C24:1 increased, while that of short C18:0 and C20:0 decreased after spontaneous dome formation. Since not only fatty acid composition of neutral GSL, sulfatide and phospholipid but also composition of long-chain bases (LCB) did not change, it was suggested that only C24 fatty acid of GM3 specifically increased in relation to dome formation. The spontaneous dome formation has been reported to be related with induction of cellular differentiation in many transporting epithelial cells. We thus assume that the change of fatty acid composition of GM3 is involved in cellular differentiation of SGE1 cells.     

DNA receptor sites for the intercalation of nogalamycin

The intercalation of the planar chromophoric moiety of nogalamycin between two base pairs of duplex DNA has been evidenced by means of low-dichroism measurements. The possible presence of specific binding sites for mogalamycin on DNA has been suggested by studies on the denaturation and renaturation of DNA complexed with nogalamycin. A clear evidence was obtained by investigating the interaction of nogalamycin with polydeoxyribonucleotides containing known, regularly repeating sequences, used as model compounds. The results obtained with these polymers and the DNA suggest that the segment containing both purine (A,G) anf pyrimidine (T,C) bases in alternate sequences is the preferential receptor site on the DNA. A decreasing affinity is exhibited by poly d(A--T)-poly d(A--T), poly d(G--C)-poly d(G--C) and poly dG-poly dC segments, in the order. The poly dA-poly dT sequence appears to be closed to the interaction of nogalamycin.     

A sensitive postcolumn derivatization/UV detection system for HPLC determination of antitumor divalent and quadrivalent platinum complexes

A sensitive postcolumn derivatization/UV detection system has been developed for HPLC analysis of antitumor divalent and quadrivalent platinum complexes. It is based on the derivatization of platinum complexes by reaction with sodium bisulfite to corresponding product(s) which has enhanced absorptivity at 280-300 nm. Platinum complexes examined in this study were cisplatin, carboplatin and oxaliplatin (divalent platinum complexes) and oxoplatin and tetraplatin (quadrivalent ones). The proposed detection system was sensitive to all these complexes. Under the detection conditions optimized for individual complexes, the HPLC gave linear relationships between the complex concentration and the peak height. Detection limits at 290 nm with 100 microliters injection were 20 nM for cisplatin, 40 nM for oxoplatin, 60 nM for carboplatin and tetraplatin and 100 nM for oxaliplatin (S/N = 3 at 0.005 AUFS). The proposed system was successfully applied for the determination of cisplatin and oxoplatin in plasma and urine. Pharmacokinetic behavior of oxoplatin and its reduced product cisplatin following a single intravenous injection of oxoplatin in rabbits has been discussed.     

Bioaccumulation of nickel by intercalation into polycrystalline hydrogen uranyl phosphate deposited via an enzymatic mechanism

A Citrobacter sp. accumulates uranyl ion (UO2(2+)) as crystalline HUO2PO4.4H2O (HUP), using enzymatically generated inorganic phosphate. Ni was not removed by this mechanism, but cells already loaded with HUP removed Ni2+ by intercalative ion-exchange, forming Ni(UO2PO4)2.7H2O, as concluded by x-ray diffraction (XRD) and proton induced x-ray emission (PIXE) analyses. The loaded biomass became saturated with Ni rapidly, with a molar ratio of Ni:U in the cellbound deposit of approx. 1:6; Ni penetration was probably surface-localized. Cochallenge of the cells with Ni2+ and UO2(2+), and glycerol 2-phosphate (phosphate donor for phosphate release and metal bioprecipitation) gave sustained removal of both metals in a flow through bioreactor, with more extensively accumulated Ni. We propose 'Microbially Enhanced Chemisorption of Heavy Metals' (MECHM) to describe this hybrid mechanism of metal bioaccumulation via intercalation into preformed, biogenic crystals, and note also that MECHM can promote the removal of the transuranic radionuclide neptunium, which is difficult to achieve by conventional methods.     

Clinical development of platinum complexes in cancer therapy: an historical perspective and an update

The vast amount of basic research on platinum coordination complexes has produced, over the past 25 years, several thousand new molecules for preclinical screening and 28 compounds which have entered clinical development. The goals of these research activities have been to identify compounds with superior efficacy, reduced toxicity, lack of cross-resistance or improved pharmacological characteristics as compared with the parent compound, cisplatin. After the remarkable therapeutic effects of cisplatin had been established, only a few other platinum compounds succeeded in reaching general availability. Whereas carboplatin is an analogue with an improved therapeutic index (mostly driven by reduced organ toxicity) over that of cisplatin, new compounds clearly more active than or non-cross-resistant with cisplatin have not yet been identified. The platinum analogues that remain under investigation are focusing on expanding the utilisation of platinum therapy to tumour types not usually treated with, or responsive to, cisplatin or carboplatin. In addition, novel routes of administration constitute another avenue of research. The clinical development of platinum coordination complexes, with emphasis on those compounds still under active development, is reviewed.     

Effects of new triphenylethylene platinum(II) complexes on the interaction with phosphatidylcholine liposomes

In a previous work we synthesized a class of new antineoplastic drugs by coupling a cisplatin derivative to a triphenylethylene moiety similar to the antiestrogen, tamoxifen. These drugs differ in the number of hydroxy functions on the triphenylethylene rings and in the length of the linking arm. To gain more insight into the cellular mechanism by which these new drugs act on cells, we studied, using differential scanning calorimetry, the effects of these compounds on the phase transition of membrane phospholipid (distearoyl phosphatidyl choline (DSPC)), and correlated these effects to drug cytotoxicity. The drugs without hydroxy function showed the highest cytotoxicity and induced little change on the thermogram of DSPC. Contrarily, the drugs bearing two or three hydroxy groups were less toxic, but induced important modifications of the thermogram. We suggest that the drugs with no hydroxy group enter the membrane, with the triphenylethylene moiety localized deep within the hydrophobic core of the bilayer and do not affect the cooperativity region (C2-C8). In contrast, drugs which bear hydroxy groups on the triphenylethylene rings system perturb the phospholipid molecular arrangement; this may be due either to the additional steric hindrance of the hydroxy functions in the core of the bilayer, or to their hydrophilic effect on the polar head of the lipid. In vitro, the cytotoxic effect of these drugs seems not to be related to their affinity for the estrogen receptor. We suggest that the addition of a triphenylethylene moiety to the platinum(II) complexes increases the hydrophobicity, and consequently the resulting drugs become more permeable to the membrane, particularly the non-hydroxylated triphenylethylene derivatives.     

Lack of nephrotoxicity of oral ammine/amine platinum (IV) dicarboxylate complexes in rodents

The comparative nephrotoxicity of i.v. cisplatin, i.v. carboplatin and six p.o. ammine/amine Pt(IV) dicarboxylates was studied in rodents following single MTD treatments. In mice, i.v. cisplatin caused proteinuria (1 g l-1), glycosuria (16.7 mM) and decreased GFR at 4 days, and histological kidney damage with onset at 6 days. In contrast, mice treated with i.v. carboplatin or p.o. ammine/amine Pt(IV) dicarboxylates had urinary glucose, urinary protein, GFR and kidney histology within the control range. In rats, i.v. cisplatin caused 5-fold elevations in plasma creatinine (188 +/- 33 microM) and urea (30.4 +/- 8.9 mM), a 10-fold fall in creatinine clearance (0.54 +/- 0.31 ml min-1 kg-1), a 25-fold elevation in urine/plasma glucose concentration ratio (3.28 +/- 0.17), a 20% increase in kidney weight (7.9 +/- 0.56 mg gm-1 body weight) and extensive histological damage 4 days after treatment. In contrast, i.v. carboplatin and p.o. JM216 (the lead compound of this series) caused neither abnormalities in renal function nor histological damage in rats. The nephrotoxicity of single MTD treatments of p.o. ammine/amine Pt(IV) dicarboxylate complexes appears less than i.v. cisplatin and comparable to i.v. carboplatin.     

Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA

The central role of nucleic acids in biosciences has effectuated the rapid development of numerous techniques for their isolation, separation, characterization and quantitation. Advances in high-performance liquid chromatography, particularly the introduction of novel microparticulate sorbents, have greatly promoted the separation and quantitation of nucleic acids. Because of their favorable mass transfer properties, micropellicular packing materials are advantageous for fast and high-resolution separations of double-stranded (ds) DNA molecules. With micropellicular packings, anion-exchange and ion-pair reversed-phase chromatography are the most popular chromatographic separation modes for dsDNA. The effective separation mechanisms in both chromatographic techniques are preferably described by nonstoichiometric models, that are founded on a better physicochemical background than traditional stoichiometric models. Column efficiency, retention characteristics, and size or sequence dependency of retention of dsDNA are greatly influenced by the chosen operational variables in both chromatographic modes. The applicability of HPLC with micropellicular stationary phases nucleic acids research includes preparative DNA fractionation, DNA restriction mapping, analysis of polymerase chain reaction products and purification of plasmid DNA.     

Investigation into sorption processes of pyrazole-containing resins with respect to metals of the platinum group

The results of investigations into increasing the capacitive characteristics of pyrazole-containing sorbents for the recovery of palladium and platinum from their acidic solutions are presented. For these purposes, a sorbent of the SVPD grade with a microporous structure based on N-vinyl-3(5)-methylpyrazole and divinylbenzene is synthesized and tested and its capacitive characteristics by palladium and platinum are determined. The comparative tests of the SVPD resin with a pyrazole-containing sorbent of the SDP-d grade with a macroporous structure obtained based on styrene divinylbenzene and 3(5)-methylpyrazole in the presence of the deprotonating additive are performed at various acidities of the starting solutions. It is shown that the sorbent of the SVPD grade is preferable due to its capacitive characteristics when compared with the macroporous SDP-d sorbent. Optimal acidities of the starting solutions for the recovery of palladium and platinum by the sorbents under study are established. Two samples of bifunctional resins containing groups of N-vinyl-3(5)-methylpyrazole and organic sulfides are synthesized. The results of their tests showed that the introduction of organic sulfides into the resin not only leads to an increase in the sorbent capacity by palladium, but also considerably widens the working acidity range of the solutions.     

Stereochemical influence on the stability of radio-metal complexes in vivo. Synthesis and evaluation of the four stereoisomers of 2-(p-nitrobenzyl)-trans-CyDTPA

Distinct differences in in vivo stability of the two diastereomeric C-Functionalized CyDTPA chelating agents, (CHX-A DTPA and CHX-B DTPA, both racemates), as recently reported prompted further investigation as to why differences in configuration produced striking effects on the in vivo stability of their yttrium complexes. To this end, the four individual component stereoisomers of CHX-A and CHX-B were synthesized and ability to bind yttrium was investigated both in vitro and in vivo.