Search

Here, we investigated the mechanism of antiproliferative action in cancer cells of new compounds structurally derived from oxaliplatin, namely a pair of enantiomers [Pt(OXA)(1R,2R-DACHEX)] (1) and [Pt(OXA)(1S,2S-DACHEX)] (2) (OXA = oxalate, DACHEX = trans-1,2-diamino-4-cyclohexene). While oxaliplatin is used almost exclusively to treat colorectal and other gastrointestinal cancers, new complex 1 shows instead high potency in malignant pancreatic adenocarcinoma PSN1 cells including superior selectivity for pancreatic cancer over noncancerous cells. Utilizing a multi-platform biochemical approach to study the unique features of the mechanism of action of this new platinum-based drug, we show that 1 has a much greater ability to penetrate pancreatic tumors than its S,S enantiomer 2 and oxaliplatin, and to be transported into cells as bound to plasma proteins. Additionally, the mechanism of action of 1 and, to a lesser extent, oxaliplatin in pancreatic cancer cells involves alterations of the lipogenesis pathway, namely inhibition of de novo lipid synthesis, acting by a new mechanism not yet considered for anticancer action of clinically used antitumor platinum drugs. These data highlight the functional diversity of platinum anticancer compounds and the potential benefits of finding new anticancer drugs applying a mechanism-based rationale.

PDF file - 65KB, Peaks observed for ESI+ mass spectra of Pt complexes dissolved in DMSO, associated with Pt-DMSO adducts.

PDF file - 1000KB, Scheme demonstrating the aquation of cisplatin in water, and prevention when stabilized in high chloride solution.

XLS file - 106KB, Use of solvent for assessing cytotoxicity of platinum complexes in the journal Molecular Cancer Therapeutics (S1). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Public Library of Science (PLoS)(S2). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Journal of Pharmacology and Experimental Therapeutics (JPET) (S3). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Cancer Research (S4). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Molecular Pharmacology (S5). Use of solvent for assessing cytotoxicity of platinum complexes (S6).

Six enantiomerically pure, oxaliplatin-like, platinum compounds (two platinum(II) and four platinum(IV)), all containing unsaturated cyclic diamine trans-1,2-diamino-4-cyclohexene (DACHEX) as a substitute for the trans-1,2-diaminocyclohexane used in oxaliplatin, were investigated. The complexes were characterized by elemental analyses, ESI-MS, and 1H-NMR spectroscopy. For the four Pt(IV) complexes the electrochemical redox behaviour, investigated by cyclic voltammetry, showed that all complexes possess reduction potentials suitable for activation in vivo. The antiproliferative activity was assessed in vitro on human cancer cell lines, also selected for resistance to platinum-based drugs or belonging to the MultiDrug-Resistant (MDR) phenotype. All complexes exhibited antiproliferative activity superior to that of cisplatin and almost equivalent to or better than that of oxaliplatin; moreover, most complexes were also capable of overcoming both the cisplatin- and the oxaliplatin-resistance. By comparing the effectiveness of the enantiomerically pure compounds with the racemic one, the R,R enantiomer emerged as the most effective in the case of Pt(II) complexes whereas the S,S enantiomer was the most effective in the case of the Pt(IV) derivatives. From the results obtained also against 3D spheroid tumor models, cis,trans,cis-[Pt(OXA)(OBz)2(1S,2S-DACHEX)] (OBz = benzoate) emerged as the most promising candidate for further preclinical investigation.

The year 2023 marks the 45th year since FDA approval of cisplatin as an anticancer drug, and, at present, it is widely used against a spectrum of human tumors, including early-stage ovarian cancer, non-small cell lung cancer (typically developed by smokers), head and neck, and advanced bladder cancer [...]

The search for more effective platinum anticancer drugs has led to the design, synthesis, and preclinical testing of hundreds of new platinum complexes. This search resulted in the recognition and subsequent FDA approval of the third-generation Pt(II) anticancer drug, [Pt(1,2-diaminocyclohexane)(oxalate)], oxaliplatin, as an effective agent in treating colorectal and gastrointestinal cancers. Another promising example of the class of anticancer platinum(II) complexes incorporating the Pt(1,n-diaminocycloalkane) moiety is kiteplatin ([Pt(cis-1,4-DACH)Cl

The use of Pt(iv) complexes as potential anticancer drugs is attractive, because they have higher stability and less side effects than Pt(ii) compounds. Moreover, some Pt(iv) complexes can also be activated with light, opening an avenue to photochemotherapy. Our purpose is to widen the library of photoactivatable Pt(iv)-based prodrugs and here we report on the oxidation of the Pt(ii) compound [PtCl(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (1) with PhICl2 or H2O2. The synthetic procedure avoids the formation of multiple species: the treatment with PhICl2 produces the Pt(iv) complex with axial chlorides, [PtCl3(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (2), while H2O2 oxidation and post-synthesis carboxylation produce [Pt(OCOCH3)2Cl(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (3), bearing acetates in the axial positions. 2 and 3 are stable in physiological-like buffers and in DMSO in the dark, but undergo photoreduction to 1 upon irradiation at 365 nm. Their stability toward reduction is a fundamental parameter to consider: cyclic voltammetry experiments show that the 2 electron reduction Pt(iv) → Pt(ii) occurs at a more negative potential for 3, because of the greater stabilization provided by the acetate axial groups; noteworthily, 3 is stable for hours also in the presence of mM concentration of glutathione. The cytotoxicity of 2 and 3 toward A2780 and A2780cis cell lines reveals that 3 is the least toxic in the dark, but is able to produce cytotoxic effects far higher than cisplatin when irradiated. To shed light on the mechanistic aspects, the interaction with protein and DNA models has been explored through high-resolution mass spectrometry revealing that 2 and 3 behave as prodrugs, but are able to bind to biological targets only after irradiation.

Kiteplatin, [PtCl2(cis-1,4-DACH)] (DACH = diaminocyclohexane), contains an isomeric form of the oxaliplatin diamine ligand trans-1R,2R-DACH and has been proposed as a valuable drug candidate against cisplatin- and oxaliplatin-resistant tumors, in particular, colorectal cancer. To further improve the activity of kiteplatin, it has been transformed into a Pt(IV) prodrug by the addition of two benzoato groups in the axial positions. The new compound, cis,trans,cis-[PtCl2(OBz)2(cis-1,4-DACH)] (1; OBz = benzoate), showed cytotoxic activity at nanomolar concentration against a wide panel of human cancer cell lines. Based on these very promising results, the investigation has been extended to the in vivo activity of compound 1 in a Lewis Lung Carcinoma (LLC) model and its suitability for oral administration. Compound 1 resulted to be remarkably stable in acidic conditions (pH 1.5 to mimic the stomach environment) undergoing a drop of the initial concentration to ~60% of the initial one only after 72 h incubation at 37 °C; thus resulting amenable for oral administration. Interestingly, in a murine model (2·106 LLC cells implanted i.m. into the right hind leg of 8-week old male and female C57BL mice), a comparable reduction of tumor mass (~75%) was observed by administering compound 1 by oral gavage and the standard drug cisplatin by intraperitoneal injection, thus indicating that, indeed, there is the possibility of oral administration for this dibenzoato prodrug of kiteplatin. Moreover, since the mechanism of action of Pt(IV) prodrugs involves an initial activation by chemical reduction to cytotoxic Pt(II) species, the reduction of 1 by two bioreductants (ascorbic acid/sodium ascorbate and glutathione) was investigated resulting to be rather slow (not complete after 120 h incubation at 37 °C). Finally, the neurotoxicity of 1 was evaluated using an in vitro assay.

The Pt(IV) prodrug of kiteplatin, cis-trans-cis-[PtCl2(RS-Ibuprofen-H)2(cis-1,4-DACH)], having in the axial positions two molecules of Ibuprofen, has been synthesised, characterized and tested in vitro. The aim was to potentiate the cytotoxic effect of kiteplatin with the anti-inflammatory activity of Ibuprofen. The reduction potential of the conjugate resulted comparable to those of other reported Pt(IV) carboxylate complexes, ensuring in vivo stability in blood during transport and intracellular reduction with release of the active species. The cytotoxic activity of the complex resulted remarkably potentiated reaching nanomolar activity. It is possible that the coordinated Ibuprofen molecules promote the transport and the accumulation of the complex in tumor cells by increasing its lipophilicity. In addition to the increased uptake, Ibuprofen could also exert an anti-inflammatory action mediated by inhibition of the COX enzymes which are overexpressed in tumors. The use of a conjugate with Pt-bound Ibuprofen can ensure similar cellular uptake and biodistribution for both the anti-inflammatory and the cytotoxic drugs in the exact ratio of 2:1.

Six platinum(IV) compounds derived from an oxaliplatin analogue containing the unsaturated cyclic diamine trans-1,2-diamino-4-cyclohexene (DACHEX), in place of the 1,2-diaminocyclohexane, and a range of axial ligands, were synthesized and characterized. The derivatives with at least one axial chlorido ligand demonstrated solvent-assisted photoreduction. The electrochemical redox behavior was investigated by cyclic voltammetry, all compounds showed reduction potentials suitable for activation in vivo. X-ray photoelectron spectroscopy (XPS) data indicated an X-ray-induced surface reduction of the Pt(IV) substrates, which correlates with the reduction potentials measured by cyclic voltammetry. The cytotoxic activity was assessed in vitro on a panel of human cancer cell lines, also including oxaliplatin-resistant cancer cells, and compared with that of the reference compounds cisplatin and oxaliplatin, all IC50 values were remarkably lower than those elicited by cisplatin and somewhat lower than those of oxaliplatin. Compared to the other Pt(IV) compounds of the series, the bis-benzoate derivative was by far (5&ndash, 8 times) the most cytotoxic showing that low reduction potential and high lipophilicity are essential for good cytotoxicity. Interestingly, all the complexes proved to be more active than cisplatin and oxaliplatin even in three-dimensional spheroids of A431 human cervical cancer cells.

A new family of anticancer compounds has been derived from oxaliplatin by inserting a double-bond between carbons 4 and 5 of the 1,2-diaminocyclohexane ring. Testing against a panel of human tumour cell lines including cervical (A431), ovarian (2008), and colon carcinomas (HCT-15 and LoVo), and two oxaliplatin-resistant clones (LoVo OXP and LoVo MDR) has shown that the new compounds have, in general, equal if not better cytotoxic activity and are able to overcome the oxaliplatin-resistance. Moreover, the oxalato derivative induced lipid droplets increase in LoVo OXP cells thus suggesting the involvement of metabolism stress in its mechanism of action.

Glutathione (GSH) plays an important role in the development of resistance to platinum-based chemotherapy, since it can prevent drug binding to DNA and resulting apoptosis of tumor cells. The recently re-discovered drug candidate kiteplatin was found active toward cisplatin- and oxaliplatin-resistant tumor cells, and this could be related to a different interplay of drug-inactivation/DNA-interaction processes. In this study GSH and GMP have been chosen as simple models of platinophiles and DNA, respectively, and the reactivity of kiteplatin has been tested toward GMP, after previous interaction with GSH; toward GSH, after previous interaction with GMP; and toward GMP and GSH simultaneously.

In this paper we report the synthesis and characterization of the complex c,t,c-[Pt(dihydrogenpyrophosphate)(OH)2(cis-1,4-DACH)] (2), a Pt(IV) derivative of kiteplatin, which could be characterized by high bone tropism (due to the pyrophosphate ligand), enough stability to be administered orally, and activatable at the tumor site where the strong reduction conditions can promote its reduction to the corresponding Pt(II) counterpart with known cytotoxic activity. The new complex was characterized by ESI-MS, multinuclear NMR, X-Ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CV). 2 is very soluble in aqueous environment, very stable at physiological pH and quite stable also in acidic conditions with the possibility of oral administration. The in vitro cytotoxicity assays, performed against a panel of four human cancer cell lines, have shown that complex 2 has a cytotoxic activity comparable to that of cisplatin and slightly lower than that of kiteplatin and the pyrophosphate Pt(II) precursor. This behavior can be attributed to its reduced ability to enter cancer cells. However, the cytotoxic activity of 2 is very promising considering that generally Pt(IV) complexes have IC50 values greater than those of the Pt(II) counterparts.

With the aim to obtain dual acting drugs able to target both nuclear DNA and mitochondria, Pt(iv) kiteplatin derivatives having dichloroacetate (DCA) ligands in axial positions have been synthesized. The rather fast hydrolysis (t1/2 of ca. 1 h) and reduction (by ascorbic acid) of these Pt(iv) derivatives did not impede a potent pharmacological effect on tumor cells. Moreover, similarly to kiteplatin, also the Pt(iv)-DCA compounds proved to be capable of overcoming oxaliplatin-resistance, which is particularly important in view of the fact that metastatic colorectal cancer is the third most common cancer in males and the second in females. The possible role of DCA released by the Pt(iv) compounds in eliciting the antiproliferative activity has also been investigated. Pt(iv)-DCA compounds determine a substantial increase of ROS production, blockage of oxidative phosphorylation, hypopolarization of the mitochondrial membrane, and caspase-3/7 mediated apoptotic cell death.

Hydroxyapatite (HA) nanocrystals are important inorganic constituents of biological hard tissues in vertebrates and have been proposed as a bone substitute or a coating material for prostheses in biomedicine. Hydroxyapatite is also amenable for its capacity to bind to a great variety of biomolecules and therapeutic agents. As drug carriers, apatite nanoparticles also have the advantage of pH dependent solubility and low toxicity. Thus HA nanoparticles are negligibly soluble at physiological pH but their dissolution is accelerated at lower pH such as that typically found in the vicinity of tumors. In the present study we have investigated the adsorption on and the release from biomimetic HA nanoparticles of two platinum derivatives of cis-1,4-diaminocyclohexane ([PtX2(cis-1,4-DACH)], X2 = Cl2 (1) and 1,1-cyclobutanedicarboxylate (CBDCA, 2)). The first of the two compounds proved to be active against colon cancer cells also resistant to oxaliplatin. The release has been investigated as a function of pH to mimic the different physiological environments of healthy tissues and tumors, and the in vitro cytotoxicity of the releasates from the HA matrices has been assessed against various human cancer cell lines. The results fully confirmed the potential of 1-loaded HA nanoparticles as bone-specific drug delivery devices.

Platinum complexes of the type [Pt(PL)(AL)]2+ where PL is a derivative of 1,10-phenanthroline and AL is cis-1,4-diaminocyclohexane (1,4-dach), have been synthesised and characterised by ultraviolet spectroscopy, elemental microanalysis, nuclear magnetic resonance and X-ray crystallography. The calf-thymus DNA binding affinity of these complexes was determined by isothermal titration calorimetry, revealing higher DNA affinity than their 1S,2S-diaminocyclohexane analogues. In vitro cytotoxicity was assessed in eleven human cell lines, revealing unexpectedly low activity for the 1,4-dach complexes.

A microwave-assisted synthesis of cisplatin, cis-[Pt(NH3)2Cl2], has been developed and optimized on both a 0.2 and 0.05 millimolar scale. The optimized synthetic procedure was modeled after the Lebedinskii-Golovnya method and is suitable for incorporating the radionuclide, (195m)Pt, into cisplatin for biological studies. Highest yields (47%) and purity are obtained using a K2PtCl4 : NH4OAc : KCl molar ratio of 1 : 4 : 2 at a temperature of 100 °C. The entire synthesis and purification procedure requires approximately 80 min. At a reaction temperature of 150 °C, the trans isomer is the exclusive product, suggesting that complexes of the general form, trans-[Pt(RNH2)2Cl2], can be synthesized directly from K2PtCl4 using [RNH3]OAc (R = alkyl or aryl moieties) via a microwave process. Two novel separation procedures have been developed which efficiently remove the major impurity (1 : 1 Magnus-type salt) from the crude reaction product, yielding a product of purity comparable to that obtained by the Dhara method and suitable for biological studies. These procedures are applicable to both the micro- and macro-scale of synthesis. The question of whether this microwave-assisted synthesis of cisplatin will be a preferred method for incorporating (195m)Pt into cisplatin is yet to be determined.

The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence are extensively studied in the laboratory setting. In this study, we examined the literature and found a significant number of studies (11%–34%) in prominent cancer journals utilizing cisplatin dissolved in DMSO. However, dissolving cisplatin in DMSO for laboratory-based studies results in ligand displacement and changes to the structure of the complex. We examined the effect of DMSO on platinum complexes, including cisplatin, carboplatin, and oxaliplatin, finding that DMSO reacted with the complexes, inhibited their cytotoxicity and their ability to initiate cell death. These results render a substantial portion of the literature on cisplatin uninterpretable. Raising awareness of this significant issue in the cancer biology community is critical, and we make recommendations on appropriate solvation of platinum drugs for research. Cancer Res; 74(14); 3913–22. ©2014 AACR.

Kiteplatin, (cis-1,4-DACH)dichloridoplatinum(ii), contains an isomeric form of the carrier ligand present in the successful antitumor drug oxaliplatin and has been recently found to be very active against oxaliplatin-resistant colon cancers, confirming that, by changing the nature of the amine ligand, it is possible to obtain platinum drugs that are not cross-resistant to those already in clinical use. Apart from interaction with DNA, another factor that can affect the activity of platinum drugs is their metabolic fate in the cellular environment. Therefore, kiteplatin has been reacted with S-donor biomolecules, such as glutathione, cysteine, and methionine. The investigation has further confirmed the different reactivity of methionine as compared to cysteine-containing peptides and has unraveled the possibility of cis-1,4-DACH to become mono-coordinated with one free end (a situation never seen for isomeric 1,2-DACH ligands) and to labilize cis ligands as a consequence of its large steric hindrance. The reaction of kiteplatin-GSH adducts with 5'-GMP has also shown how the reaction products can be different depending upon the aerobic or anaerobic reaction conditions used.

The immediate visual comparison of platinum chemotherapeutics’ effects in eukaryotic cells using accessible plant models of transgenic Arabidopsis thaliana is reported. The leading anticancer drug cisplatin, a third generation drug used for colon cancer, oxaliplatin and kiteplatin, promising Pt-based anticancer drugs effective against resistant lines, were administered to transgenic A. thaliana plants monitoring their effects on cells from different tissues. The transgenic plants’ cell cytoskeletons were labelled by the green fluorescent protein (GFP)-tagged microtubule-protein TUA6 (TUA6-GFP), while the vacuolar organization was evidenced by two soluble chimerical GFPs (GFPChi and AleuGFP) and one transmembrane GFP-tagged tonoplast intrinsic protein 1-1 (TIP1.1-GFP). The three drugs showed easily recognizable effects on plant subcellular organization, thereby providing evidence for a differentiated drug targeting. Genetically modified A. thaliana are confirmed as a possible rapid and low-cost screening tool for better understanding the mechanism of action of human anticancer drugs.

This tribute to Dr Rosenberg is especially fitting, as an introduction to this themed issue, since the discovery of Cisplatin has not only had an enormous impact on the practice of modern day cancer chemotherapy, it has also promoted the explosive growth and maturity of the field of contemporary bioinorganic chemistry.

Although the encouraging antitumor activity of [PtCl2(cis-1,4-DACH)] (1; DACH = diaminocyclohexane) was shown in early studies almost 20 years ago, the compound has remained nearly neglected. In contrast, oxaliplatin, containing isomeric 1R,2R-DACH carrier ligand, enjoys worldwide clinic application as a most important therapeutic agent in the treatment of colorectal cancer. By extending the investigation to human chemo-resistant cancer cells, we have demonstrated the real effectiveness of 1 in circumventing cisplatin and oxaliplatin resistance in LoVo colon cancer cells. The uptake of compound 1 by the latter cells was similar to that of sensitive LoVo cells. This is not the case for all other compounds considered in this investigation. Interaction with ds-DNA, investigated by a biosensor assay and by QM/MM geometry optimization of the 1,2-GG intrastrand cross-link, does not show significant differences between 1 and oxaliplatin. However, the DNA adducts of 1 are removed from repair systems with lower efficiency and are more effective in inhibiting DNA and RNA polymerase.

Biodegradable, PEG-coated, nanoparticles (NPs) have gained therapeutic application as injectable colloidal systems for the controlled and site-specific release of drugs. In this paper, encapsulation in PLGA-PEG polymer NPs has been exploited to lower the toxicity and to increase the antitumor activity of kiteplatin ([PtCl2(cis-1,4-DACH)]). Kiteplatin contains an isomeric form of the diamine ligand present in oxaliplatin and proved to be particularly active against ovarian and colon cancers. To favor encapsulation of the platinum drug in the hydrophobic core of the polymeric micelles, Pt(iv) prodrugs having hydrophobic carboxylic ligands at the axial positions were used in place of hydrophilic Pt(ii) complexes (compounds 1-4). The size, size distribution, and zeta potential (ZP) were measured by dynamic light scattering (DLS) and laser Doppler velocimetry (LDV), and drug encapsulation efficiency (EE) correlated to the alkyl chain length of the different Pt(iv) prodrugs. The number of the Pt atoms per NP (in the range of 1.3-2.4 × 10(6)) is comparable to that of polysilsesquioxane-based NPs and higher than that found for other nanoparticle platforms. The platinum-loaded PLGA-PEG NPs, tested in vivo in a syngeneic murine solid tumor (LLC), had a higher antitumor effect and, most importantly, were markedly less toxic than kiteplatin.

Kiteplatin, the neglected drug analogous of cisplatin but containing cis-1,4-DACH in place of the two ammines, has been recently reevaluated for its activity against cisplatin- and oxaliplatin-resistant tumors, in particular colo-rectal cancer. With the aim of further improving the pharmacological activity of this drug, Pt(IV) prodrugs were derived by addition of two, differently substituted, benzoate groups in axial positions (X-ray structure). The cytotoxic activity of both compounds resulted markedly potentiated reaching nanomolar concentration against a wide panel of human cancer cells. The ability of benzoate ligands to enhance the activity of kiteplatin most likely originates from their lipophilicity promoting a higher drug accumulation in cancer cells; however, it is to be noted that the increase in pharmacological effect is far greater than the increase in cellular uptake. Overcoming cisplatin- and oxaliplatin-resistance by kiteplatin derivatives appears to relate to the inability of membrane extrusion pumps to remove active Pt species from tumor cells.

Author(s): Muggia, Franco M; Bonetti, Andrea; Hoeschele, James D; Rozencweig, Marcel; Howell, Stephen B

The anticancer activity of cisplatin is triggered by its formation of intrastrand adducts involving adjacent G residues of DNA. To obtain information on the different conformers that can be formed{,} carrier ligands such as 2{,}2[prime or minute]-bipiperidine{,} which provide large steric bulk near the platinum coordination plane and decrease the dynamic motion about the Pt-N7 bonds{,} were introduced ({"}retro-modelling{"} approach). In the present study we investigate the effect of cis-1{,}4-diaminocyclohexane (cis-1{,}4-DACH) on the formation{,} stability{,} and stereochemistry of (cis-1{,}4-DACH)Pt(ss-oligo) adducts (ss-oligo = d(GpG) with 3[prime or minute]- and/or 5[prime or minute]-substituents). Interesting features of this ligand{,} absent in previous retro-modelling studies{,} include the large bite angle (expected to impede the ease of interconversion between possible conformers){,} the presence of two protons on each nitrogen (a characteristic associated with antitumor activity){,} and the absence of chiral centres. The use of cis-1{,}4-DACH has made it possible to detect different conformers in a system containing a primary diamine carrier ligand associated with anticancer activity and to confirm the previous hypothesis that the coexistence of different conformers established in studies of retro models having relatively bulky ligands is not an artefact resulting from carrier-ligand bulk. Moreover{,} the data for the (cis-1{,}4-DACH)Pt(d(GpG)) and (cis-1{,}4-DACH)Pt(d(GGTTT)) adducts indicate that at a temperature close to the physiological one (40 [degree]C) HH1 and [capital Delta]HT1 conformers are present in comparable amounts. In contrast{,} at low temperature (close to 0 [degree]C) the equilibrium shifts dramatically toward the more stable HH1 conformer (for the (cis-1{,}4-DACH)Pt(d(TGGT)) adduct the HH1 conformer is always dominant{,} even at high temperature). Notably{,} (cis-1{,}4-DACH)PtCl2 (Kiteplatin) has been recently reinvestigated and found to be particularly active against colorectal cancer (including oxaliplatin-resistant phenotypes).

Although the encouraging antitumor activity of [PtCl(2)(cis-1,4-DACH)] (1; DACH = diaminocyclohexane) was shown in early studies almost 20 years ago, the compound has remained nearly neglected. In contrast, oxaliplatin, containing the isomeric 1(R),2(R)-DACH carrier ligand, enjoys worldwide clinic application as a most important therapeutic agent in the treatment of colorectal cancer. By extending the investigation to human chemotherapy-resistant cancer cells, we have demonstrated the real effectiveness of 1 in circumventing cisplatin and oxaliplatin resistance in LoVo colon cancer cells. The uptake of compound 1 by the latter cells was similar to that of sensitive LoVo cells. This is not the case for all other compounds considered in this investigation. Interaction with double-stranded DNA, investigated by a biosensor assay and by quantum mechanical/molecular mechanical geometry optimization of the 1,2-GG intrastrand cross-link, does not show significant differences between 1 and oxaliplatin. However, the DNA adducts of 1 are removed from repair systems with lower efficiency and are more effective in inhibiting DNA and RNA polymerase.

A wide range of asymmetric Schiff base ligands containing primary amine functions can be prepared in one step and in high yield by the reaction of VO(OEt) 3 with salicylaldehyde and the appropriate diamine. The resulting VO 2 L complexes contain tridentate ligands with phenolate, imine, and amine coordination to the dioxovanadium(V) complex ion. An X-ray structure of one compound ([VO 2 (1,2-pnSAL), 5A, where 1,2-pnSAL=1(N-salicylideneamina)-2-aminopropane) demonstrates that it is a dimer in the solid state with a V 2 O 4 score

Forty-one compounds of the general formula V v OLL', where L is a tridentate ligand (HSALIMH = [4-(2-salicylideneaminato)ethyl]imidazole; H 2 SHED = N-(salicylideneaminato)-N'-(2-hydroxyethyl)ethylenediamine; HENSAL = N-(salicylideneaminato)ethylenediamine) and L' is a bidentate, noninnocent ligand (e.g., catechol (H 2 CAT), pyrogallol (H 2 PYR), or salicylhyddroxamic acid (H 3 SHI) have been prepared and spectroscopically characterized

Selected physical and inorganic concepts and factors which might be important in assessing and/or understanding the fate and disposition of metal compounds in a biological environment are reviewed. The stereochemistry (geometry), thermodynamic stability, redox properties, and intrinsic reactivity/lability are properties of metal compounds which can have a major influence on metal-target binding and on the eliciting of activity/toxicity. Concepts and factors are illustrated with appropriate examples where possible. Efforts to correlate the toxicity of metal compounds with a suitable indicator of toxicity should be expanded to include other parameters or combinations of parameters.

The organometallic half-sandwich Ru(II) arene anticancer complex [(eta(6)-fluorene)Ru(en)Cl]PF(6) () has been synthesized in high yield and purity on a micromole scale with incorporation of the beta-emitting radioisotope (106)Ru (half-life = 1.01 y) using a refined procedure involving conversion of RuCl(3) into [(eta(6)-fluorene)RuCl(2)](2), and then [(eta(6)-fluorene)Ru(CH(3)CN)(2)Cl]PF(6) as intermediates. Distribution studies 0.25 h post i.v. injection of (106)Ru- at a dose of 25 mg kg(-1) show that (106)Ru is well distributed throughout the tissues of a rat. This appears to be the first report of the radiolabelling of a potential ruthenium antitumour agent for distribution/biological studies.

Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl(2)] or cis-[PtCl(2)(NH(3))(2)] have been investigated in solution via observation of [(1)H,(15)N] NMR resonances of the Pt complexes, [(1)H,(13)C] resonances of the eCH(3) groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.

The first two analogs 5a,b of a new class of neutral large-ring square-planar Pt(II) chelate complexes of the generic structure [Pt(cis-1,4-dach)X2] were synthesized via a refined technique, structurally characterized by NMR (1H, 13C, 195Pt), FAB mass spectrometry, and X-ray crystallography, and evaluated for antitumor activity in vitro and in vivo in sensitive and Pt-resistant murine leukemia cell systems. An X-ray crystal structure analysis confirmed that [Pt(cis-1,4-dach)malonate] 5b is monomeric and that the cis-1,4-diaminocyclohexane (dach) ligand is incorporated in a unique and previously unknown locked boat conformation. Complex 5b crystallized as colorless rectangular plates in the orthorhombic space group Pcmn with Z = 4 and the lattice parameters a = 6.239(1) A, b = 9.965(2) A, and c = 18.437(4) A. Important structural parameters are Pt-O = 2.024(5) A, Pt-N = 2.021(6) A, N-Pt-N = 100 degrees, and N-Pt-O = 85 degrees; R = 0.0515, Rw = 0.0635. Antitumor results in murine tumor models show that the parent molecule 5a (X2 = 2 Cl) (a) is more dose potent than cisplatin against the leukemias and solid tumors examined, (b) possesses significant activity against cisplatin-resistant leukemias, but exhibits partial cross-resistance with cisplatin, and (c) may possess a spectrum of activity different from that of cisplatin. Antitumor test results in vitro indicate that (a) 5a is at least equivalent to cisplatin in dose potency and effectiveness in the leukemia cell systems studied except in the [Pt(1,2-dach)Cl2]-resistant L1210 cell line, (b) the cisplatin-resistant leukemia cell systems exhibit partial cross-resistance to 5a, (c) 5a possesses either comparable or greater cytotoxicity than the reference complexes, CI-973 (3) and bis(platinum) complex 4, and (d) 5a is more effective (approximately 18-fold) than [Pt(1R,2R-dach)Cl2] 2 in inhibiting growth in the Pt(1,2-dach)-resistant L1210 cell line, suggesting that [Pt(cis-1,4-dach)Cl2] is either not recognized as or is not acting as a "typical" Pt(dach) complex. The encouraging antitumor activity of 5a, coupled with a 10-fold higher aqueous solubility compared to [Pt(1R,2R-dach)-Cl2] 2 warrants the following future studies: synthesis of selected analogs, elucidating the nature of Pt-DNA binding sites, the mechanism of action, and the mechanistic basis for the lack of cross-resistance of [Pt(cis-1,4-dach)Cl2] against the [Pt(1,2-dach)Cl2]-resistant L1210 cell line.

Comparative Nephrotoxicity of a Novel Platinum Compound, Cisplatin, and Carboplatin in Male Wistar Rats. Wolfgang, G. H. I., Dominick, M. A., Walsh, K. M., Hoeschele, J. D., and Pegs, D. G. (1994). Fundam. Appl. Toxicol. 22, 73-79. The nephrotoxicity of three platinum-containing antitumor agents was compared at doses that approximate the LD10 (cisplatin) or the LD50 (CI-973, carboplatin) doses. Male Wistar rats were administered single iv doses of 45 mg/kg CI-973, 6.5 mg/kg cisplatin, or 65 mg/kg carboplatin and observed for 4 days. Cisplatin treatment increased blood urea nitrogen (4×), creatinine (3×), glucose, and fractional electrolyte excretions, and decreased creatinine clearance by Day 4. These parameters were not significantly altered in CI-973- and carboplatin-treated animals. Cisplatin increased urinary excretion of LDH (sixfold), GGT (twofold), and NAG (twofold); CI-973 and carboplatin increased GGT excretion (approximately twofold). Cisplatin induced the following functional changes as a consequence of direct nephrotoxicity: decreases in GFR (84%), ERPF (97%), ERBF (96%), and ERTS (95%), and increases in FF (fivefold), Functional changes, attributed to prerenal effects of CI-973, included a decrease in ERPF (35%) and an increase in FF (48%). No changes were seen following carboplatin treatment. All cisplatin-treated rats had proximal tubular necrosis in the outer stripe of the outer medulla, extending multifocally into inner cortical medullary rays. No renal lesions were detected by light or electron microscopy in the control or CI-973- or carboplatin-treated rats. Cisplatin produced marked nephrotoxicity as determined by biochemical, functional, and histopathologic endpoints. CI-973 and carboplatin were significantly less nephrotoxic than cisplatin.

The biological activity of a series of dinuclear bis(platinum) complexes of formula [(cis-PtX2-(NH3)]2(NH2(CH2)nNH2)] (X = Cl, n = 4-9, compounds 6-11; X2 = malonate, n = 5 or 6, compounds 12 and 13) is described in selected murine leukemia, murine solid tumor, and human tumor cell lines and in murine leukemia cell lines rendered resistant to cisplatin (cis-[Pt(NH3)2Cl2]). The bis(platinum) compounds showed greater activity in vitro against murine tumor cell lines resistant to either cisplatin or DACH ([Pt(DACH)Cl2]). The resistance factor is dependent on chain length of the diamine, and the structural feature of a dinuclear complex is of general use in reducing cross-resistance with cisplatin. In vivo [(cis-PtCl2(NH3)]2(NH2(CH2)5NH2)] (7) showed a % T/C of 204 against murine L1210 leukemia resistant to cisplatin compared to a % T/C of 104 for cisplatin itself at optimal doses. The complex [(Pt(mal)(NH3)]2(NH2(CH2)6NH2)] (13) was highly active in the colon 26 tumor line with 3/10 tumor-free survivors (dose of 186 mg/kg, ip D1,5,9); however, 13 was subject to substantial cross-resistance in the cisplatin resistant L1210 leukemia (% T/C 139 versus % T/C of 223 in the sensitive line). In four selected human tumor lines in vitro, compounds 6-11 were uniformly more potent than cisplatin. In the corresponding xenografts, compound 7 showed greater activity in the HCT-8 (coloadenocarcinoma) and H23 (nonsmall cell lung), but diminished potency in AH125 and H520 (both nonsmall cell lung) lines in comparison to cisplatin. Retention of activity against cisplatin-resistant cell lines and a different spectrum of activity compared to cisplatin in some human tumor cell lines suggest that this class of complexes is mechanistically different from mononuclear complexes and worthy of further development toward clinical trials.

F040 STUDIES OF THE ADDUCT FORMED ON INTERACTING CISPLATIN WITH HUMAN APOTRANSFERRIN: SYNTHESIS, IN VITRO BINDING KINETICS, IN VIVO PHARMACOKINETICS AND BIODISTRIBUTION IN THE RAT. J.D. Hoeschelea L.R. Whitfield,b W.R. Leopold,b W. McNally,b J. Nawrocki,b J. Phillips,C a Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109-I 055 b Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48105 and C Cancer Therapy and Research Foundation of South Texas, San Antonio, TX 78229. Transferrins are glycoproteins which are important in the cellular transport and storage of iron and other metal ions into cells [ 11. Since many tumor cells overproduce transferrin receptors, an attractive concept is to consider using transferrin as a delivery system to target cancer drugs specifically to tumor cells. We have undertaken a test of this concept using a cisplatin(cisPt)apotransferrin(aTf) adduct which was (1) synthesized, isolated (via dialysis), and analyzed via 1g5mPt radioactivity, (2) tested in vivo for antitumor activity vs implanted M5076 ovarian sarcoma tumors in the mouse, and (3) used to study the pharmacokinetic and biodistribution properties in vivo for comparison with those of cisplatin. The results and conclusions are as follows: (1) CisPt reacts with human aTf with an apparent first-order rate constant of -2x10-5 set-l. Since this rate constant is comparable to that for the aquation reaction of cisPt, it is likely that the reaction proceeds via an aquation intermediate. (2) The isolated adduct is non-stoichiometric with an approximate empirical formula of Pt~.410_75Tf. (A Pt/Tf ratio of 1.8-2.0 would be expected if the cisPt-aTf reaction went to completion and cisPt occupied both available Fe@) binding sites.) (3) The cisPt-aTf adduct is inactive against murine M5076 ovarian sarcoma over a wide range of dose levels (799-3318 mg as Tf/Kg body weight), indicating that active cisPt is not being delivered via the adduct. (4) The cisPtaTf adduct is a unique entity which exhibits pharmacokinetic and biodistribution properties distinctly different from those of cispt.

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammineplatinum(II]. Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl2 significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl2 pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.

The reactions of bovine serum albumin (BSA) with (trimethylphosphine)(2,3,4,6-tetra-O-acetyl-1-thio-..beta..-D-glucopyranosato-S)gold(I), Me/sub 3/PAuSAtg, and its chloro analogue, Me/sub 3/PAuCl, were studied to develop insights into the role of the phosphine ligand in the serum chemistry of the related antiarthritic drug auranofin (triethylphosphine)(2,3,4,6-tetra-O-acetyl-1-thio-..beta..-D-glucopyranosato-S)gold(I). /sup 31/P NMR spectroscopy, protein modification, and gel-exclusion chromatography methods were employed. Comparison of the reactions of the methyl derivatives to the previously reported reactions of auranofin and Et/sub 3/PAuCl with BSA demonstrated that similar chemical species are formed but revealed three major differences. Despite these differences, the results for the methyl analogues provide important confirmation for previously developed chemical models of auranofin reactions in serum. Me/sub 3/PO was not observed in reaction mixtures lacking tetraacetylthioglucose (AtgSH); this result affirms the role of AtgSH, displaced by the reaction of Me/sub 3/PAuSAtg at Cys-34, in the generation of the phosphine oxide (an important metabolite in vivo). The weak binding sites on albumin react with Me/sub 3/PAuCl, but not Me/sub 3/PAuSAtg, demonstrating the importance of the strength and reactivity of the anionic ligand-gold bond on the reactions of auranofin analogues. The gold binding capacity of albumin is enhanced after Me/sub 3/PO is formed, consistent with reductive cleavage of albumin disulfide bonds bymore » trimethylphosphine. 24 references, 2 figures, 3 tables.« less

The binding of cis(c)- and trans(t)-Pt(NH3)2Cl2 to DNA at platinum/DNA-nucleotide ratios (Ri) of 0.1 or less has been studied by means of radioactive 195mPt-labeled compounds. Kinetic data are consistent with the following scheme: (Formula: see text). At 25 degrees C and pH 5-6 in 5 mM NaClO4, the values for the rate constants in the above scheme for the c-isomer are k2 = 2.2 X 10(-5) sec -1, k7 = 0.32 (sec M)-1, and k8 = 143 (sec M)-1; for the t-isomer the values are k2 less than 0.5 X 10(-5) sec-1 and k7 = 0.95 (sec M)-1. Platinum-DNA adducts do not undergo detectable exchange for 3 days at 37 degrees C, indicating the absence of a dynamic equilibrium. For both isomers the rate of binding is the same for single- and double-stranded DNA. The conclusions derived from Ag+ and H+ titration studies are consistent with binding at guanine N(7) for Ri less than 0.1. The reaction rate is competitively inhibited by various salts and buffers and is suppressed by raising the pH (50% inhibition of initial rates at pH 7.3). At 37 degrees C and pH 7 in 0.15 M NaCl, 6-8% of both the c- and t-isomers bind to DNA in 24 h, suggesting that both compounds should bind to DNA under biological conditions.