James D. Hoeschele

Studies on the antitumor activity of group VIII transition metal complexes. Part I. Platinum (II) complexes

Abstract A wide variety of Platinum(II) complexes have been investigated for antitumor activity against Sarcoma 180 in Swiss white female mice. In general, only neutral complexes exhibit activity while charged species are inactive and relatively nontoxic. A series of complexes of the type cis -[PtA 2 X 2 ] (where A 2 is either two monodentate or one bidentate amine ligand and X 2 is either two monodentate or one bidentate anionic ligand) have been studied with A and X being systematically varied. This has resulted in the identification of at least ten potentially active antitumor drugs. Trans isomers are inactive in comparison with active cis complexes and the presence of cis -reactive ligands appears to be a necessary parameter for antitumor activity. Complexes with highly reactive ligands such as cis -[Pt(NH 3 ) 2 (H 3 O) 2 ] (NO 3 ) 2 are highly toxic. Palladium(II) complexes, which are analogs of the active Platinum(II) compounds, have been found to be inactive.

Kinetic analysis of the in vitro binding of radioactive cis- and trans-dichlorodiammineplatinum(II) to DNA.

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.

In vivo and in vitro binding of platinum to metallothionein.

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.

Chemical softness and acute metal toxicity in mice and Drosophila

Correlations reported by Jones and Vaughn between chemical softness parameters and LD50s for metal compounds in rodents have been confirmed and extended. In the present study 14-day LD50s for mice were determined under uniform experimental conditions for 24 metal salts injected ip. For certain ions the correlation between the LD50 and the Pearson and Mawby softness parameter, sigma/sup p/, is better than that obtained by Jones and Vaughn using data from diverse sources. Notably, however, we found no correlation for the alkaline-earth metals either with sigma/sub p/ or with other softness parameters. In parallel experiments with the same metal compounds, we also found correlations between sigma/sub p/ and 4-day LC50s for Drosophila exposed to the salts in their food. For both the mouse and Drosophila, soft ions were generally found to be more toxic than hard ions such that their rankings are significantly correlated. Several differences in relative toxicities of the metal ions were found to occur between mice and Drosophila, and some interpretations of these findings are given. Correlations between toxicity and physicochemical parameters of toxic materials can offer insights into biochemical mechanisms of toxicity and may be useful in predicting toxicity.

Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Abstract Reactions between various apo and metal-bound forms of human serum transferrin (80 kDa) and the recombinant N-lobe (40 kDa) with [Pt(en)Cl2] or cis-[PtCl2(NH3)2] have been investigated in solution via observation of [1H,15N] NMR resonances of the Pt complexes, [1H,13C] resonances of the eCH3 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.

Revisiting [PtCl2(cis-1,4-DACH)]: An Underestimated Antitumor Drug with Potential Application to the Treatment of Oxaliplatin-Refractory Colorectal Cancer

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.

Investigation of correlations between chemical parameters of metal ions and acute toxicity in mice and drosophila

We are studying correlations between physicochemical properties associated with metal ions and observed toxicity. In order to test correlations, we obtained, under uniform conditions, LD50 values for acute toxicity in mice for 24 metal ions. The new data show a better correlation between LD50 and Pearson and Mawbys softness parameter sigma p, defined using chemical concepts of hard and soft acids and bases, than had been obtained by others. From a wide range of physicochemical parameters, the electrode potential can give almost as good a correlation as sigma p. Better correlations might exist for parameters more relevant to biological systems.

Inorganic concepts relevant to metal binding, activity, and toxicity in a biological system

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.

Distribution of platinum chemotherapeutic complexes in normal tissues and in tumors

Abstract Pharmacokinetic studies in animals and in man have reported poor penetration of the parent platinum complex, cis -dichloroammineplatinum ( cis -DDP), into the brain. In this study, levels of a 195m Pt-labeled analog, malonato-diaminocyclohexaneplatinum(II) (PHM), were measured in tissues of normal and brain tumor-bearing rats between 1 and 30 hr after injection. Higher concentrations of platinum were recorded in tissues of tumor-bearing rats compared to tissues of normal animals and in intracerebral brain tumors compared to normal brain. A peak of a secondary maximum of uptake was observed at 24 hr. Results predict that it should be possible to obtain levels of platinum in the brain tumors on the order of levels that produced enhanced radiation effects in previous studies using cultured mammalian cells.

Adducts formed between deoxyguanosine and cis-Dichlorodiammineplatinum(II): Separation by means of cation-exchange chromatography

Abstract The interaction between deoxyguanosine (dG) and cis -dichlorodiammineplatinum(II) ( cis -Pt) leads to the 2:1 and the 1:1 dG-Pt adducts. These adducts were separated on an Aminex A6 cationexchange column by use ot 0.01 M K 2 CO 3 (pH 11) as an eluent. The stoichiometry of the adducts was determined from the 195m Pt radioactivity and from the absorbance of the guanine chromophore at 280 nm. Time-course studies show that dG reacts initially with cis -Pt to form the 1:1 adduct, which then interacts with a second molecule of dG to form the 2:1 adduct. Acid hydrolysis (100°C in 88% formic acid for 5–15 min) of the 1:1 and 2:1 adducts results in their conversion to two new products, which elute differently from the column but which still contain Pt bound in the same stoichiometric ratio to dG as in the nonhydrolyzed adducts. The hydrolyzed adducts show a negative diphenylamine reaction indicative ot cleavage of the glycosidic bond. It is concluded that mild acid hydrolysis converts the 1:1 and 2:1 dG-Pt adducts into the corresponding guanine-Pt adducts, which are chromatographically distinguishable. This acid hydrolysis-high pressure liquid chromatography (HPLC) procedure has application to the identification of the Pt adducts formed in DNA.