Development of HPLC conditions for valid determination of hydrolysis products of cisplatin.

In water, the antineoplastic drug cisplatin, cis-[PtCl2(NH3)2] (1) hydrolyses slowly to the aqua complexes cis-[Pt(NH3)2Cl(H2O)]+ (2) and, to a small extent, cis-[Pt(NH3)2(H2O)2]2+ (3), which are thought to play an important role in the metabolism of cisplatin. HPLC is a useful technique for monitoring 2 and 3, but only if the components of the mobile phase used in the reverse phase HPLC technique are unreactive toward these aqua complexes under the conditions of the experiment. 15N Nuclear magnetic resonance (NMR) with samples highly enriched (>98%) in 15N has been used to check the reactivity of 2 and 3 toward substances commonly used as components of the mobile phase. The results reported herein indicate that acetonitrile, often used as an organic modifier, reacts readily with 2 and 3. Methanol, also commonly employed, is much less reactive. Carboxylic acids RCO2H (R = CH3, H, CF3), which are frequently used to adjust pH of the mobile phase, also react readily with 2 and 3. Trifluoromethanesulfonic acid ("triflic acid"), CF3SO3H, is unreactive. Neither hexanesulfonic acid nor sodium dodecyl sulfate (SDS), used as "ion-pairing agents", reacts significantly with 2 or 3 under the experimental conditions, but SDS gives better peak separation. Commercial SDS must, however, be purified from chloride contamination. From our studies, optimal conditions for HPLC separation of 1, 2, and 3, with a C18 stationary phase at 37 degrees C, require an aqueous mobile phase with 3% v/v methanol, 0.05 mM SDS, and pH 2.5 (adjusted with triflic acid). This technique was then used to measure levels of 1, 2, and 3 in ultrafiltered serum after incubation for various times with cisplatin at 37 degrees C.

Reactions of cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques.

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.

Effects of geometric isomerism and ligand substitution in bifunctional dinuclear platinum complexes on binding properties and conformational changes in DNA.

The DNA binding profile of a series of dinuclear platinum complexes [{trans-PtCl-(L)2}2H2N(CH2)nNH2]2+ (L = NH3 or py; 1,1/t,t/NH3 and 1,1/t,t/py, respectively) and [{cis-PtCl-(NH3)2H2N(CH2)nNH2]2+ (1,1/c,c/NH3) was examined to compare the effects of geometrical isomerism and the presence of ligands other than NH3 in the coordination sphere. Steric effects, because of the geometry of the leaving groups cis to the diamine bridge or the presence of planar pyridine ligands, result in diminished binding to calf thymus DNA for these isomers. In contrast, the pyridine derivative shows a distinct binding preference for poly(dG-dC).poly(dG-dC) in comparison to both NH3 isomers. Both NH3 complexes induced the B-->Z transition in poly(dG-dC).poly(dG-dC), but the presence of a pyridine ligand stabilized the B conformation. The bifunctional binding of the NH3 isomers results in unwinding of supercoiled pUC19 plasmid DNA equivalent to cis-DDP, while the unwinding of the pyridine derivative is approximately twice that of the mononuclear trans-[PtCl2(py)2]. DNA-DNA interstrand cross-linking is very efficient for all three agents, but sequencing studies indicated that only the 1,1/t,t/NH3 derivative is capable of forming a (Pt,Pt) intrastrand cross-link to the adjacent guanines of a d(GpG) sequence. The effects on DNA caused by bifunctional binding of dinuclear complexes are compared with those from the mononuclear [PtCl2(NH3)2] isomers. The results are discussed with respect to the antitumor activity of the dinuclear series.