Cisplatin Carbonato Complexes. Implications for Uptake, Antitumor Properties, and Toxicity

The reaction of aquated cisplatin with carbonate which is present in culture media and blood is described. The first formed complex is a monochloro monocarbonato species, which upon continued exposure to carbonate slowly forms a biscarbonato complex. The formation of carbonato species under conditions that simulate therapy may have important implications for uptake, antitumor properties, and toxicity of cisplatin

Stability of Carboplatin and Oxaliplatin in their Infusion Solutions is Due to Self-Association

Carboplatin and oxaliplatin are commonly used platinum anticancer agents that are sold as ready-to-use aqueous infusion solutions with shelf lives of 2 and 3 years, respectively. The observed rate constants for the hydrolysis of these drugs, however, are too large to account for their long shelf lives. We here use electrospray-trap mass spectrometry to show that carboplatin and oxaliplatin are self-associated at concentrations in their ready-to-use infusion solutions (∼27 mM and 13 mM, respectively) and, as expected, when the drug concentration is reduced to more physiologically relevant concentrations (100 μM and 5 μM, respectively) the association equilibrium is shifted in favor of the monomeric forms of these drugs. Using 1H NMR we measure the intensity of the NH resonance of the two symmetry-equivalent NH 3 molecules of carboplatin, relative to the intensity of the γ-methylene CH resonance, as a function of total drug concentration. Then, by fitting the data to models of different molecularity, we show that the association complex is a dimer with a monomer-dimer association constant of K (M -1) = 391 ± 127. The work presented here shows that carboplatin and oxaliplatin mainly exist as association complexes in concentrated aqueous solution, a property that accounts for the long term stability of their ready-to-use infusion solutions, and that these association complexes may exist, to some extent, in the blood after injection

Pt (IV) Complexes as Prodrugs for Cisplatin

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl 2(OH) 2(NH 3) 2], 3, and a carboxylate-modified analog, c,t,c-[PtCl 2(OH)(O 2CCH 2CH 2CO 2H)(NH 3) 2], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using 195Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, 195Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, 13C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed

Activation of Carboplatin by Carbonate

Carboplatin, [Pt(NH3)2(CBDCA-O,O\u27)], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, is in wide clinical use for the treatment of ovarian, lung, and other types of cancer. Because carboplatin is relatively unreactive toward nucleophiles, an important question concerning the drug is the mechanism by which it is activated in vivo. Using [1H,15N] heteronuclear single quantum coherance spectroscopy (HSQC) NMR and 15N-labeled carboplatin, we show that carboplatin reacts with carbonate ion in carbonate buffer to produce ring-opened products, the nature of which depends on the pH of the medium. The assignment of HSQC NMR resonances was facilitated by studying the reaction of carboplatin in strong acid, which also produces a ring-opened product. The HSQC NMR spectra and UV-visible difference spectra show that reaction of carboplatin with carbonate at pH \u3e 8.6 produces mainly cis-[Pt(NH3)2(CO3(-2))(CBDCA-O)]-2, 5, which contains the mono-dentate CBDCA ligand and mono-dentate carbonate. At pH 6.7, the primary product is the corresponding bicarbonato complex, which may be in equilibrium with its decarboxylated hydroxo analogue. The UV-visible absorption data indicate that the pKb for the protonation of 5 is approximately 8.6. Thus, the reaction of carboplatin with carbonate produces a mixture of ring-opened species that are anions at physiological pH. HSQC NMR studies on 15N-labeled carboplatin in RPMI culture media containing 10% fetal bovine serum with and without added carbonate suggest that carbonate is the attacking nucleophile in culture media. However, because the rate of reaction of carbonate with carboplatin at physiological pH is small, NMR peaks for ring-opened carboplatin were not detected with HSQC NMR. The rate of disappearance of carboplatin in culture medium containing 9 x 10(8) Jurkat cells is essentially the same as that in carbonate buffer, indicating that the ring-opening reaction is not affected by the presence of cells. This work shows that carbonate at concentrations found in culture media, blood, and the cytosol readily displaces one arm of the CBDCA ligand of carboplatin to give a ring-opened product, which at physiological pH is a mixture of anions. These ring-opened species may be important in the uptake, antitumor properties, and toxicity of carboplatin

Modification of Carboplatin by Jurkat Cells

Using [1H,15N] heteronuclear single quantum coherance (HSQC) NMR and 15N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt2+ anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k1 = kc [CO32 -] + km + ku N, where kc is the rate constant for reaction of 1 with carbonate in the medium, km is the rate constant for reaction of 1 with all other components of the medium, and ku is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium

Role of Carbonate in the Cytotoxicity of Carboplatin

Carboplatin, [Pt(NH3)2(CBDCA-O,O\u27)], 1, where CBDCA is cyclobutane-1,1-dicarboxylate, is used against ovarian, lung, and other types of cancer. We recently showed (Di Pasqua et al. (2006) Chem. Res. Toxicol. 19, 139-149) that carboplatin reacts with carbonate under conditions that simulate therapy to produce carbonato carboplatin, cis-[Pt(NH3)2(O-CBDCA)(CO3)]2-, 2. We use 13C and 1H NMR and UV-visible absorption spectroscopy to show that solutions containing carboplatin that have been aged in carbonate buffer under various conditions contain 1, 2, and other compounds. We then show that aging carboplatin in carbonate produces compounds that are more toxic to human neuroblastoma (SK-N-SH), proximal renal tubule (HK-2) and Namalwa-luc Burkitt\u27s lymphoma (BL) cells than carboplatin alone. Moreover, increasing the aging time increases the cytotoxicity of the platinum solutions as measured by the increase in cell death. Although HK-2 cells experience a large loss in survival upon exposure to carbonato forms of the drug, they have the highest values of IC50 of the three cell lines studied, so that HK-2 cells remain the most resistant to the toxic effects of the carbonato forms in the culture medium. This is consistent with the well-known low renal toxicity observed for carboplatin in therapy. The uptake rates for normal Jurkat cells (NJ) and cisplatin-resistant Jurkat cells (RJ), measured by inductively coupled plasma mass spectrometry (ICP-MS), are 16.6 +/- 4.2 and 12.3 +/- 4.8 amol of Pt h-1 cell-1, respectively, when exposed to carboplatin alone. However, when these cells are exposed to carboplatin that has been aged in carbonate media, normal Jurkat cells strongly bind/take up Pt at a rate of 14.5 +/- 4.1 amol of Pt h-1 cell-1, while resistant cells strongly bind/take up 5.1 +/- 3.3 amol of Pt h-1 cell-1. Collectively, these studies show that carboplatin carbonato species may play a major role in the cytotoxicity and uptake of carboplatin by cells

Modification of carboplatin by Jurkat cells

Using [1H,15N] heteronuclear single quantum coherance (HSQC) NMR and 15N-labeled carboplatin, 1, we show that Jurkat cells affect the rate of disappearance of the HSQC NMR peak in culture medium for this Pt2+ anticancer drug. The decay or disappearance rate constant for 1 in culture medium containing cells is k1=kc[CO32-]+km+kuN, where kc is the rate constant for reaction of 1 with carbonate in the medium, km is the rate constant for reaction of 1 with all other components of the medium, and ku is the rate constant for reaction of 1 with cells having a number density N in the medium. Since Jurkat cells only take up a small amount of the platinum present in the medium (\u3c1%), the observed disappearance of the HSQC NMR peak for 1 cannot be due to uptake of carboplatin by the cells

New Extracellular Resistance Mechanism for Cisplatin

The HSQC NMR spectrum of 15N-cisplatin in cell growth media shows resonances corresponding to the monocarbonato complex, cis-[Pt(NH3)2(CO3)Cl]-, 4, and the dicarbonato complex, cis-[Pt(NH3)2(CO3)2]-2, 5, in addition to cisplatin itself, cis-[Pt(NH3)2Cl2], 1. The presence of Jurkat cells reduces the amount of detectable carbonato species by (2.8 ± 0.7) fmol per cell and has little effect on species 1. Jurkat cells made resistant to cisplatin reduce the amount of detectable carbonato species by (7.9 ± 5.6) fmol per cell and also reduce the amount of 1 by (3.4 ± 0.9) fmol per cell. The amount of detectable carbonato species is also reduced by addition of the drug to medium that has previously been in contact with normal Jurkat cells (cells removed); the reduction is greater when drug is added to medium previously in contact with resistant Jurkat cells (cells removed). This shows that the platinum species are modified by a cell-produced substance that is released to the medium. Since the modified species have been shown not to enter or bind to cells, and since resistant cells modify more than non-resistant cells, the modification constitutes a new extracellular mechanism for cisplatin resistance which merits further attention

Modification and Uptake of a Cisplatin Carbonato Complex by Jurkat Cells

The interactions of Jurkat cells with cisplatin, cis-[Pt( 15NH3)2Cl2] (1), are studied using 1H-15N heteronuclear single quantum coherence (HSQC) NMR and inductively coupled plasma mass spectrometry. We show that Jurkat cells in culture rapidly modify the monocarbonato complex cis-[Pt(15NH 3)2(CO3)Cl]- (4), a cisplatin species that forms in culture media and probably also in blood. Analysis of the HSQC NMR peak intensity for 4 in the presence of different numbers of Jurkat cells reveals that each cell is capable of modifying 0.0028 pmol of 4 within ∼0.6 h. The amounts of platinum taken up by the cell, weakly bound to the cell surface, remaining in the culture medium, and bound to genomic DNA were measured as functions of time of exposure to different concentrations of drug. The results show that most of the 4 that has been modified by the cells remains in the culture medium as a substance of molecular mass \u3c3 kDa, which is HSQC NMR silent, and is not taken up by the cell. These results are consistent with a hitherto undocumented extracellular detoxification mechanism in which the cells rapidly modify 4, which is present in the culture medium, so it cannot bind to the cell. Because there is only a slow decrease in the amount of unmodified 4 remaining in the culture medium after 1 h, -1.1 ± 0.4 μM h -1, the cells subsequently lose their ability to modify 4. These observations have important implications for the mechanism of action of cisplatin