Understanding How the Platinum Anticancer Drug Carboplatin Works: From the Bottle to the Cell

Carboplatin, a platinum anticancer drug used to treat many types of human cancer, contains a bidentate dicarboxylate chelate leaving ligand, a structural feature that makes it much less chemically reactive than the first-generation platinum anticancer drug cisplatin, which contains two monodentate chloride leaving ligands. In water, carboplatin exists in a monomer-dimer equilibrium with an association constant of K (M -1) ≈ 391, a property that accounts for the long-term stability of its ready-to-use infusion solution. When administered in the clinic, carboplatin is believed to exert its biological effects by interacting with genomic DNA and proteins. The slower substitution kinetics of carboplatin, compared to cisplatin, has prompted investigators to focus on mechanisms by which the compound can be activated in vivo. Carbonate, which is in equilibrium with hydrogen carbonate, carbonic acid, and dissolved carbon dioxide, is ubiquitous in biological systems, and is found in high concentrations in the blood, the interstitial fluid, and the cytosol. Activation of carboplatin by carbonate, CO 3 2- (k 1 = 2.04 ± 0.81 × 10 -6 in 24 mM carbonate buffer, pH 7.5 at 37 °C), for example, leads to the formation of platinum species that are more cytotoxic than the parent drug. This short review focuses on the reason for the unusual stability of carboplatin in its aqueous ready-to-use infusion solution, describes the reactions of the drug with biologically common nucleophiles and summarizes the activation chemistry that make the drug more reactive toward substances present in the biological system

Understanding how the platinum anticancer drug carboplatin works: From the bottle to the cell

Carboplatin, a platinum anticancer drug used to treat many types of human cancer, contains a bidentate dicarboxylate chelate leaving ligand, a structural feature that makes it much less chemically reactive than the first-generation platinum anticancer drug cisplatin, which contains two monodentate chloride leaving ligands. In water, carboplatin exists in a monomer–dimer equilibrium with an association constant of K (M−1) ≈ 391, a property that accounts for the long-term stability of its ready-to-use infusion solution. When administered in the clinic, carboplatin is believed to exert its biological effects by interacting with genomic DNA and proteins. The slower substitution kinetics of carboplatin, compared to cisplatin, has prompted investigators to focus on mechanisms by which the compound can be activated in vivo. Carbonate, which is in equilibrium with hydrogen carbonate, carbonic acid, and dissolved carbon dioxide, is ubiquitous in biological systems, and is found in high concentrations in the blood, the interstitial fluid, and the cytosol. Activation of carboplatin by carbonate, CO32− (k1 = 2.04 ± 0.81 × 10−6 in 24 mM carbonate buffer, pH 7.5 at 37 °C), for example, leads to the formation of platinum species that are more cytotoxic than the parent drug. This short review focuses on the reason for the unusual stability of carboplatin in its aqueous ready-to-use infusion solution, describes the reactions of the drug with biologically common nucleophiles and summarizes the activation chemistry that make the drug more reactive toward substances present in the biological system

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

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

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+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

Species-dependent effective concentration of DTPA in plasma for chelation of 241AmI

Diethylenetriaminepentaacetic acid (DTPA) is a chelating agent that is used to facilitate the elimination of radionuclides, such as americium, from contaminated individuals. Its primary site of action is in the blood, where it competes with various biological ligands, including transferrin and albumin, for the binding of radioactive metals. To evaluate the chelation potential of DTPA under these conditions, the competitive binding of 241Am between DTPA and plasma proteins was studied in rat, beagle and human plasma in vitro. Following incubation of DTPA and 241Am in plasma, the 241Am-bound ligands were fractionated by ultrafiltration and ion-exchange chromatography, and each fraction was assayed for 241Am content by gamma scintillation counting. Dose-response curves of DTPA for 241Am binding were established, and these models were used to calculate the 90% maximal effective concentration, or EC90, of DTPA in each plasma system. The EC90 were determined to be 31.4, 15.9 and 10.0 μM in rat, beagle and human plasma, respectively. These values correspond to plasma concentrations of DTPA that maximize 241Am chelation while minimizing excess DTPA. Based on the pharmacokinetic profile of DTPA in humans, after a standard 30 μmol kg−1 intravenous bolus injection, the plasma concentration of DTPA remains above EC90 for approximately 5.6 h. Likewise, the effective duration of DTPA in rat and beagle were determined to be 0.67 and 1.7 h, respectively. These results suggest that species differences must be considered when translating DTPA efficacy data from animals to humans and offer further insights into improving the current DTPA treatment regimen

Solid dispersions of the penta-ethyl ester prodrug of diethylenetriaminepentaacetic acid (DTPA): formulation design and optimization studies

The penta-ethyl ester prodrug of diethylenetriaminepentaacetic acid (DTPA), which exists as an oily liquid, was incorporated into a solid dispersion for oral administration by the solvent evaporation method using blends of polyvinylpyrrolidone (PVP), Eudragit® RL PO and α-tocopherol. D-optimal mixture design was used to optimize the formulation. Formulations that had a high concentration of both Eudragit® RL PO and α-tocopherol exhibited low water absorption and enhanced stability of the DTPA prodrug. Physicochemical properties of the optimal formulation were evaluated using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). In vitro release of the prodrug was evaluated using the USP Type II apparatus dissolution method. DSC studies indicated that the matrix had an amorphous structure, while FTIR spectrometry showed that DTPA penta-ethyl ester and excipients did not react with each other during formation of the solid dispersion.. Dissolution testing showed that the optimized solid dispersion exhibited a prolonged release profile, which could potentially result in a sustained delivery of DTPA penta-ethyl to enhance bioavailability. In conclusion, DTPA penta-ethyl ester was successfully incorporated into a solid matrix with high drug loading and improved stability compared to prodrug alone

Preparation of alginate beads containing a prodrug of diethylenetriaminepentaacetic acid

A penta-ethyl ester prodrug of the radionuclide decorporation agent diethylenetriaminepentaacetic acid (DTPA), which exists as an oily liquid, was encapsulated in alginate beads by the ionotropic gelation method. An optimal formulation was found by varying initial concentrations of DTPA pentaethyl ester, alginate polymer, Tween 80 surfactant and calcium chloride. All prepared alginate beads were ~1.6 mm in diameter, and the optimal formulation had loading and encapsulation efficiencies of 91.0 ± 1.1 and 72.6 ± 2.2%, respectively, and only 3.2 ± 0.8% water absorption after storage at room temperature in ~80% relative humidity. Moreover, Fourier transform infrared spectroscopy showed that DTPA penta-ethyl ester did not react with excipients during formation of the DTPA penta-ethyl ester-containing alginate beads. Release of prodrug from alginate beads was via anomalous transport, and its stability enhanced by encapsulation. Collectively, these data suggest that this solid dosage form may be suitable for oral administration after radionuclide contamination