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Metallomics for drug development: an integrated CE-ICP-MS and ICP-MS approach reveals the speciation changes for an investigational ruthenium(III) drug bound to holo-transferrin in simulated cancer cytosol† Svetlana S. Aleksenko,a Magdalena Matczuk,b Xifeng Lu,zb Lidia S. Foteeva,a Katarzyna Pawlak,b Andrei R. Timerbaev*a and Maciej Jaroszb A method based on combining inductively coupled plasma mass spectrometry (ICP-MS) with capillary electrophoresis (CE) or an ultrafiltration step was developed to study the speciation of the serumprotein adducts of a ruthenium anticancer drug under in vitro intracellular conditions. The formation of a reactive Ru species in the cell, following the metal release from the protein, is thought to play an important role in the drug’s mode of action. Glutathione and ascorbic acid at their cancer cytosol concentrations were shown to be capable of altering the metal speciation in the drug adduct with holo-transferrin but not that with albumin. The appearance of the additional peaks in ICP-MS electropherograms (by recording both Ru- and Fe-specific signals) was found to be dependent on time which allowed for kinetic assessment of the evolution of novel metal species. On the contrary, after the

Received 24th March 2013, Accepted 5th June 2013 DOI: 10.1039/c3mt00092c

addition of citric acid the ruthenium ion (within the appropriately complexed scaffold) remained sequestered in the adduct. This was inferred as a proof of the speciation changes taking place by a virtue of a redox mechanism rather than due to ligand-exchange transformations. The protein-bound metallodrug was further characterized by direct ICP-MS assaying so as to confirm a partial release of

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ruthenium induced by glutathione.

Introduction The knowledge of the exact mode of action is critical for improving the efficacy of chemotherapeutic treatment using the existing drugs but perhaps more importantly, for the rational design of future compounds with superior pharmacological properties. This issue appears to be vital for lead drug candidates that have shown the most important biological results and have progressed toward clinical trials. Of the investigational anticancer metallodrugs, this

stage of development has successfully been reached by indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (I; Scheme 1).1,2 In phase I studies, the ruthenium-based drug showed a proven efficacy in the treatment of a range of primary tumors, particularly colorectal carcinomas. Furthermore, there is much consent

a

Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, Kosygin Str. 19, 119991 Moscow, Russian Federation. E-mail: [email protected]; Fax: +7-495-938-2054; Tel: +7-495-939-7035 b Chair of Analytical Chemistry, Warsaw University of Technology, Noakowskiego 3, 00664 Warsaw, Poland † Electronic supplementary information (ESI) available: description of binding kinetic experiments; figure showing evolution of the ruthenium–holo-transferrin adduct; table listing ICP-MS instrumental and operating parameters; table with typical levels of the components of reaction mixtures. See DOI: 10.1039/ c3mt00092c ‡ Permanent address: College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou, China.

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Scheme 1

Structural formula of the ruthenium drug.

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Paper regarding the mechanisms by which I executes its way to the cancer cell and enters it; here interactions with serum transport proteins play a central role.3,4 Also, some solid speculation found in the literature points to possible cell interaction pathways for the ruthenium drug, leading to the inhibition of cell proliferation.5–7 However, the full picture of drug’s cell processing is still a matter of debate.8,9 In particular, there is an as yet lack of understanding of how the drug, arriving into the cell predominantly in the form of transferrin adduct via the transferrin receptor, is activated by liberating the Ru moiety from the protein, and what the nature of the metal functionality is, when it is released intracellularily and then participates in drug targeting. Another issue that requires attention of pharmaceutical developers is a meaningful correlation between transferrin binding and biological activity of I. Most of the reasons – at least from the point of view of analytical chemists – is that while there are a number of established analytical techniques used to probe protein–metallodrug interactions in vitro,3,10 only a few of them are suitable to conduct a challenging metallomic study centered on providing detailed insight into cellular fates and the mode of activation for I, as well as related ruthenium-based anticancer agents. In this context, our choice fixed upon inductively coupled plasma mass spectrometry (ICP-MS) interfaced with capillary electrophoresis (CE). During recent years, a combined CE-ICPMS technique has emerged as a promising tool for biospeciation analysis.11–13 Herein, its use takes advantage of gentle, species friendly conditions, good tolerance to biological samples, and great resolving power from the separation side and high sensitivity, element specificity, and multi-elemental detection capability from the detection side. One of the appealing application domains of CE-ICP-MS in the field is metalloproteomic analysis related to direct monitoring of protein-mediated metabolism of anticancer metallodrugs (see a recent brief overview14), including the ruthenium drug under scrutiny.15–19 Two cited reports, in which the method was shown to be feasible for the estimation of metallodrug–protein adducts regarding their stability in simulated physiological environment, deserve special consideration in view of the topic of concern. No detectable alterations of I bound to albumin or transferrin were demonstrated to occur under near-extracellular conditions, viz., in the presence of the major blood reducing agent, ascorbic acid.16 We note that this is not surprising as the adduct formation with serum proteins was documented to make metal-based drugs protected from the redox reactions.20 On the other hand, the following kinetic assaying revealed that the drug adduct with transferrin (but not with albumin) experienced a change in ruthenium speciation in the capillary electrolyte mimicking intracellular fluid of tumor cells (with respect to pH and chloride concentration).19 Along with its hybrid configuration, standalone ICP-MS holds great promise as a reliable technique to assess the distribution of metal-based drugs between different blood compartments and to quantify the protein adducts formed in real samples. However, a separation step should be incorporated for the isolation of the protein-bound fractions from the excess drug in order to characterize a given adduct (e.g., by implementing a simple and

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Metallomics robust ultrafiltration step). Comprehensive information can be located in the corresponding reviews.10,21 However, to our knowledge, no application of ICP-MS for probing the reactivity of the ruthenium–protein adducts in the presence of cytosolic constituents has been reported. Within this frame, the present study is focused on elaborating the ICP-MS-based platform for in vitro screening of the possible bioconversion, accompanying the intracellular activation of the ruthenium drug after its transferrin-mediated uptake. Different cytosol components, displaying both reductive and complexformation functions, were tested, both alone and in a mixture, with due account for their relevant concentration levels. By using the complementary measurement protocols and integrating the CE-ICP-MS and ICP-MS information, we were able to ascertain that the adduct formed between I and transferrin (in its ironsaturated form) releases both ruthenium and iron moieties under the action of glutathione and ascorbic acid. This conceptual result is believed to have important implications regarding the drug’s availability for further reactions in the cell, including those that encounter the druggable cell targets.

Experimental Chemicals Holo-transferrin human (498%), glutathione (498%), sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from Sigma (USA). Sodium hydroxide and citric acid (499.5%) were the products of Fluka (Switzerland). L-Ascorbic acid (499.5%) was purchased from Sigma-Aldrich (China). Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] was donated by Prof. B. K. Keppler (University of Vienna, Austria). High-purity water used throughout this work was obtained with Elix Water Purification system (Millipore, France). Sample and electrolytes A 0.1 M stock solution of I was prepared just before analysis. The holo-transferrin adduct of I was prepared by the incubation of a mixture of 0.1 mM I and 5  105 M protein at 37 1C for 2.5 h to ensure the complete formation prior to reactivity testing. The required incubation time was determined according to the kinetic procedure outlined in the ESI.† The background electrolyte (BGE) for the CE experiments was 10 mM phosphate buffer, pH 6.0 prepared by mixing the appropriate volumes of 10 mM NaH2PO4 and 10 mM Na2HPO4 and adding 4 mM NaCl. For analyses in which cytosol additives to the BGE were employed, this buffer contained also 10 mM glutathione, 10 mM ascorbic acid or 50 mM citric acid, or a mixture of these constituents. In addition, acidic phosphate buffer was used to dissolve the drug–protein adducts in the case where intracellular conditions were simulated in the sample (followed by the addition of a respective reductive or complexing agent; see below). The incubation buffer (10 mM phosphate buffer, pH 7.4) was made up similarly and contained 100 mM NaCl to mimic extracellular conditions. Ultrafiltrates were obtained by centrifuging the samples through a 10 kDa cut-off filter (Amicon Ultracel, Millipore, France) for 40 min (10 000 rpm, 37 1C).

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Table 1

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ICP-MS instrument and interface settings and operational CE conditions

ICP-MS Plasma gas flow rate, L min1 Auxiliary gas flow rate, L min1 Sample cone Skimmer cone Plasma RF power, W Isotopes monitored Dwell time, ms

15 0.9 Pt (1.0 mm orifice) Pt (0.4 mm orifice) 1290 102 Ru, 57Fe 100

Interface Spray chamber volume, mL Nebulizer gas flow rate, L min1

5 1.0

CE Capillary BGE Sample injection, mbar  s Voltage, kV Current, mA Sheath liquid

Fused-silica, inner diameter 75 mm, length 70 cm 10 mM NaH2PO4–10 mM Na2HPO4, 4 mM NaCl, pH 6.0 300 25 28–32 1 mM phosphate buffer (pH 6.0), 0.4 mM NaCl and 20 mg L1 Ge

Instrumentation An Agilent 7500 and an X-7 (Thermo Elemental) mass spectrometers were used for ICP-MS measurements, employing the conditions listed in Table 1 and Table S1 (ESI†), respectively. Fitted with a microconcentric nebulizer CEI-100 (CETAC, Omaha, NE, USA), the mass spectrometer was interfaced to an Agilent HP3D CE system. Instrument control and data analysis were performed using ChemStation software (Agilent). Fused-silica capillaries of 70 cm total length (60 cm to UV detection window) and inner diameter of 75 mm were obtained from CM Scientific Ltd. (Silsden, UK). The capillary cassette and sample tray were thermostatted at 37 1C. The basic operation parameters for the CE-ICP-MS interface are also assembled in Table 1. The nebulizer performed in the self-aspiration mode using the sheath liquid to provide closing the electrical connection and to produce a fine aerosol. All signal quantifications were done in the peak-area mode by monitoring the total ion current of the major ruthenium isotope, 102Ru, during each CE run. The stability of CE-ICP-MS performance was controlled by measuring the normalized 72Ge signal during the post-run conditioning of the CE capillary as well as throughout the analysis. Analysis was only initiated when the signal was sufficiently high (cps 44000) and stable (RSD o2%). All electropherograms were smoothed employing a running average mathematical method,22 with 11-point periods of average (equivalents of 6 s of analysis). Such a treatment does not change peak shape but smooth the baseline noises. Samples were incubated at 37 1C in a WB 22 thermostat (Memmert, Germany). Ultrafiltration was carried out using a MPW-350R centrifuge (JW Electronic, Poland). Experimental procedures The capillary was conditioned with 0.1 M NaOH (10 min), a mixture of 0.1 M NaOH, methanol, and water (25/50/25, v/v/v) (5 min), water (5 min), and BGE (10 min). After each analysis, the capillary was rinsed with the same solutions and in the same sequence for 1, 1, 1, and 3 min, respectively. After flushing, the current level was checked by applying a constant voltage of 25 kV for 30 s. The BGE was degassed in an ultrasonic bath and passed

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through a 0.45 mm membrane filter before analysis. Samples were introduced into the capillary hydrodynamically by applying a pressure of 30 mbar for 10 s (injection volume 25.5 nL). A voltage of 25 kV was applied (with a positive polarity placed at the inlet end of the capillary) to generate the separations. UV-detected acetone was used as a marker of electroosmotic flow. Prior to analysis, the Ru–protein adducts were separated from the unbound Ru species, as well as from high chloride, using ultrafiltration through a 10 kDa cut-off filter. The highmolecular-mass fraction was constituted in 10 mM phosphate buffer (pH 6.0) containing 4 mM NaCl and then subjected to reverse ultrafiltration in order to be quantitatively removed from the membrane filter. The subsequent analysis was carried out in accordance with one of the three following procedures (see also a scheme in Fig. 1). After the addition of 10 mM glutathione, 10 mM ascorbic acid, or 50 mM citric acid to the adduct solution (5  105 M), the mixture was incubated at 37 1C and its aliquots were continuously taken for CE-ICP-MS analysis over 24 h. Alternatively, the sample solution was directly introduced at the inlet of the capillary filled with a BGE which contained the same additives, comprising the same concentration levels as in the off-line CE mode. With both CE modes, the total ion current of 102Ru and 57Fe isotopes was monitored. In the direct assaying, the adduct sample was fortified with 10 mM glutathione, thermostatted at 37 1C, and after ultrafiltration analyzed by ICP-MS. The concentrations of all the components of reaction mixtures chosen with due account for their physiological (or therapeutical) levels or ranges are gathered in Table S2, available in the ESI.†

Results and discussion Approach taken Fig. 1 is a schematic illustration of the basic concept of acquiring information on the reactivity of ruthenium(III) bound to serum proteins using ICP-MS as a metal-specific assaying tool.

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Fig. 1 ICP-MS-based strategies used to investigate the reactivity of the protein adducts of I in the presence of cancer cytosol components. Holo-transferrin is depicted as the most relevant example. I – CE-ICP-MS analysis of the reaction mixture containing the adduct and the cytosol additive; II – CE-ICP-MS analysis using the cytosol additive to the capillary electrolyte; III – ultrafiltration of the reaction mixture and the ICP-MS analysis of the ultrafiltrate. The color of streams and the BGE is related to a metal release (green for Ru and red for Fe).

After the adduct formed under extracellular conditions is isolated by extensive ultrafiltration (this step not shown in the figure), it is subject to exposure by a cytosol component (or a mixture of several components), followed by the separation of reactants and potential reaction products (or at least some of these). In the work presented here, CE and ultrafiltration were implemented for this purpose. Basically, there are two experimental setups that can be applied to monitor the CE profiles of the protein–drug adducts in the presence of pertinent cytosol constituents.14,16 One is based on the off-line measurement of the reaction kinetics when it proceeds outside the CE system (see Fig. 1, Chart 1). This implies that the reaction components are present in a properly incubated sample and its aliquots are continuously taken for analysis. The ICP-MS signal of relevant metal species is recorded provided that they are spatially resolved before entering the detector (actually, the interface). This kinetic CE mode is particularly suitable for the examination of reactions with a moderate to a slow speed. Faster processes can hardly be measured accurately, as the time, required to complete the CE run and to condition the capillary for the next run, is usually no less than 15 min. This limits the number of measurements needed for establishing a representative kinetic profile. Besides, it is difficult to account for the development of the reaction during sample introduction and electrophoresis. Another, in situ (or in-capillary) mode (Chart II) assumes that the course of the respective reaction can be followed when the adduct is introduced into the capillary filled with a BGE that contains the bioadditive of interest. This means that the adduct is supposed to undergo possible chemical changes during the

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migration along the capillary. In this setup, the capillary serves not only as a separation chamber but also as a microreactor. The consequent benefits are in that the reaction occurs completely within the capillary (without a contact with the atmosphere which is especially important for monitoring redox processes) and that there is no time delay from the time when reaction starts to the time of analysis of the reaction mixture. However, only reactions that are fast enough to progress appreciably within the time scale of typical CE run (about 15 min) are appropriate for kinetic characterization. With ultrafiltration as the separation method of choice (Chart III), the reaction mixture is centrifuged through the cut-off filter and the ultrafiltrate is collected for the ICP-MS analysis (following a proper dilution). If the ultrafiltrate contains a detectable Ru, then we can conclude that a given cytosol component cleaves the drug–protein adduct and converts the metal into a certain low-molecular-mass species. In this case, quantification data afford the degree of drug transformation to be calculated (by taking the Ru concentration in residual proteinaceous fraction as a difference between the total and the released metal). On the other hand, any change in the Ru speciation toward the formation of a species with a molecular mass comparable to that on the intact adduct cannot be distinguished by this approach. Selection of the target protein After intravenous administration, I undergoes extensive protein binding and the drug’s delivery and uptake involves at least two transport proteins, albumin and transferrin. Although albumin does act as the major binding partner to I in blood serum, the

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mechanism on how the drug–protein adduct comes into the cell remains elusive. Furthermore, to our knowledge, there is no experimental evidence of chemical changes in the albumin adduct at physiological conditions, this study being no exception (see below). The fraction of I bound to transferrin was found to be much smaller, constituting less than 2% in vitro15 and 1% in plasma samples of patients.23 On the other hand, it is well documented5,24 that after binding to transferrin, I is released from the protein inside the cell, supposedly after reduction to Ru(II) by biological reductants, and as such an active form presumably interferes with iron metabolism. Although transferrin is capable of binding the two iron(III) ions, it is normally saturated with iron in blood only by 30%.25 It is likely for this reason why a greater part of transferrin binding studies has been executed for I using apo-transferrin (i.e., the iron-devoid form). In our opinion, the following facts do not justify the abandonment of holo-transferrin as binding partner for the Ru-based drug. First, almost one third of protein molecules occur at physiological conditions as bound to iron(III). Second, the diferric transferrin possesses a higher affinity to transferrin receptors than other forms of the protein.26 Last but not least, there is a requirement of iron bound to transferrin to achieve the most effective uptake of I to the cytoplasm.27 Seemingly, because of this inadequacy, there are no convincing data to ensure whether the formation of the Ru(III)–transferrin conjugate results in no displacement of Fe(III). Moreover, if binding to other sites than those involved in the Fe bonding is possible, the composition of drug adducts (and possibly their reactivity) would differ for the transferrin forms different in the number of iron atoms attached. To avoid such an uncertainty, the effects of cancer cytosol components were essentially assayed here for the drug adduct with holotransferrin. Since no published accounts on the interaction of I with this protein can be traced in the literature, it was deemed obligatory to investigate binding kinetics with respect to the complete adduct formation. Binding to holo-transferrin Shown in Fig. 2 is a representative panel of electropherograms demonstrating time-dependent changes in the Ru speciation upon the interaction of I with holo-transferrin. The both 57Fe and 102Ru signals were simultaneously monitored in order to recognize the peak due to the protein-bound drug fraction in a fairly complicated migration pattern observed as long as the reaction progressively develops. Importantly, the 57Fe signal, indicating the migration of the protein, has not been subject to any alteration upon attaching the ruthenium moiety, at least at the level of the limit of detection of the method (1  107 M). In order to increase clarity, the Fe trace is not shown in other electropherograms. The observed electrophoretic behavior unequivocally verifies that no displacement of Fe(III) from its two binding sites in transferrin occurs. Likewise, in ultrafiltrates obtained after the incubation of I with holo-transferrin, no iron was traced by standalone ICP-MS. These findings have a solid thermodynamic confirmation, if one compares the protein-binding constants for iron(III) and I, which are in range of

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Fig. 2 Electropherograms illustrating the kinetics of drug binding to holotransferrin. Sample: protein (5  105 M) and I (1  104 M) incubated at 37 1C. CE and ICP-MS conditions, see Table 1. Peak identification: 1  an intermediate product(s); 2  Ru–holo-transferrin adduct; 3  trans-[RuCl4(1Hindazole)2]. The ‘‘UF’’ trace was obtained after ultrafiltration.

(1–6)  1022 28 and (5.6–6.5)  103 M1,15,29 respectively. To emphasize, human serum transferrin contains other 17 accessible histidine residues that may accommodate ruthenium without impairing the iron–protein bonds, and binding to other sites (not involved in the iron transportation) is not excluded.9 As can be judged from Fig. 2, before reaching a (pseudo)equilibrium state, the reaction goes through the formation of at least one intermediate form. As this intermediate migrates faster than the electroosmotic flow, it can be assumed that it is a positively charged aquated form of I, with the chlorido ligands substituted by water molecules. Indeed, this assumption is in accordance with the hydrolysis (aquation) rate for I which was reported to be faster than adduct formation.23 After ultrafiltration of the reaction mixture through a cut-off filter of 10 kDa (to remove the unbound Ru species, albeit these represent less than 0.3% of the total metal amount, according to ICP-MS data), the 102Ru signal was detected for the protein fraction remaining on the filter. This result, shown in the upper trace of Fig. 2, proved that the adduct can be purified by ultrafiltration. Furthermore, due to such a treatment the intermediate

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Paper product can be assigned to a species with a fairly low molecular mass ({10 kDa). Complete formation (490%) of the adduct takes from 1 to 2.5 h, depending on the drug-to-protein ratio. For a molar ratio of 2 : 1, the binding curve is shown in Fig. S1 (ESI†). In order to compare the reactivity of I toward holo- and apo-transferrin in a persuasive way, the reaction rate constant was calculated, as described in the ESI.† The obtained value, k = 0.101  0.008 min1 (n = 9), appears to be a close approximation of the most recent reference datum, 0.093  0.002 min1 measured for the drug interaction with apo-transferrin (using the same technique).17 This similarity allows perhaps only one conclusion, that is, loading with iron does not discriminate the protein from binding the Ru drug with the same reactivity as exhibited the iron-void transferrin form. The observed compatibility of ruthenium with iron provides also further evidence that ruthenium is involved in iron metabolism and uptake30 and may enter the cells – within the same molecular entity – via transferrin receptors.30 Interaction of the drug–transferrin adduct with cytosol components The reducing environment of solid tumors, as compared to healthy tissues, is in part due to relatively high concentrations of glutathione (0.510 mM) as well as ascorbic acid present in cancer cells at r10 mM. As a matter of fact, the redox potential of I is physiologically accessible, and reduction of Ru(III) to Ru(II) by both bioreductants is supported by several studies.31–34 However, in most of these reports, including that performed in cell culture setting,33 the Ru drug was examined as its initial molecular entity. This would never happen in a real-world situation, as I meets the intracellular compartment being greatly altered by virtue of hydrolytic and protein-binding mechanisms. Another important comment is that both glutathione and ascorbic acid hold also complexing properties. Therefore, along with a stronger cellular complexing agent, citric acid, they could eventually compete with the protein and the carrier indazole ligands for the central metal and change the coordination of the Ru active site. CE-ICP-MS. In a pilot experiment devised to test the possibility of exploiting the in-capillary CE mode as apparently more suitable for (redox) kinetic measurements, all three cytosol constituents were added to the BGE at their maximum estimated concentration levels (see Table S2, ESI†). The electrolyte buffer pH and sodium chloride concentration were also adjusted to the respective values found in cancer cytosol, i.e., pH 6.0 and 4 mM NaCl. As was mentioned above, in this setup the in-capillary reaction is expected to be initiated after introduction of the sample, containing the adduct, and the application of voltage. The electrophoresis commenced causes the adduct to penetrate the electrolyte zone and to encounter the mixed reactants. Dramatic changes in speciation were in fact visible in the case of iron (Fig. 3). These were the result of its complete removal from the protein after the exposure of the tested mixture, accompanied by the formation of two new iron species.

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Fig. 3 Iron species (1 and 2) released from the Ru–holo-transferrin adduct (3) upon migrating across the BGE containing glutathione (10 mmol L1), ascorbic acid (10 mmol L1), and citric acid (50 mmol L1).

However, the adduct remained unaffected with respect to the rutheniumprotein framework, none of the BGE additives being able to exert its reducing (or complexing) action over 15 min of examination (regardless of sample injection volume possibly affecting the reaction rate). This observation is in accord with the known fact that ascorbate does not effectively influence ruthenium in I under physiological conditons5 and in simulated extracellular environment (after the drug is bound to transferrin or albumin).16 It is worth nothing that the adduct between I and albumin behaved similarly, retaining a single-peak migration profile (data not shown). This does not mean that no releasing event is possible but rather that the kinetics of any of the possible reactions is slow to be followed over the amount of time the analyte remains in the capillary. Next, the pre-capillary CE mode was attempted to delineate whether the cytosol components could discharge the Ru moiety or form conjugates with the Ru–transferrin adduct at longer reaction times. The adduct was first mixed with 10 mM glutathione and the reaction was analyzed by CE-ICP-MS over time. As can be seen from Fig. 4A, even after 30 min of incubation at 37 1C the electropherogram showed significant transformations. Both the Ru- and Fe-traces contained the second peak at migration times smaller than that of the adduct, which corresponds to positively charged metal species. Furthermore, a peak due to another (anionic) form of Ru was observed (Fig. 4A, peak 4). After an incubation period of 2.5 h, this species became the most abundant form of Ru and also importantly, glutathione entirely removed iron from transferrin. In contrast, for ruthenium the release rate is rather slow; it took about 24 h to have the peak of the parent adduct disappearing from the electropherograms. This can be clearly viewed from Fig. 5 that portrays the full-time kinetic changes in the ruthenium speciation. Despite the fact that cells are much more complex than simulated in vitro system, the slow Ru discharge observed might have important implications on the drug behavior. The transferrin adduct could serve as a cellular depot from which small amounts of an active cytotoxic species are gradually released in order to exhibit its anticancer activity.

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Fig. 5 Distribution of the ruthenium species after the action of glutathione as a function of time. The species correspond to peak 2 (circles), 3 (squares), and 4 (triangles) in Fig. 4A.

Fig. 4 Effect of (A) glutathione and (B) ascorbic acid on the ruthenium and iron speciation after incubation with the holo-transferrin adduct of I. CE and ICP-MS conditions, see Table 1. Peak identification: 1, 2, and 4  the released species; 3  Ru–holo-transferrin adduct.

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The general trend of alterations resulted from the adduct exposure to ascorbic acid (10 mM) was the same but the rate of the effect was different. On average the adduct tends to be more stable (Fig. 4B), regarding the retaining of both metals attached to the protein, with a degree of decomposition of only 50% after 20 h-incubation (cf. the data of Fig. 5). On the other hand, the number of the fast-migrating peaks for each metal (due to cationic transformation products) was not one but two. Remarkably, both sets of species have the same migration times (Fig. 4B, upper traces). These findings, as well as the fact that there was no marked negative species characteristic to the reaction of the adduct with glutathione, indicate a different mechanism of action for ascorbic acid. Citric acid, an intracellular chelator with high affinity toward both Fe(III) and Ru(III) (the complex-formation constants are as large as 3.2  1011 35 and 2.5  108,36 respectively), surprisingly exerted no influence on the adduct integrity. After more than 8 h of observation at a 1000-fold molar excess of citric acid, no new signals were observed in ICP-MS electropherograms. This means that the protein scaffold is capable of protecting the (coordinatively) bound metals from ligandexchange interactions, a conclusion in favor of a redox mechanism of drug transformation under simulated cytosol conditions tested. ICP-MS. Finally, additional evidence to support the observed pattern of adduct reactivity was obtained by direct ICP-MS study. It showed substantial differences in metal release behavior responding to 10 mM glutathione. Whereas only about 3% Ru turned into a low-molecular-mass fraction and found its place in the ultrafiltrate, ultrafilterable iron was found predominant (97%). Both metals exhibited fairly fast release rates, with the indicated degree of conversion attained already after 1 h. Since the main fraction of Ru exceeds 10 kDa (yet after 45 h of examination), the rutheniumprotein framework could barely be altered by glutathione. Also, taking into account the CE-ICP-MS results, we cannot rule out the reduction of Ru(III) (even in the bound state). However, proper characterization of the release (or transformation) process was beyond the scope of

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Paper this work. Therefore, the present observations have only qualitative character regarding molecular mechanisms involved in such changes in speciation.

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Conclusions ICP-MS is a powerful method for ascertaining possible metabolic transformations of metal-based drugs and in this context, contributes well in the development of lead drug candidates. For one of the promising nominees for clinical use, I, the evolution of novel Ru species at simulated intracellular conditions was noted for the first time. The concurrent change in speciation of iron originated from the transferrin adduct of I can be hypothesized as ruthenium is involved in iron metabolism and may even disrupt it. Unfortunately, the speciation capability of ICP-MS coupled to CE does not extend to situations where the elucidation of molecular structures is needed. ICP-MS totally destroys molecular information thus reducing electropherograms to simple ‘‘elementograms’’. Therefore, an exact mechanism of drug transformation (or activation) must await in-depth structural studies to be undertaken using CE with a molecule-specific detector or an alternative technique. In any case, the results of our research provide a striking example of the drug’s speciation changes that might take place upon entering cancer cells.

Acknowledgements This work was performed while author S.S.A. held a Mianowski Fund Fellowship at Warsaw University of Technology. Experiments using standalone ICP-MS were done at the Institute of Microelectronics Technology and High-Purity Materials, Russian Academy of Sciences (Chernologolovka, Russia) under leadership of Dr. Vasilii Karandashev. The authors would like to thank Dr. Hooshmand Sheshbaradaran (Niiki Pharma Inc., USA) for his critical review of the manuscript. Financial support of this research by European Union (in the framework of Regional Development Fund through the Joint UW and WUT International PhD Program of Foundation for Polish Science – ‘‘Towards Advanced Functional Materials and Novel Devices’’; MPD/2010/4) and the Russian Foundation of Basic Research is gratefully acknowledged.

References 1 M. A. Jakupec, V. B. Arion, S. Kapitza, E. Reisner, A. Eichinger, M. Pongratz, B. Marian, N. Graf v. Keyserlingk and B. K. Keppler, KP1019 (FFC14A) from bench to bedside: Preclinical and early clinical development – An overview, Int. J. Clin. Pharmacol., 2005, 43(12), 595–596. 2 F. Lentz, A. Drescher, A. Lindauer, M. Henke, R. A. Hilger, C. G. Hartinger, M. E. Scheulen, C. Dittrich, B. K. Keppler and U. Jaehde, Pharmacokinetics of a novel anticancer ruthenium complex (KP1019, FFC14A) in a phase I doseescalation study, Anticancer Drugs, 2009, 20(2), 97–103.

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