9 Platinum(IV) Anticancer Complexes

PLATINUM(IV) ANTICANCER COMPLEXES

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9 Platinum(IV) Anticancer Complexes Matthew D. Hall, Rachael Dolman and Trevor W. Hambley Centre for Heavy Metals Research, School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia

1. 2. 3. 4.

5. 6. 7.

8.

INTRODUCTION HISTORY MECHANISM OF ACTION DESIGN 4.1. Electrochemistry 4.2. Lipophilicity SYNTHESIS IN VITRO INTERACTIONS WITH BIOMOLECULES BIOLOGICAL FATE 7.1. Extracelllar Biotransformations and Metabolites 7.2. Intracellular Fate CONCLUSIONS ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES 297

297 298 300 301 302 306 307 310 311 311 314 316 316 316 317

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1.

HALL, DOLMAN AND HAMBLEY

INTRODUCTION

Platinum anticancer drugs, including cisplatin, carboplatin and oxaliplatin are amongst the most widely used class of anticancer drugs, being used in the treatment of up to half of all patients undergoing chemotherapy. Despite the potential advantages offered by Pt(IV) complexes, relatively few have been prepared and tested and only three have undergone clinical trials. In this review we describe the work that has been done towards understanding the biological chemistry and toward rational design and selection of Pt(IV) complexes as anticancer agents. Platinum(IV) based anticancer drugs have a number of potential advantages over their platinum(II) analogues. In particular, they are less reactive and therefore will undergo fewer reactions on route to the tumour, resulting in fewer side effects and reduced drug loss due to deactivation. They also offer more possibilities for structural variation than Pt(II), allowing chemical and physical properties such as reduction potential and lipophilicity to be tuned. In recent years, the authors have developed principles for the selection of Pt(IV) complexes as anticancer agents based on their own work and that of others. In this chapter we describe how the chemical and physical properties can be tuned using the work that led up to the development of the selection criteria as a context. 2.

HISTORY

The anticancer activity of Pt(IV) complexes has been known of since the discovery of cisplatin (cis-[PtCl2(NH3)2]) by Rosenberg and colleagues [1-3]. When an electric potential was applied across platinum electrodes in an E. coli culture, the bacterial replication was inhibited. It was determined that this was due to dissolution of Pt from the electrodes forming a number of complexes, at least two of which were Pt(IV) complexes. The complexes were trialled for anticancer activity, and both Pt(II) and Pt(IV) compounds were found to be active. However, a Pt(II) compound, cisplatin, was selected for clinical trials, and now forms the basis of one of the world’s largest selling classes of anticancer drugs, with sales in excess of US $ 1 billion per year.


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Since then, thousands of platinum complexes, most of them Pt(II), have been synthesized in the pursuit of analogues with improved activity and diminished side-effects. Tobe and co-workers synthesized a large number of Pt(II) and Pt(IV) complexes and examined their toxicity and solubility [4]. Based on its very high solubility, the Pt(IV) complex iproplatin (cis,trans,cis-[PtCl2(OH)2(ipa)2]) was selected for preclinical and clinical trials. Iproplatin was sufficiently well tolerated to enter phase II and III trials, but was ultimately found to be less active than carboplatin and so was not registered for clinical use [5,6]. Tetraplatin ([PtCl4(R,Rchxn)]) showed great promise in preclinical studies but caused severe peripheral neuropathy in treated patients and phase I trials were subsequently abandoned [7]. JM216 (cis,trans-[PtCl2(OAc)2(cha)(NH3)]), a more lipophilic drug, was recently in phase III trials as an orally active agent, but these were abandoned due to variability in drug uptake [8].

It is disappointing that none of the Pt(IV) complexes that have entered clinical trials have revealed improved activity over cisplatin, especially in light of promising preclinical indicators for a range of Pt(IV) complexes. The potential of Pt(IV) anticancer agents to reduce side effects, increase activity, and tumor specificity, has not yet been fully realised. Why have complexes which demonstrated promising preclinical activity failed in the clinic? An improved understanding of the chemical and pharmacological parameters affecting Pt(IV) complexes as drugs is helping to elucidate the mechanism of action of this class of complexes and will allow the rational design of future Pt(IV) agents.

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3.


MECHANISM OF ACTION

Pt(IV) complexes (octahedral, d 6) are inert relative to their Pt(II) congeners. It is widely believed that reduction to the more kinetically labile Pt(II) is essential for the anticancer activity of Pt(IV) complexes to be effected (Figure 1) [9-11]. If this is the case then the ease with which a Pt(IV) complex is reduced can be expected to influence its biological activity, and a requirement of activity would be that the Pt(II) analogue is active [12]. While it is generally agreed that reduction is required for activity, the extent of reduction extra- and intra-cellularly has not been well characterized, nor has the distribution of platinum drugs in tumour cells. Direct binding of DNA by Pt(IV) complexes has been reported in vitro without reductants present [13-15], but mechanistically these reactions can be dis

FIG. 1. Generic octahedral Pt(IV) complex, containing cis amine (A) and leaving (Y) groups in the equatorial plane, and axial (X) ligands. The two electron reduction to square planar Pt(II), with loss of axial ligands is shown.

FIG. 2. Depiction of the potential extra- and intracellular fate of Pt(IV) complexes in the body.


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counted as they have half-lives far exceeding that of reduction. Hence, it is unlikely that Pt(IV) can survive the milieu of reductants in vivo to arrive at the nucleus intact [16]. Pt(IV) can be reduced extracellularly and enter the cell as Pt(II), or intracellularly having entered as Pt(IV) (Figure 2). Pt(IV) complexes may, however, be distributed differently to Pt(II). An understanding of the relative importance of these steps, and the factors which govern them, should allow for improved design. 4.

DESIGN

As mentioned earlier, the reduction of Pt(IV) complexes to their active Pt(II) analogues is ultimately required for activity. Drug design then should ensure that the equatorial moiety of the pro-drug Pt(IV) complex will yield an efficacious Pt(II) complex. This is supported by the observation of Rotondo and co-workers that Pt(IV) complexes such as mer[PtCl3(dien)] which yield inactive Pt(II) species are themselves inactive, while the reverse is true for those based on active Pt(II) analogues [17]. The structure-activity rules employed in the design of Pt-based chemotherapeutics have been reviewed elsewhere [12]. Rational design is crucial, and this necessitates an understanding of the properties that determine the effectiveness of the complex. There are a number of factors to consider when assessing the possible effects of administering the Pt(IV) analogue of a Pt(II) complex. The kinetic inertness of Pt(IV) complexes means that there is increased opportunity for the complex to arrive at the cellular target intact. Modifying the ligands1 of Pt(IV) complexes alters the solubility of the complex (lipophilic versus hydrophilic) and thus its ability to enter tumor cells before being reduced to yield the active Pt(II) drug. Preventing side reactions by administering Pt(IV) complexes has the potential to lead to a drug that has reduced toxicity and higher activity. The axial ligands 1Herein,

the ligands that lie above and below the plane of the am(m)ine ligands in platinum(IV) complexes will be referred to as the ‘axial’ ligands, and those that lie in the plane of the am(m)ine ligands will be referred to as the ‘equatorial’ ligands. This is in keeping with common usage in literature when discussing platinum(IV) complexes [18, 19].

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can be modified to alter the reduction potential of the pro-drug, thus varying the rate of activation. Changing the axial ligands also modifies the pharmacological parameter log P (lipophilicity). 4.1. Electrochemistry

Cyclic voltammagrams for Pt(IV) complexes reveal that reduction is an irreversible two electron single step process associated with the loss of axial ligands, and as such reduction potentials reported in the literature are peak values for the forward wave (Ep) [20]. The reduction potentials of Pt(IV) complexes are dependent on the coordination environment of the complex, and it has been shown that the axial ligands generally exert a greater influence on reduction potentials than the equatorial ligands, with equatorial amine ligands exerting the least effect [20]. This is advantageous as the activity of the complex is not compromised during design.

TABLE 1 Reduction potentials of Pt(IV) complexes with a range of axial (X) and leaving (Y) ligands.

Ligand (X) Cl– –OC(O)CH 3 –OC(O)CH CH 2 3 –OC(O)CH CH CH 2 2 3 OH–

OH–

Ligand (Y)

EP (MV)

Ref.

Cl– Cl– Cl– Cl– Cl–

–4 –326 –301 –273 –664

18 18 18 18 18

–789

22


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The most commonly examined and synthetically accessible (vide infra), axial ligands are Cl–, OH–, and carboxylato ligands. Reduction occurs most readily for complexes with axial chloro ligands, intermediate for acetato ligands, whilst those containing hydroxo ligands are the least readily reduced, as shown in Table 1. This trend has been confirmed by Choi et al. [24], who also reported that reduction rates generally correlate with reduction potentials. Modifying the length of the carboxylato chain has a minimal effect on Ep, but changing the nature of the donor has a large effect. Further tuning of reduction potentials can be achieved by the addition of electron withdrawing groups to axial ligands; for example, the bromoacetato ligand increases the reduction potential with respect to the acetato ligand by 120 mV, despite the ligand variation being distant from the metal [21]. Varying the equatorial leaving group(s) (Y) is possible, however only a small number have been employed in effective Pt(II) complexes. Table 1 demonstrates that changing from chloro to ethylmalonato ligands results in a more inert complex, and in general four oxygen donors stabilize Pt(IV) better than any other ligand combination. Battle et al. varied the equatorial leaving group (Y) using a number of bidentate oxygen donor ligands and attributed the small variation in reduction potential to steric strain variation in the bidentate ligands [22]. Unlike the axial and equatorial leaving groups, variation of the non-leaving am(m)ine ligands does not have a large effect on the reduction potential, as the ligand donor atom is not altered. An early study demonstrated that variation of the equatorial amine ligands in complexes of the general form cis-[PtCl4(NRH2)2] had a small, but measurable effect on Ep [23]. Both the s-donor ability and steric strain of the ligands were considered to be factors influencing Ep. Choi and coworkers also suggested greater bulk as the reason for JM216 being more readily reduced than its ethane-1,2-diamine analogue [24]. Small structural variations at the axial, leaving and amine ligands have been demonstrated to have little effect on reduction potential. Altering the nature of the ligand donor, however, results in complexes incorporating a wide range of reduction potentials. Thus, given a desired reduction potential window for activation, it is feasible that appropriate modification of the ligands can allow for alteration of the log P and other physical properties.

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Experience in the clinic has demonstrated that a general relationship between reduction potential and in vivo activity of Pt(IV) complexes cannot be expected. While tetraplatin is readily reduced to its active form, it results in high toxicities, yet the reductively inert iproplatin has also been shown to be highly active. Despite this, extensive preclinical and clinical trials do provide an insight into how reduction potential influences biological behavior. Tetraplatin, with axial chloro ligands, is rapidly reduced in vivo, and all excreted metabolites are Pt(II) species [25]. Similarly, JM216 is reduced, but some aquated Pt(IV) species have been detected suggesting the complex does remain intact for a period of time [26]. In contrast, iproplatin, the most difficult to reduce of the three complexes, remains intact in vivo, and large amounts are excreted. Pendyala and coworkers have proposed that the complex enters the cell and is reduced intracellularly, though no direct evidence is provided [27]. A complicating factor when assessing the effect of reduction potentials on activity is the differing Pt(II) complexes yielded by tetraplatin, JM216 and iproplatin. It is difficult to discern, for example, whether the high toxicity of tetraplatin is due to its rapid reduction, or the subsequent toxicology of the Pt(II) active species yielded, though it is notable that most chxn containing species exhibit neurotoxicity [28]. Examination of cytotoxicity data do not reflect the clinical experience; namely that difficult to reduce species can be as active as others. Kratochwil and Bednarski found no relationship between the reduction potentials and cell growth inhibition activity for a number of Pt(IV) complexes [29], nor did Hambley et al. when examining a series of organometallic Pt(IV) complexes [20]. Choi et al. did observe that more difficult to reduce compounds are generally less cytotoxic [24]. The difficulty in interpreting these observations again lies in the structural diversity of the resultant Pt(II) moieties acting to complicate the analysis. The cytotoxicity of a set of Pt(IV) complexes which yield the same Pt(II) species on reduction (in this instance, cisplatin) allows for a simplified understanding of the effect of Ep on cytotoxicity, as shown in Table 2. An apparent relationship between reduction potential and cytotoxicity in vitro can be observed in Table 2; as the reduction potential increases, cytotoxicity increases. There are a number of important implications for drug design and screening here. From a preclinical perspective the


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trans-dihydroxo complex appears to be relatively inactive compared to cisplatin, yet the trans-dihydroxo complex iproplatin in fact progressed the furthest in clinical trials of any of the three drugs tested, and oxoplatin (cis,trans,cis-[PtCl2(OH)2(NH3)2]) is known to be highly active in vivo [30]. It may be fortuitous that iproplatin was selected for further pre-clinical screening based on its high solubility rather than from cytotoxicity assay screening, and the potential exists for a number of effective complexes to have been passed up in this fashion. Siddik and coworkers examined a series of Pt(IV) complexes and selected a number for further studies based on their IC50 values [31], choosing those with axial trifluoroacetato ligands, the most readily reduced complexes of those examined [24]. It seems likely that the robustness of the dihydroxo complexes which render it relatively inactive in in vitro assays confers the improved activity in vivo. This allows for a greater proportion of the drug to arrive at the target site intact, and would lower side effects – a major limitation in platinum drug development. Certainly cytotoxicity assays do not appear to be a sound basis alone for Pt(IV) drug selection.

TABLE 2 Comparison of Ep against the cytotoxicity (IC50) of a series of Pt(IV) complexes in the A2780 parental ovarian cancer cell line [82].

Ligand (X)

Ep

IC50a

(cisplatin)

-

2.5

–260

8.4

–635

17.9

–880

22.0

Cl– –OC(O)CH

OH–

3

a

Values are mean for data from three independent experiments with four values for each. Standard deviations were within the range 5 – 40%.

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4.2. Lipophilicity

Lipophilicity, as measured by relative solubilities in water and an organic solvent (chloroform, or more typically 1-octanol) or partitioning between the two solvents has been investigated as a property relevant to the activity of platinum complexes, particularly as one of the primary mechanisms of resistance to cisplatin is decreased drug accumulation [32]. More lipophilic drugs should allow for increased cellular accumulation via passive diffusion through the cell membrane, allowing for activity against resistant tumor phenotypes. Early studies examining the relationship between the structure and solubility of Pt(II) and Pt(IV) diamine complexes revealed that as the amine chain length increases, aqueous solubility decreases [33]. However, this was not necessarily coupled with increasing solubility in chloroform. The Pt(IV) trans-dichloro and dihydroxo analogues were also prepared, and the trans-dihydroxo complexes displayed markedly increased aqueous solubility [33]. In the instance of iproplatin, solubility was two orders of magnitude greater than that of its Pt(II) analogue, and on the basis of this it was selected for further trials. Yoshida et al. examined a series of cis-ammine/cycloalkylamine Pt(IV) complexes with differing alicyclic ring sizes [34]. A correlation between increasing cellular drug accumulation, intracellular DNA binding, cytotoxicity and the increasing partition coefficient of the complexes for the series was observed. This is not entirely unexpected, as all complexes had the same axial ligands, and therefore similar reduction potentials, so the correlation demonstrated may in fact be due to the reduced species, not the Pt(IV) complexes. However, variation of amine ligands to alter lipophilicity presents limited opportunities, as they are an essential feature for activity and modification could result in altered efficacy. Modifying the axial ligands of Pt(IV) complexes also alters the solubility of the complex (lipophilic versus hydrophilic) and therefore its ability to enter tumor cells. Kidani et al. [50] showed that for a series of dicarboxylato Pt(IV) analogues based on the [Pt(oxalato)(chxn)] moiety, log Poct values increased with increasing carboxylato ligand chain length. This feature has been employed, for example, in the design of JM216 as a lipophilic, orally active drug. Analogues of JM216 were found to have


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high cellular uptake and cytotoxicity in vitro [35]. The quest for increasingly lipophilic drugs has not translated into correlations between log P and biological activity [36], or more effective drugs in the clinic. A complicating factor in the design of lipophilic drugs is their reduction to Pt(II) in vivo, which may account for some disparity in cellular uptake and activity between in vitro and in vivo systems. A number of researchers have made efforts to produce even more lipophilic complexes such as tetracarboxylato Pt(IV) complexes; Lee et al. synthesized complexes with a wide range of log P values with carboxylato ligands of increasing chain length [37]. The lower than expected activity of lipophilic Pt complexes may be due to membrane partitioning of drugs, similar to that reported by McKeage et al. for a series of gold phosphine complexes [38]. Efforts to develop a model for the prediction of partition coefficients have been made, and a systematic study of the log Poct values of Pt(II) complexes was undertaken by Souchard et al. [39]. While lipophilicity of the complexes was shown to be linearly related to that of their respective amines, variation of leaving groups did not result in a similar relationship. While analysis of fragments could not be used as a predictive model, accurate log P prediction using calculations of polar surface area have recently proven to be more reliable [40]. The experimental log Poct for 39 platinum compounds with a variety of structures were modelled, and an excellent fit with experimental data was achieved. 5.

SYNTHESIS

The three types of axial ligands employed in the Pt(IV) complexes which have entered clinical trials, chloro, hydroxo and acetato, have been the most widely synthesised and tested in the pursuit of active Pt(IV) compounds. The syntheses of these three complex types are closely related to one another, and the limited range of ligands employed can be understood from their synthesis. Oxidation of Pt(II) complexes is commonly achieved by addition of hydrogen peroxide in an aqueous solution, resulting in the trans-dihydroxo complex (Figure 3) [41]. Alternatively, chlorine gas may be bubbled through an aqueous solution of the Pt(II) compound to achieve the trans-dichloro complex [42]. However a cheaper

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FIG. 3. Synthetic scheme for the oxidation and synthesis of trans-dihydroxo, dichloro and dicarboxylato complexes from platinum(II).

and more commonly employed route is available by adding concentrated HCl to the dihydroxo complex [18]. In this instance, the ligand substitution is achieved by protonating the hydroxo ligands at very low pH to yield neutral, labile aqua ligands which are replaced by the chloride anions. These two ligand types dominated early Pt(IV) drug development, until the carboxylate complexes were synthesised for biological testing in the 1990’s [43-45], though one example of that type had been reported earlier [46]. The starting material for their synthesis are again the trans-dihydroxo Pt(IV) complexes; however, rather than a substitution reaction, the carboxylato ligands are generated through nucleophilic attack by the coordinated hydroxo ligand on an anhydride or acyl chloride [47], as originally reported for cobalt(III) hydroxo complexes [48]. The nucleophilic nature of the kinetically inert hydroxo ligand on Pt(IV) has also been used to generate carbonate ligands from pyrocarbonates and carbamates from isocyanates [45], but little has been reported of their activity or reduction potentials. Kim and Sohn reported that the electrophilic reaction of trialkylsilyl chloride with hydroxo ligands results in siloxy ligands coordinated to Pt(IV) [49]. Despite these more recent synthetic advances, the quest for efficacious drugs has recently focussed on structure variation rather than incorporation


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of new ligand types. Kim et al. have produced a series of tetracarboxylato complexes of the form cis-[PtA2(OCOR)4], encompassing a large range of lipophilicities, by reacting the tetrahydroxo species with an excess of anhydride [19]. Testing in mouse models showed better oral activity than for JM216 [37]. Hetero tetracarboxylato complexes have also been synthesized by stepwise addition of stoichiometric equivalents of anhydrides, and the isomers were purified using silica columns. Another mode of ligand variation is the development of ‘mixed’ axial ligands, which would have intermediate reduction potentials. For example Kizu et al. synthesised trans-carboxylatochloro complexes by two routes; by reaction of HCl with the trans-dicarboxylato ligand to replace a carboxylato ligand with chloride, and reaction of a silver carboxylate salt with the trans-dichloro complex [50]. The silver salt method was found to result in much higher yields, and reduction of the carboxylatochloro species was found to be much faster than the dicarboxylato species. A trans-hydroxomethoxy Pt(IV) complex has been reported, along with the trans-acetatomethoxy Pt(IV) analogue [51]. While a huge number of Pt(IV) complexes have been generated for screening of antineoplastic activity, the synthetic variation has not been great, and the few novel axial ligand arrangements reported have not generally been characterized with respect to chemistry and activity. An exception to this is the oxidative addition of dithiobis(formamidium) cation to Pt(II) complexes yielding a 1,1,3,3-tetramethylthiourea axial ligand bound to the Pt(IV) complexes, which displayed cytotoxicity comparable to cisplatin, though resistance was not circumvented in a cisplatin-resistant cell line [52]. While the inertness of Pt(IV) renders ligand substitution impractical, other means have not been pursued beyond variation of the alkyl component of carboxylato ligands; for example, there have been few reports on ligand modification on Pt(IV) [45]. A recent novel development is the attachment of a bioactive moiety, oestrogen, as estradiol-3-benzoate axial ligands in order to exploit the observation that ER(+) cells exposed to estrogen are sensitized to cisplatin treatment [53]. A concerted effort on new methodologies for Pt(IV) complexes is required to take full advantage of the potential that Pt(IV) chemistry offers for rational design and targeting of tumors.

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6.


IN VITRO INTERACTIONS WITH BIOMOLECULES

There is a wide range of small and protein biomolecules present in blood and cells, partitioned in both aqueous and membrane environments. Any of these biomolecules have the potential to interact with Pt(IV) complexes. However, unlike most drugs, the main reactive pathway is not necessarily binding, but reduction to Pt(II), as Pt(IV) complexes are sufficiently inert not to react. The interaction of biomolecules with Pt(IV) complexes in vitro has been extensively examined and reviewed elsewhere [16], and it is intended here to provide an overview of important interactions. Reduction is the primary fate of Pt(IV) complexes, but aquation and substitution reactions have been reported, though the half-life of these is on a time scale of days or weeks. It is important to note that the physiological relevance of very slow reactions is questionable, as the clearance of platinum drugs occurs within days [54-56]. Given the requirement of reduction of Pt(IV) to yield the labile Pt(II) species, research has inevitably focussed on redox active molecules, along with DNA and simpler analogues. Despite this, many endogenous small molecules such as NADH and ATP have received little attention, nor have reductases and other electron donor enzymes. Subsequent to reduction of Pt(IV) complexes (irrespective of the bioreductant), the Pt(II) complex yielded would undergo further biotransformation processes, which have been extensively examined and reviewed elsewhere [57,58]. The reductant that has received most attention is the amino acid L-cysteine, a constituent of many enzymes and proteins such as metallothionein and albumin, and the tripeptide glutathione. The thiol functional group RSH is readily oxidized to the disulfide bridged cystine, as shown in Equation (1). This two electron couple serves as a redox balance through a number of biomolecules [59], and as a structural motif in proteins. Thiols have been shown to reduce Pt(IV) complexes with a stoichiometry of [RSH]:[Pt(IV)] = 2:1 [60]. 2 RSH [ RSSR + 2 H+ + 2 e–

(1)

The amino acid L-methionine contains a thioether side chain (R-SCH3) and is also a component of many biomolecules (e.g., albumin). The reduction of Pt(IV) complexes by methionine is reported to be a 1:1 reac-


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tion [61]. Ascorbate (vitamin C) has also been shown to be an important reductant in vitro [62], undergoing two one electron steps, reacting in a 1: 1 ratio with Pt(IV). The protein metallothionein contains 20 cysteine residues, and while its primary role is metal ion scavenging [63], it has also been shown to be capable of reducing Pt(IV) complexes[64]. Albumin is a small protein responsible for controlling blood pH, serves as a detoxicant, and is one of the major thiols in blood [65]. The protein has one free thiol (Cys34) and six methionine residues, and its interaction with Pt(IV) has received considerable attention [66,67]. Reduction of Pt(IV) by albumin is not entirely clear even at this stage, though the cysteine residue has been shown to be responsible for reduction of Pt(IV)-iodo complexes [66], and reduction by cysteine should be kinetically favored over methionine [16]. Glucose has also been shown to be capable of reducing Pt(IV) complexes under physiological conditions [25,68]. 7.

BIOLOGICAL FATE

7.1. Extracellular Biotransformations and Metabolites

While the contributions towards reduction of Pt(IV) complexes in vitro may be easily characterized, determining the fate of Pt(IV) in complex systems, and discerning whether Pt(IV) is reduced or non-covalently bound with proteins in vivo is more difficult. A knowledge of the contributions of individual components to biotransformation is also important for rational drug design in order to avoid deactivation and side effects. Evaluation of the reduction of tetraplatin in RPMI tissue culture medium and rat plasma was performed by Chaney and co-workers, and revealed much of the basic understanding of Pt(IV) reduction in blood plasma [25,68]. It was shown that reduction is the only metabolic pathway for the Pt(IV) drug in both tissue culture medium with 15% foetal calf serum (FCS) and rat plasma. By comparing the reduction of tetraplatin by the individual components of the cell growth medium under identical conditions, it was shown that 15% FCS reduced the greatest proportion (82.6%) followed by the growth medium (16.8%), glutathione (7%), and glucose (4.6%). While glutathione is a potent reducing agent, its concentration in cell growth medium is low (3.1 µM in this instance)

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accounting for the low amount of reduction. Protein thiols in FCS were proposed to be responsible for reduction, and the second-order rate constant for reduction by albumin sulfhydryl was far greater than that of ascorbate, supporting the notion that thiols are the primary reductants of Pt(IV) in vivo [68]. Rat (and human) plasma contain much higher levels of sulfhydryl groups than does cell growth medium, and reduction of tetraplatin was found to be far more rapid, tetraplatin having a half-life of 3 s [25]. On pre-treating rat plasma with N-methylmaleimide (NEM) to block the thiol groups, the rate of tetraplatin reduction was slowed threefold. However, this did not eliminate reduction completely, revealing that 20-30% of the reducing potential of rat plasma is non-thiol based. The nature of these other reductants was not determined. Pharmacokinetic experiments in humans showed that a high degree of Pt-protein binding was demonstrated, probably subsequent to reduction [69]. Following reduction, a number of Pt(II) biotransformation products arise from slow substitution reactions with biomolecules such as glutathione and methionine [70]. In humans, no unreacted tetraplatin is excreted following treatment, the major biotransformation product being the Pt(II) analogue [PtCl2(chxn)] (t1/2 = 13 min), followed by a number of ‘unreactive’ Pt(II) products, similar to the analogous cisplatin excreted biotransformation products [54]. It is important to note that the reduction of tetraplatin using growth medium and animal plasma in vitro correlates well with observed metabolism in vivo. This observation allows for reasonable conclusions to be drawn from in vitro analysis of developmental Pt(IV) complexes. In contrast to tetraplatin, iproplatin is difficult to reduce, and therefore displays a remarkably different spectrum of reactivity. In vitro protein binding assay reveals that no protein binding occurs in human plasma after 48 h [71], while as expected nearly all of the Pt(II) analogue cis-[PtCl2(ipa)2] is bound, with a half-life of 2.7 h [72]. Animals studies revealed that iproplatin was excreted unchanged, with a halflife of approximately 0.3 - 0.5 h, followed by the emergence of Pt(II) metabolites with a longer half-life of 39 h [56]. In humans, the half-life of iproplatin in plasma (0.67 - 1.27 h) was found to be longer than in animals, and after 12 h all Pt present was in the form of metabolites, most of which is protein bound [73]. It is interesting that after 48 h in


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vitro iproplatin is intact in human plasma studies, whereas reduction occurs far more rapidly in vivo, and this may be partially due to the intracellular reduction and efflux proposed by Pendyala and coworkers, as discussed below [74]. As with tetraplatin, no Pt(IV) biotransformation products were found, and the primary metabolite is the Pt(II) analogue [27], followed by other Pt(II) products. JM216 is intermediate in reduction potential, having diacetato ligands in the axial sites. Its metabolism is complex, and includes production of a number of Pt(IV) biotransformation products in plasma binding assays in vitro [26,75]. Following incubation of JM216 in human plasma, 93% of Pt was found to be protein bound [75], with the remaining 7% of Pt in the plasma ultrafiltrate as unbound or free Pt. Analysis of the free Pt species revealed a number of Pt(IV) biotransformation products, in contrast to tetraplatin and iproplatin. While the Pt(II) analogue of JM216, JM118 (cis-[PtCl2(cha)(NH3)]) predominated, three aquation products were also detected in the plasma ultrafiltrate, shown in Figure 4. These metabolites were shown to be active in cytotoxicity screening, as they are ultimately reduced to Pt(II) complexes [76]. The Pt(IV) metabolites were also observed in mouse blood plasma following oral and i.v. administration [76], and it is interesting to note that human blood plasma from phase trial patients only resulted in JM118 and the diaquated species JM383 [77]. Neither of the monoaquated species JM559 and JM518 were detected. Conversely, JM216 was also detected intact in mouse plasma, but not in human plasma. Studies in our laboratory have shown that protein binding of Pt(IV) complexes correlates with their reduction potential [67]. It was discovered that the binding of [PtCl4(en)] with albumin was almost identical to that of its Pt(II) analogue [PtCl2(en)], as the axial chloro groups of the complex are very labile, facilitating rapid reduction, followed by

FIG. 4. The three aquation products of JM216 following incubation for 4 h in human plasma ultrafiltrate; JM559, JM518 and JM383.

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binding. The other three complexes tested; cis,trans-[PtCl2(OAc)2(en)], cis,trans-[PtCl2(OH)2(en)] and trans-[Pt(ethmal)(OH)2(en)] exhibited little if any binding. The protein binding of [PtCl4(en)], cis,trans-[PtCl2(OAc)2(en)], cis,trans-[PtCl2(OH)2(en)], trans-[Pt(OH)2(ethmal)(en)] in RPMI medium supplemented with 15% FCS correlated with their reduction potentials; the more readily reduced complex ([PtCl4(en)]) binding to the greatest extent, with progressively less binding for the complexes containing acetato and hydroxo axial ligands. 7.2. Intracellular Fate

That JM216 and iproplatin could persist in plasma intact, and JM216 survives long enough to undergo ligand substitution (aquation) supports the notion that Pt(IV) complexes can indeed arrive at their physiological target intact. Direct observation of Pt(IV) complexes entering cells, and their ultimate intracellular fate is more difficult to achieve than simpler solution studies, due to the difficulties involved in working with cells, the more limited range of techniques available, and probably the interdisciplinary nature of the problem. Despite the short lifetime of tetraplatin in biological fluids (described earlier), Chaney and co-workers examined the intracellular metabolism of tetraplatin versus its Pt(II) analogue [PtCl2(chxn)] and observed two Pt(II) biotransformation products in tetraplatin treated cells not produced in the Pt(II) treated cells [78]. Pt levels in tetraplatin treated cells were also higher, though tetraplatin was not detected intracellularly. It was proposed that the metabolites formed, despite being Pt(II), were formed by Pt(II)catalyzed substitution of Pt(IV), though the authors concede there is no evidence for this. Conversely to tetraplatin, Pendyala and co-workers detected intracellular iproplatin after incubation with murine L1210 cells, along with its primary metabolite, the Pt(II) analogue cis-[PtCl2(ipa)2] and an unidentified metabolite [74], demonstrating that iproplatin can be reduced intracellularly. To aid the experiments, the drug incubation was carried out in Hank’s balanced salt solution to minimize extracellular reduction. Intracellular JM216 has also been detected by HPLC [79], and when glutathione was depleted in the cells (which would lower the rate of reduction), the Pt(IV) aquated species JM383 was also detected.


315

Recent experiments in our laboratories employing X-ray absorption near edge spectroscopy (XANES) have allowed us to develop a technique for determining the proportion of Pt(IV) and Pt(II) oxidation states in a mixed system by analysis of the XANES spectrum white line height.[80] By collecting XANES spectra from pelleted parental A2780 ovarian cells treated with Pt(IV) complexes, the percentage of Pt(IV) remaining at a number of time-points has been determined, as shown in Table 3. The proportion of Pt(IV) complexes remaining after 2 h corresponds well with their reduction potentials, and as described for in vitro experiments with biological media, there is little or no Pt(IV) remaining after 24 h. That the Pt observed is intracellular has been confirmed using an elemental imaging technique (SRIXE) [81]. TABLE 3 The proportion of intracellular Pt(IV) (±5%) remaining after incubation of complexes with A2780 ovarian cancer cells. Complex

Incubation period 2 h

24 h

cis-[PtCl2(NH3)2]

1%

–5 %a

cis-[PtCl2(NH3)4]

5%

–3 %a

cis,trans,cis-[PtCl2(OAc)2(NH3)2]

33 %

2%

cis,trans,cis-[PtCl2(OH)2(NH3)2]

54 %

0%

a

Negative values have no physical significance but are within the estimated error from zero.

That Pt(IV) complexes can enter cells prior to reduction is important, as it justifies the design and development of Pt(IV) drugs in the quest for Pt drugs with improved efficacy, and a lack of cross-resistance with currently employed Pt drugs.

316

8.


CONCLUSIONS

The biological behavior of Pt(IV) complexes is clearly related to how readily and rapidly they are reduced. However, even some of the most inert complexes are reduced intracellularly and are active anticancer agents. Thus tuning of these and other properties to minimize side effects and maximize tumor targeting is feasible. Therefore, the Pt(IV) oxidation state merits further attention, not only for classical cisplatin analogues, but also for multinuclear and trans complexes. ACKNOWLEDGEMENTS Thanks to Dr. Timothy W. Failes, Ms. Rebecca Alderden and Ms. Mei Zhang for useful discussions. Thanks to the Australian Research Council and the University of Sydney Cancer Research Fund for financial support. ABBREVIATIONS ATP cha chxn carboplatin CBDCA cisplatin Cys dien DNA en EP ER(+) ethmal FCS h HPLC i.v.

adenosine 5'-triphosphate cyclohexylamine cyclohexyl-1,2-diamine [PtCl2(CBDCA)] 1,1-cyclobutanedicarboxylic acid cis-[PtCl2(NH3)2] cysteine diethylenetriamine deoxyribose nucleic acid ethylenediamine cathodic reduction potential estrogen receptor positive ethylmalonato fetal calf serum hour high performance liquid chromatography intravenous


IC50 ipa iproplatin mV JM118 JM216 log P log Poct NADH NEM OAc oxoplatin RPMI s SRIXE tetraplatin XANES

317

cytotoxicity isopropylamine cis,trans,cis-[PtCl2(OH)2(ipa)2] millivolt cis-[PtCl2(cha)(NH3)] cis,trans-[PtCl2(OAc)2(cha)(NH3)] partition coefficient partition coefficient in octanol nicotinamide adenine dinucleotide (reduced) N-methylmaleimide acetato cis,trans,cis-[PtCl2(OH)2(NH3)2] Roswell Park Memorial Institute second synchrotron resonance induced X-ray emission [PtCl4(R,R-chxn)] X-ray absorption near edge spectroscopy

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