Medicinal Inorganic Chemistry - American Chemical Society

Chapter 8

Mechanistic Studies of Motexafin Gadolinium (Xcytrin ): A Redox Active Agent That Reacts with Electron-Rich Biological Substrates Downloaded by UNIV MASSACHUSETTS AMHERST on September 2, 2012 | http://pubs.acs.org Publication Date: August 25, 2005 | doi: 10.1021/bk-2005-0903.ch008

®

1

1

1

Darren J . Magda , Nikolay Gerasimchuk , Zhong Wang , Jonathan L . Sessler , and Richard A. Miller 2

1

1

Pharmacyclics, Inc., 995 East Arques Avenue, Sunnyvale, CA 94085 Department of Chemistry and Biochemistry, 1 University Station, The University of Texas at Austin, Austin, TX 78712-0165 2

Motexafin gadolinium (MGd, Xcytrin®) is a water soluble gadolinium(III) texaphyrin complex that is currently undergoing a Phase III clinical trial as an anticancer agent used in combination with radiation therapy of metastatic non-small cell lung cancer. It is also in clinical trials for use in a number of other oncological applications. Its mechanism of action is thought to rely, at least in part, on its ability to function as a redox cycling agent, capturing electrons from electron-rich endogenous species, so-called reducing metabolites, and transferring them to oxygen to produce various reactive oxygen species that are known to trigger apoptosis. In this chapter, the reaction of MGd and various other congeneric metallotexaphyrin complexes with the prototypical reducing species NADPH, ascorbate, and dithiothreitol, is summarized.

110

© 2005 American Chemical Society In Medicinal Inorganic Chemistry; Sessler, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

Ill

Downloaded by UNIV MASSACHUSETTS AMHERST on September 2, 2012 | http://pubs.acs.org Publication Date: August 25, 2005 | doi: 10.1021/bk-2005-0903.ch008

Introduction The texaphyrins are pentaaza, Schiff base-derived expanded porphyrins [1] that are known to form stable 1:1 complexes with many metal cations, including those of the trivalent lanthanide series. The gadolinium(III) complex of one particular texaphyrin derivative, motexafin gadolinium (MGd; Xcytrin®; 1, Chart 1), has been extensively studied as an MRI detectable anti-cancer agent [2-9]. Clinical studies have confirmed that this agent is taken up into and retained in tumors with high specificity [7-9]. Currently, MGd is the focus of numerous clinical trials, including ones in advanced head and neck cancer, multiple myeloma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and recurrent glioma.

(1) M = G d , n = 2 ( M G d ) (2) Μ = Lu, η = 2 (MLu) (3) Μ = Υ , η = 2 (Y-Tex)

(13) Μ = G d , η = 2 (DAGd-Tex) (14) Μ = L u , η = 2 (DALu-Tex) (15) Μ = Μ η , η = 1 (DAMn-Tex)

(4) Μ = Dy, η = 2 (Dy-Tex) (5) Μ = Er, η = 2 (Er-Tex) (6) Μ = E u , η = 2 (Eu-Tex) (7) M = S m , n = 2 ( S m - T e x ) (8) Μ = N d , η = 2 (Nd-Tex) (9) M = C d , n = 1 (Cd-Tex) (10) Μ = Μ η , η = 1 (Mn-Tex) (11) M = C o , n = 1 (Co-Tex) (12) Μ = F e ( 0 )

1 / 2

, η = 1 (Tex-Fe-O-Fe-Tex)

Chart 1. Structure of motexafin gadolinium 1 and its congeners. Because of its advanced clinical status, efforts are ongoing to study in detail the chemical and biological properties of MGd. Initial mechanistic studies revealed a clearance pathway that was largely hepatic [3] and provided support for a very novel mechanism of action [10,11]. This latter mechanism, manifest

In Medicinal Inorganic Chemistry; Sessler, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.


112 in terms of increased apoptosis, is believed to involve redox modulation processes [12] that take place outside of the nucleus [13]. In this chapter we review the chemical attributes of motexafin gadolinium (MGd; 1), focusing on those that make it attractive as a potential drug candidate. Particular attention will be paid to reactions of MGd and related metallotexaphyrin systems with electron-rich species, so-called reducing metabolites, since such reactions are thought critical to its biological activity. Here, key questions we are seeking to answer include specifically i) whether similar reactivity patterns are observed for different classes of reducing metabolites and ii) whether other metallotexaphyrins show greater or lesser levels of reactivity. The most defining feature of motexafin gadolinium and, indeed, all texaphyrins, is their greater size relative to the porphyrins. Not only do they contain five, rather than four, inward-pointing nitrogen atoms, they are characterized by a central coordinating core that is roughly 20% larger than that of the porphyrins. As a consequence, they form stable, nonlabile 1:1 complexes with most trivalent cations of the lanthanide series, including gadolinium(III) [5, 14, 15]. Another salient feature of the lanthanide(III) texaphyrins is exhibited in their redox behavior. While not particularly easy to oxidize, these complexes are easy to reduce [15-17]. This stands in marked contrast to what is true for freebase and metalated porphyrins and is thought to have important mechanistic implications (as described in more detail later on in this chapter). At low proton concentrations, the first reduction wave, corresponding to a single electron reduction process, is found to be quite reversible. In the specific case of MGd in DMSO the first reduction wave is seen at about -294 mV vs. Ag/AgCl or about 72 mV vs. NHE (cf. Figure 1) [17]. The E value associated with this reduction wave is seen to vary from complex to complex, with a greater ease of reduction being seen in the case of the smaller lanthanide cations; this is as would be expected based on considerations of size, charge density, and the fact that these smaller cations are better accommodated within the texaphyrin core [14]. In all cases, however, the reduction process remains facile and, in analogy to what is seen for metalloporphyrin complexes containing redox inactive metal centers, is thought to be ligand based. 1/2

Biolocalization Studies Studies of tissue and sub-cellular localization have been carried out with MGd and other texaphyrins [13,18]. These have provided support for the notion that texaphyrins are taken up into cells via an active transport process and largely co-localize with endosomes, lysosomes, and, eventually, mitochondria. By contrast, no evidence of significant incorporation into the nucleus was obtained over the full multi-hour time scale associated with these studies. Such


113

findings support the notion that MGd does not mediate its primary biological effect via a mechanism that involves the direct modification of nuclear DNA. Rather, alternative modes, involving perturbation of cytoplasmic processes, are thought to be operative (see below) [10].


Ο

-220-,

-OOU Η

,

0.86

1

0.88

1

0.90

•

1

0.92

J

•

0.94

1

0.96

1

r-

0.98

Lanthanide(lll) ionic radius, A.U. Figure 1. Plot of first reduction potential vs. Ag/AgCl in DMSOfor various lanthanide(III) texaphyrin complexes vs. ionic radius. This figure was constructed using datafirstpublished in ref 17.

Early Mechanistic Studies Prior to the completion of the sub-cellular localization studies, it was thought that MGd could be mediating its radiation enhancing effect by acting as an "electron sponge" [19]. Specifically, it was considered likely that it would serve to "trap" the solvated electrons produced in conjunction with hydroxyl radicals when water is subject to ionizing radiation. To the extent such "trapping" occurs within the nucleus, it would serve to increase the effective concentration of hydroxyl radicals, highly reactive species that are known to engender both reparable and irreparable modifications of DNA [20]. While such a direct mode of action now seems unlikely on the basis of the sub-cellular localization studies, ease of MGd reduction was identified as a key chemical property in the course of this early mechanistic work. In particular, it was found that solvated electrons, generated by pulse radiolysis, would serve to effect the one-electron reduction of


114 MGd. Furthermore, it was found that this reduced species would react with oxygen in a fast, reversible process to produce superoxide anion (cf. Scheme 1). This species is rapidly converted to hydrogen peroxide under biological conditions. The rate constant for non-enzymatic disproportionation of superoxide is estimated, for instance, to be 1 χ 10 M ' s" at pH 7.4 [21]. 5


MGd

+ 0 ~ 2

NADPH+ 0 Ascorbate

+ 0

2

^

MGd""" + 0 M

G

2

—

M

G

d

d

»»

1

1

0)

2

+

NADP + H 0 2

2

()

2

Dehydroascorbate +

H 0 2

2

(3)

Scheme 1. Key redox reactions of MGd and reducing metabolites. Independently, it was found that thiols, ascorbate, and NADPH could serve as a source of reducing equivalents, producing hydrogen peroxide and the oxidized forms of these key metabolites under conditions designed to mimic those present in typical cellular assays [10,11]. Since peroxide and the oxidizing metabolites formed in this process (disulfides, dehydroascorbate, etc.) are known to induce apoptosis and potentiate the effects of ionizing radiation in vitro [22,23], it was thought that these redox processes could play a key role in mediating the biological activity of MGd. This led to a working proposal, embodied in the Scheme 2, wherein this redox active agent acts in the presence of oxygen to generate the oxidized forms of these key metabolites and peroxide site-specifically within cancerous lesions in vivo. Initial evidence for this mechanism included i) the loss of MGd spectral features under hypoxic conditions in the presence of NADH and ii) the formation of N A D in the presence of catalytic amounts of MGd in the presence of oxygen. Subsequent chemical findings supporting this hypothesis involved quantifying the amount of hydrogen peroxide produced under the assay conditions and measuring the oxygen consumption in the presence and absence of the antioxidant enzymes superoxide dismutase and catalase. This work was complemented by biological studies demonstrating: i) Cytotoxicity of MGd in the presence of NADPH or ascorbate; ii) formation of intracellular peroxide by oxidation of dichlorofluorecin diacetate (DCFA) to fluorescent dichlorofluorescein (DCF); Hi) potentiation of the effects of ionizing radiation by the combination of MGd and ascorbate; and iv) synergistic biological effects with agents known to be involved in thiol metabolism, namely buthionein sulfoximine (BSO, an inhibitor of glutathione synthesis), diamide (a thiol oxidant), and antimycin A (a superoxide generator). +


115 ascorbate

„ H 0 * Q

X

2

superoxide

2

^ -

hydroxyl nyoroxy. radical

ascorbate radical

i 1

ς

=

X

superoxide ζ

R

T

metabolism

MGd


ascorbate

V

vitamin Ε G

S

S

G

J

2

2

^\ MGd ^ 0

2

glutathione

^

r e d u £ e d

^ / ^ H 0

2

dehydroascorbate

glutathione (GSH) 2

NAD

G

S

S

G

\ thioredoxin 'glutathione^ reductase p+reductase N ^ D P H thioredoxin

„

MGd

Λ

H 0 2

2

Λ

0

2

glucose

Scheme 2. Abridged description of the cellular antioxidant system and the effect that ionizing radiation and the presence of Xcytrin (MGd, 1) can have on this system. This scheme is modified from one that originally appeared in ref 10. In this chapter we present further evidence in favor of the generalized mechanism shown in Scheme 2 while reviewing briefly some of the previously published findings. It is important to note that Scheme 2 does not distinguish between global oxidative stress as opposed to any specific biological "target" as the basis for MGd activity. The in-depth studies to be described herein were undertaken in an effort to identify the more important biological targets of MGd. The focus will be on three classes of reducing metabolites of critical biological importance, namely NADPH, ascorbate, and thiols. In the course of this discussion, we will compare MGd to other related metallotexaphyrins (e.g., 212). All of these compounds share an identical equatorial ligand, and differ only in the identity of the coordinated metal cation. As might be anticipated, several of these analogues, e.g., those derived from transition metal cations, display properties markedly different from those of MGd. Other congeners, such as those drawn from the lanthanide series, differ in more subtle ways. In this regard, it is useful to note that the atomic radius of the lanthanide cation contracts, as the series is traversed from left to right. As a result, the corresponding texaphyrin complexes exhibit a graduated increase in stability, ease of reduction, and lipophilicity. It would thus be predicted that any structure activity relationships that are developed in the course of comparing lanthanide


116


derivatives, however revealing, may be unable to distinguish the relative contributions to activity made by each of these individual properties. Additional data, e.g., derived from texaphyrin complexes bearing altered solubilizing substituents on the ligand, are invaluable in making this latter determination. Nonetheless, this review will focus primarily on compounds 1-12, and make mention of other derivatives (i.e., 13-15) only where most germane.

Reaction with Reducing Metabolites

NADH/NADPH NADH and NADPH are important sources of reducing equivalents within cellular milieus. These chemically related species both have redox potentials of -315 mV vs. NHE (Ε°', i.e., at pH 7) [24]. As such, they represent some of the most reducing species present in cells. NADH is formed via the Krebs cycle and is principally involved in ATP generation through oxidative phosphorylation which occurs in the mitochondria. By contrast, NADPH is for the most part formed via the pentose cycle and is an important cofactor in a number of cellular enzymatic processes, including those involved in maintaining the cell in a reduced state as shown in Scheme 2 above. These two cofactors can be interconverted, albeit indirectly, via their oxidized forms through the action of NAD kinase [25]. Both species possess a negatively charged phosphoric anhydride linkage as part of their chemical structure and thus might be anticipated to coordinate to MGd in an axial position. It was thus considered important to understand how MGd and related metallotexaphyrin complexes would interact with these cofactors.

Reactions of MGd with NADH/NADPH In initial studies, the oxidation of NADPH by MGd was quantified using HPLC methods [10]. We now favor a method based on oxygen consumption. In this approach, the decrease in dissolved oxygen concentration in a sealed vessel is measured as a function of time at a chosen concentration of texaphyrin and reducing metabolite using a commercially available ruthenium bipyridyl-based oxygen sensor (Ocean Optics) [11]. This approach has the important benefit of enabling comparisons involving a variety of redox cycling substrates using a general assay. It also allows the concentration of MGd (or other absorbing species) to be simultaneously monitored by UV-vis spectroscopy.


117

As illustrated by the time-dependent oxygen consumption curve reproduced in Figure 2, aqueous, air-saturated, pH 7.5 HEPES buffered saline solutions of NADPH are to a first approximation kinetically stable, as judged both by lack of oxygen consumption (cf. initial time points in Figure 2) and by independent HPLC-based analysis of NADP as reported in ref. 10. However, in sealed vessels, where the dissolved oxygen is not replenished, addition of MGd induces a decrease in the overall oxygen concentration. Provided both the NADPH and oxygen are initially in substantial excess of the metallotexaphyrin complex, the [0 ] vs. time plot is linear at early times (ca. an hour for the experiment shown in Figure 2). This allows an initial rate, V , of 0.70 μΜ/min to be calculated. Gratifyingly, this value is coincident with that obtained by monitoring the NADP concentration increase as a function of time by HPLC and by the decrease in NADPH absorbance at 338 nm. Furthermore, this initial rate was found to be first order in [MGd], provided both oxygen and NADPH were present in excess.


+

2

0

+

a_i— —ι— —ι— —ι—>—ι— —ι— —ι— —\—«—ι— —r* ι 0 20 40 60 80 100 120 140 160 180 Time, minutes Figure 2. Plot of dissolved [0 ] vs. time for a sealed vial containing NADPH (initial concentration: 250 μΜ) in HEPES/NaCl buffer at pH 7.5 showing the effects of MGd addition (12.5 μΜ initial concentration after dilution). From the linear portion of the decay profile, an initial reaction rate, V , of 0.70 μΜ/min was calculated. 1

1

1

1

1

1

1

2

0

Separate studies, carried out using UV-vis spectroscopy, served to demonstrate that the MGd-mediated oxidation of NADPH in the presence of air


118


produces hydrogen peroxide. Thus, consistent with the pulse radiolysis experiments described above, we propose that electron transfer from NADPH to MGd serves to produce a reduced texaphyrin species that then reacts with oxygen to regenerate the starting MGd complex and to produce superoxide (not detectable under the reaction conditions), which disproportionates to form peroxide. The net result is shown in eqn. 2 of Scheme 1.

Effect of Oxygen When the absorption spectrum of MGd, rather than the concentration of dissolved 0 , is monitored as a function of time, it is found that little change in the texaphyrin spectral features is observed during the linear phase of the [ 0 ] vs. time curve. At longer times, or under conditions where oxygen is deliberately excluded from the reaction vessel, significant bleaching in the texaphyrin absorption bands is observed. This decrease in texaphyrin absorbance is accompanied by the formation of new absorbances typical of texaphyrin synthetic precursors. In contrast to what is observed when ascorbate or dithiothreitol (DTT) was used as the reductant (vide infra), addition of air did not serve to reverse the bleaching and restore the initial MGd spectrum. Thus, the decrease in texaphyrin spectral intensity observed in the absence of oxygen (which, presumably, functions as an electron acceptor) is thought to reflect the irreversible formation of multiply reduced species, including perhaps those wherein the basic texaphyrinframeworkis ruptured. While not addressed by experiment, it is considered that the formation of these latter species is facilitated in the case of NADPH because it can, inter alia, act as a more effective hydride donor than the other reductants considered in this study (see below). 2

2

Reactions of other metallotexaphyrins with NADH/NADPH +

Monitoring both the oxygen concentration and that of NADP as a function of time allowed the efficacy of MGd as an NADPH oxidation catalyst to be compared with that of related metallotexaphyrins. As illustrated in Figure 3, such a comparison leads to the conclusion, that for this particular reductant, MGd is substantially less effective than its Lu(III)-, Y(III)-, and Dy(III)containing congeners, 2-4. One interpretation of this finding is that these other metallotexaphyrins could have a role to play as therapeutic agents, although other factors, such as bioavailability may override the benefits of greater catalytic prowess. Another possibility is that the cofactor oxidation process as studied is not directly germane to biological activity. This latter interpretation is supported by the relatively low rates of reaction involved both in an absolute


119 sense and as compared to those obtained using other reducing species (see below). In preliminary comparative tests using intact cells, the human ovarian cancer cell line MES-SA was used and the extent of proliferation was monitored by


1-,

Time, minutes +

Figure 3. Plot of the [NADP+]/[NADP + NADPH] ratio Γ fraction NADP ) vs. time for a sealed vial containing NADPH (initial concentration: 250 μΜ) in HEPES/NaCl buffer at pH 7.5 showing the effects of different metallotexaphyrins (12.5 μΜ initial concentration after dilution of MGd (1), MLu (2), Y-Tex (3), and Dy-Tex (4)) on the rate of NADPH reduction. +

tetrazolium salt (MTT) reduction [10]. RPMI 1640 with dialyzed serum was chosen as the medium in these experiments to assure the absence of adventitious ascorbate (vide infra), while NADPH was added at a concentration of 333 μΜ. Under these conditions, the presence of MGd at the 50 μΜ level caused no decrease in cell viability within error, whereas a similar concentration of MLu caused a 40 ± 10% reduction in cell survival. By contrast, replacement of NADPH by ascorbate reversed the order of complex activity. For instance, at an ascorbate concentration of 111 μΜ, 50 μΜ of MGd, 1, led to a 65 ± 5% reduction in cell survival vs. no apparent reduction in the case of MLu, 2. In all cases, the effects, where observed, were significantly (or even completely) obviated by the addition of catalase and superoxide dismutase. Such findings are consistent with the extracellular production of reactive oxygen species (i.e.,


120 outside of the cells themselves), under conditions that may or may not mimic those operative in vivo. They also serve to demonstrate that the redox cycling activity observed in buffered saline, outlined above, is recapitulated in tissue culture medium, i.e., in the presence of serum proteins.


Effect of thioredoxin reductase and other enzymes During the course of the studies described above, it was recognized that other components present in the cellular milieu would need to be considered prior to any attempt to extrapolate these measurements to biological systems. In particular, it was of interest to examine the rate of NADPH oxidation by MGd in the presence of enzymes for which NADPH is a cofactor, such as thioredoxin reductase (EC 1.6.4.5). NADPH was therefore incubated with MGd as described above at concentrations providing a low rate of chemical reaction. When thioredoxin reductase was added to this system, a dramatic enhancement of oxygen consumption was observed. As might be expected, oxygen consumption was accompanied by a corresponding bleaching of the NADPH absorbance at 338 nm and, after most of the oxygen present in the cuvette was consumed, texaphyrin absorbances at 470 and 742 nm. Similar findings were obtained where the order of addition was reversed, i.e., a slow background reaction between NADPH and the enzyme, followed by rapid reaction upon addition of catalytic amounts of MGd (cf. Figure 4). Other enzymes, such as cytochrome c reductase and cytochrome P450 reductase, could be effectively substituted for thioredoxin reductase. By contrast, glutathione reductase and other cofactor-binding enzymes investigated did not display this effect. Experiments such as these lead us to suggest that MGd can oxidize cofactors indirectly, perhaps by accepting an electron from the flavin subunit of their enzyme complexes. Although the details of MGd interaction with enzymatic processes such as these remain the subject of ongoing investigation, it is reasonable to conclude from the data available at this time that such interactions are likely to contribute to the biological activity of the drug.

Ascorbate

Reactions of MGd and congeners with ascorbate Ascorbate, with a one-electron reduction potential, E ' of 282 mV vs. NHE [26], is another critical source of cellular reducing equivalents. Regenerated in 0



121

vivo from the reduction of dehydroascorbate by NADPH either directly or via the intermediacy of glutathione, this species serves not only to maintain cells in a reduced state, it also plays a role in protecting against the deleterious effects of superoxide and other radicals. It thus complements enzymatic pathways (e.g., superoxide dismutase) that afford an alternative means of protection. Ascorbate is also of interest because it is not the product of biosynthesis in humans but rather must be obtained from the diet. This is not true, however, for other species, such as mice. Surprisingly, ascorbate is often absent in standard cell culture media. We, and others, have thus proposed that understanding the effects of MGd on ascorbate could provide important insights into the behavior of this agent both in vitro and in vivo [10,27].

300n

Ί&0 MGd

0

* 200·

2

338 nm (NADPH) 468 nm (MGd) 742 nm (MGd)

Time (minutes) Figure 4. Change in dissolved oxygen concentration as a function of time observed whenfirstthioredoxin reductase (0.6 unit) and then subsequently MGd (62.5 μΜ) is added to a sealed cuvette containing NADPH (initial concentration: 1.25 mM) in HEPES/NaCl buffer at pH 7.5. Also monitored in this experiment were the change in absorbance at wavelengths characteristic of NADPH (338 nm) and MGd (468 and 742 nm).



122 As proved true for NADPH, addition of a catalytic amount of MGd to aerated solutions of ascorbate led to rapid, catalytic oxidation and concurrent production of hydrogen peroxide in accord with Eqn. 3, Scheme 1. However, in contrast to what was observed with NADPH, in the case of ascorbate, MGd was found to be a substantially more effective oxidation catalyst than MLu or two other lanthanide(III) texaphyrin complexes containing smaller coordinated cations, namely Dy-Tex (4) and Er-Tex (5). Other lanthanide(III) texaphyrins containing larger cations, namely the Eu(III), Sm(III), and Nd(III) complexes (68, respectively) showed rates that were similar to those of MGd. Initial rates of reaction therefore appear to fall into two groups, that correlate with ionic radius, as can be seen for the seven texaphyrin lanthanide congeners shown in Figure 5. 14-i

c

12

MGd

3.

10

°

of

Sm-Tex

^

0

Nd-Tex

I

Of

c ο

i

Eu-Tex Dy-Tex 6-

MLu

S c

Er-Tex

4

2-

0-1

, 0.86

»

, 0.88

,

, 0.90

,

, 0.92

,

, 0.94

,

, 0.96

,

,

,

0.98

j 1.00

Lanthanide(lll) ionic radius, A.U. Figure 5. Plot of initial rates of ascorbate oxidation vs. lanthanoid(III) ionic radius, as determined from the change in absorbance at 266 nm. The texaphyrin complexes (0.05 molar equivalent) in question were added to solutions of ascorbate (1.25 mM) in buffered saline (100 mM NaCl, 50 mM HEPES, pH = 7.5). All reactions were conducted in 1 mm cuvettes. As mentioned earlier, we observe changes in reduction potential, stability, and solubility as the lanthanide series is traversed. Presumably, this reflects the fact that the metal is out of the plane in complexes with the lighter cations (as inferred from previous X-ray diffraction analyses) [14], whereas in complexes



123

with heavier cations, such as MLu, the cation is more centrally coordinated with the plane of the texaphyrin core. In the reaction with NADPH, it is plausible to rationalize the observed order of reactivity based on reduction potential. In the reaction with ascorbate, other factors, such as solubility, could be playing a role. In fact, a variety of experiments, including extinction coefficient studies, have led us to consider that MLu, but not MGd, is likely to be aggregated under conditions analogous to those employed in the ascorbate oxidation studies. In order to assess a possible effect of aggregation, we therefore compared the reactivity of MLu with that of its (doubly protonated) diamine congener, DALu-Tex (14). As seen in Figure 6, the hydrophilic DALu-Tex catalyzed ascorbate oxidation with an initial rate of reaction faster than that of MLu and comparable to that of MGd. On the other hand, the rate of ascorbate oxidation by MGd was essentially the same as that of its diamine derivative, DAGd-Tex (13, data not shown). This, along with the observation that essentially first order rate behavior was seen as the MGd concentration was varied over a 1.5 χ 10" to 6 χ ΙΟ" Μ concentration range (the initial rate was found to increase by a factor of 4.8 as the ascorbate:MGd ratio was varied from 80:1 to 20:1), is completely consistent with the previous findings that MGd is not aggregated under normal aqueous buffer conditions, at least at concentrations relevant to the present experiments. Perhaps not surprisingly, the initial rate of catalysis was found to increase as the concentration of oxygen was increased. For instance, when the normal air atmosphere in the cuvettes used to study the reaction dynamics was replaced by pure oxygen, the initial rate was increased by a factor of ca. 3 and the degree of completion (as judged by the loss of ascorbate) was also found to be enhanced. Conversely, when oxygen was excluded from the vessel, no reaction occurred, in contrast to the situation seen above using NADPH as the reductant. These observations are consistent with the texaphyrin undergoing a reversible 1electron reduction with the driving force for the reaction being thermodynamically unfavorable in the absence of oxygen. In the presence of oxygen, peroxide is formed via the disproportionation of superoxide with the effect that the overall reaction becomes energetically favorable. When the concentrations of oxygen and ascorbate are high relative to those of MGd, the rate of ascorbate oxidation is catalytic in MGd, as observed with NADPH above. Under these conditions, the MGd complex is stable, with no evidence of decomposition being observed. However, as the oxidation process is allowed to proceed, a new species, characterized by spectral features at 510 and 780 nm, is seen to grow in. This species undergoes precipitation when allowed to stand in saline, and reverts to MGd in organic solvent. On the basis of these observations and specific chemical analysis, it was assigned as being a coordination polymer with oxalate [11]. Oxalate is a known decomposition product of dehydroascorbate. It is also known to be unstable at physiological pH 6

6



124

and in the presences of superoxide. Carboxylate anions coordinate well to gadolinium(III) and other lanthanide(III) cations. Thus, the formation of oxalate and its subsequent reaction with MGd over time is considered reasonable. The fact that it can bridge two Gd(III) centers in side-on fashion, allowing the planar aromatic texaphyrin ligands to stack, explains the ease of formation of the polymer and its spectral characteristics. Indeed, viscosimetry measurements indicate an average molecular weight of about 100,000 dalton for the species [28]. While the importance of such polymer formation under in vivo conditions is subject to current analysis, it is noteworthy that it is taken up into A549 lung cancer cells more effectively than MGd [11]. The potential importance of the reactions with ascorbate as a possible indicator of biological function propelled us to study whether other metallotexaphyrins besides those containing lanthanide(III) cations could 14.4 μΜ/min

1.951.90-

Ε

c CO

•

MLu

ο

DALu-Tex

1.85-

CO CM

1.80-

15 Φ α c

1.75-

hm

1.70-

®

Ο

η

Medicinal Inorganic Chemistry - American Chemical Society

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