![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada
(Received May 18, 2005; accepted June 17, 2005)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Hasinoff et al., 1998; Cvetkovic and Scott, 2005). There is now considerable evidence to indicate that this toxicity may be due to iron-dependent oxygen free radical formation (Malisza and Hasinoff, 1995; Meyers, 1998) on the relatively unprotected cardiac muscle. Thus, dexrazoxane can be considered a prodrug analog of EDTA that is activated upon hydrolysis to its one-ring open intermediates B and C, and then to its fully ring-opened form ADR-925 according to the scheme in Fig. 1 (Hasinoff, 1990, 1994a,b, 1998; Schroeder et al., 2005). Thus, dexrazoxane may act through its ring-opened hydrolysis product ADR-925 (Fig. 1), which can either remove iron from the iron-doxorubicin complex (Buss and Hasinoff, 1993) or bind free iron, thus preventing iron-based oxygen radical formation.
|
,b). Given the slow rate at which dexrazoxane hydrolysis-activation occurs in vitro, it is, thus, unclear how sufficient amounts of ADR-925 could be present in heart tissue to chelate iron and prevent oxygen radical damage before dexrazoxane was eliminated (ß-phase t1/2 of 4.2 ± 2.9 h in humans) (Hochster et al., 1992). It is, however, observed that in both humans (Schroeder et al., 2003) and rats (Schroeder and Hasinoff, 2002), dexrazoxane is quickly metabolized to B and C, and then to ADR-925. The rapid rate of hydrolysis of dexrazoxane to B and C and the rapid appearance of ADR-925 in plasma in vivo suggested that, first, dexrazoxane and, then, B and C were all enzymatically metabolized.
We previously showed that the dexrazoxane metabolic intermediates B and C are rapidly formed from dexrazoxane in a primary rat hepatocyte suspension (Hasinoff et al., 1994), a result that is consistent with dexrazoxane being metabolized by the zinc hydrolase DHPase (EC 3.5.2.2
[EC]
). However, although pure DHPase enzymatically hydrolyzes dexrazoxane to B and C, it is unable to enzymatically hydrolyze B and C to ADR-925 (Hasinoff et al., 1991; Hasinoff, 1993). In other work with purified DHOase (EC 3.5.2.3
[EC]
) we showed that this zinc hydrolase is able to enzymatically hydrolyze B and C to ADR-925, but cannot act on dexrazoxane (Schroeder and Hasinoff, 2002). Thus, DHPase and DHOase may act sequentially, and in concert, to effect the full metabolism of dexrazoxane to its active metal ion-chelating form ADR-925. Whereas DHOase is present in a variety of tissues including the heart, liver, and kidney (Kennedy, 1974), DHPase is only present in the liver and the kidney, but not in the heart (Dudley et al., 1974; Hasinoff et al., 1991; Hamajima et al., 1996). Because dexrazoxane acts as a cardioprotective agent, we investigated the metabolism of its one-ring open intermediates B and C to ADR-925 in adult rats with a view to gaining a complete description of the metabolic hydrolysis-activation of dexrazoxane. More recently, we showed that C is rapidly metabolized by DHOase in both rat myocyte and hepatocyte suspensions (Schroeder et al., 2005). We also showed that C underwent a slower Ca2+- and Mg2+-promoted hydrolysis in both human and rat plasma (Schroeder et al., 2005).
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Analysis of B, C, and ADR-925. The HPLC analysis of dexrazoxane, B and C using an ion-pair reagent with the reversed-phase C18 column (detection wavelength 205 nm) has been described (Schroeder et al., 2002, 2003, 2005). We have previously described a calcein fluorescence flow-injection analysis determination of ADR-925 on an HPLC apparatus (Schroeder et al., 2002, 2003, 2005). In this study, ADR-925 was determined using the same method except that the fluorescence was determined on a BMG LABTECH (Durham, NC) Fluostar Galaxy 96-well fluorescence plate reader (
ex, 485 nm; em, 520 nm).
DHPase-Mediated Biosynthesis of the B/C Mixture. Milligram quantities of a mixture of B and C were biosynthetized using the dexrazoxane-hydrolyzing activity of DHPase. We previously showed that DHPase rapidly hydrolyzes dexrazoxane to B and C, but not to ADR-925 (Hasinoff et al., 1991; Hasinoff, 1993). DHPase is present as a minor protein contaminant in C-30 Sigma bovine catalase. We and others have previously used it as a source to obtain pure DHPase (Hasinoff et al., 1991; Hasinoff, 1993). Four milliliters of a crystalline suspension of catalase (35 mg of protein/ml) were dialyzed (molecular weight cut-off of 12,000) for 24 h at 4°C with 4 liters of distilled water to remove preservative. Two milliliters of dexrazoxane hydrochloride in water (30 mg/ml) were titrated with 5 M NaOH to pH 7 and then added to the dialyzed catalase/DHPase mixture such that the final dexrazoxane solution concentration was 10 mg/ml. The reaction was carried out at room temperature and was monitored by HPLC, and typically reached completion in 45 to 60 min. The reaction was deemed complete when the dexrazoxane levels were less then 10 µM (or less than 0.03 mol % of the initial dexrazoxane concentration). Acetonitrile (2:1, v/v) was used to precipitate proteins from the reaction mixture. After centrifugation to remove precipitated protein, the B/C mixture was dried under nitrogen and reconstituted in sterile saline (0.9% NaCl w/v) just before use. Under these reaction conditions, dexrazoxane was hydrolyzed to B and C with a B/C ratio of 4.1:1, which was consistent with our previous published enzyme kinetic reports of DHPase-mediated hydrolysis of dexrazoxane (Hasinoff, 1993). The B/C mixture obtained was also analyzed for ADR-925 and was found to contain less then 0.5 mol % ADR-925.
Preparation of ADR-925 from Dexrazoxane. ADR-925 used for the animal experiments was prepared by adding 50 µl of 5 M NaOH per ml to 10 mg/ml dexrazoxane hydrochloride solution for 48 h. We previously showed that the NaOH-catalyzed hydrolysis of dexrazoxane gave ADR-925 (Hasinoff, 1994a). At 48 h, the reaction was quenched with 45 µl of 5 M HCl per ml and was titrated to pH 7. ADR-925 prepared in this fashion contained less then 0.003, 0.006, and 0.006 mol % of dexrazoxane, B, and C, respectively. The ADR-925 was filter-sterilized prior to animal use.
Dosing and Sample Collection. The rats (male Sprague-Dawley, 300–350 g) were allowed food and water ad libitum before the study. The rats were anesthetized i.p. with a combination of ketamine (90 mg/ml) and xylazine (10 mg/kg), and the left jugular vein was cannulated. The B/C mixture and ADR-925 were administered at a dose of 20 mg/kg (5 mg/ml in saline) as an i.v. bolus infusion (2 ml/min) through the tail vein. Blood samples (300 µl) were removed from the left jugular vein, as described (Schroeder and Hasinoff, 2002), from a group of three rats at times of 2, 15, 30, 45, 60, and 90 min. One 300-µl blood sample was drawn from each rat prior to the administration of the B/C mixture or ADR-925 as a control. The blood samples were added to heparin (10 µl of 1000 units/ml) and centrifuged for 5 min at 500g. The plasma was removed, 20 µl of 5 M HCl/ml of plasma was added, and the samples were stored at –80°C to prevent further hydrolysis (Hasinoff, 1994a). To precipitate plasma proteins, acetonitrile (Fisher) was added in a 2:1 ratio (v/v). The sample was mixed, allowed to settle for 5 min, and centrifuged at 8000g for 10 min. The supernatant was removed, acidified to pH 3 with HCl, and evaporated to dryness under nitrogen. The samples for the ADR-925 analysis were reconstituted in water to their original volume just before analysis. Samples for dexrazoxane, B, and C analysis were reconstituted in 10 mM HCl/500 µM disodium EDTA to their original volume just prior to analysis. The dexrazoxane hydrochloride (40 mg/kg) for the tissue level studies was administered as described (Schroeder and Hasinoff, 2002). The animal protocol was approved by the University of Manitoba Animal Care Committee.
ADR-925 Levels in Liver, Heart, and Brain Homogenates. After the last blood collection time point (2.8 h after the infusion of dexrazoxane, or 1.5 h after the infusion of the B/C mixture or ADR-925), the heart, brain, and liver were removed and weighed. Using surgical scissors, the organs were cut into small pieces (<5 mm3), washed three times in a 50-ml centrifuge tube by adding 30 ml of 10 mM HCl to stabilize dexrazoxane and B and C (Hasinoff, 1994a), and rapidly swirled for about 1 min, after which the wash solution was discarded. After the third wash, the wash solution was visibly clear of blood. The washed minced organs were homogenized using a Polytron homogenizer (Kinematica, Basel, Switzerland) for 5 min. The homogenate was then centrifuged at 0°C for 2 h at 18,000g. The supernatant was removed and placed in a 1.5-ml microcentrifuge tube and stored at –80°C until analyzed. For the animals treated with dexrazoxane, part of the supernatant sample was treated with 250 mM NaOH for 15 h to convert dexrazoxane, B, and C into ADR-925 (Hasinoff, 1994a) and, thus, have some measure of the total amount (ADR-925tot) of all forms of dexrazoxane in the tissues.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
In an initial pharmacokinetic analysis, the plasma concentrations for B, C, and ADR-925 over the first 30 min were each individually fitted to an i.v. bolus one-compartment model with a single exponential term, where Cb, Cc, and Cadr are the concentrations of B, C, and ADR-925 at time t, respectively. CB, CC, and CADR are their respective concentrations at time 0, and kb, kc, and kADR are their respective first-order elimination rate constants in eqs. 1, 2, and 3:
 | (1) |
 | (2) |
 | (3) |
|
Because ADR-925 achieved its maximum plasma concentration at the first time point of 2 min, this result suggested that it was produced from the rapid metabolism of B and C. At 2 min, the plasma concentration of ADR-925 reached a value of 114 ± 15 µM, which was already about one-third the value of the concentration of B with a value of 315 ± 42 µM at this time. Inspection of the initial concentration-time data in Fig. 2b, plotted for times up to 30 min, also showed that the initial apparent elimination of ADR-925 paralleled that of B and C. Together, these results suggest that the appearance of ADR-925 was formation rate-limited by the rapid in vivo metabolism of B and C. Given these observations, it was decided to attempt a more complete pharmacokinetic analysis that would contain terms that described the rates at which both B and C were metabolized. Thus, the initial elimination of B and C was modeled assuming that they were eliminated solely through metabolism via a first-order process, and that the ADR-925 metabolite formed was itself eliminated via a first-order process with a rate constant, km. Thus, with these assumptions, the elimination of B and C and the first-order formation of ADR-925 for times up to 30 min can be described by eqs. 4, 5, and 6 (Jackson et al., 2004):
 | (4) |
 | (5) |
 | (6) |
|
 | (7) |
and ß are the -distributive and ß-terminal phase elimination rate constants, respectively. A and B are their respective pre-exponential factors, the sum of which gives Cmax. The average of the best-fit parameters from three rats gave and ß values of 0.40 ± 0.20 min–1 and 0.024 ± 0.011 min–1, respectively, and a Cmax value of 820 ± 230 µM. The smooth curve in Fig. 3 was calculated from the best-fit parameters for all the data in eq. 7. The average derived overall elimination rate constant, k10, had a value of 0.14 ± 0.06 min–1.
Tissue Levels of ADR-925 in Rats Treated with Dexrazoxane, the B/C Mixture, or ADR-925. Because our previous studies showed that dexrazoxane was quickly metabolized in the rat (Schroeder and Hasinoff, 2002) and in humans (Schroeder et al., 2003), it was decided to determine the tissue levels of ADR-925 in heart, liver, and brain tissues to determine whether, in fact, ADR-925 was present in these tissues. Tissues were collected 2.8 h after administration of 40 mg/kg dexrazoxane and 1.5 h after the administration of 20 mg/kg of the B/C mixture or ADR-925. In the dexrazoxane study, part of the tissue supernatants were treated with NaOH to convert any remaining dexrazoxane, B, or C in the tissue to ADR-925. Thus, the ADR-925tot values in Table 2 are the sum of the dexrazoxane, B, C, and ADR-925 levels in the tissue. The ADR-925 assay is a sensitive fluorescence assay (Schroeder and Hasinoff, 2002) and allowed a determination of low amounts of ADR-925 in these tissues. However, low levels of dexrazoxane, B, and C in these tissues precluded their direct determination by HPLC.
|
The results in Table 2 show that after administration of 40 mg/kg dexrazoxane, both the heart and liver ADR-925 levels were approximately half the total amount of all forms of dexrazoxane and its three metabolites (ADR-925tot). The presence of ADR-925 in the heart and liver indicates that dexrazoxane was metabolized in vivo to ADR-925. It is also noteworthy that ADR-925 levels in the brain were undetectable (<2 µmol/kg wet tissue), indicating that the polar dexrazoxane and its three polar metabolites were unable to detectably cross the blood-brain barrier. We previously showed that dexrazoxane, B, C, and ADR-925 were all detectable in the plasma (49, 13, 3.2, and 30 µM, respectively) of similarly dexrazoxane-treated rats at 3 h (Schroeder and Hasinoff, 2002), and thus, it is not surprising that significant levels of ADR-925 were still present in the heart and liver at 2.8 h after dexrazoxane administration.
Tissue levels of ADR-925 1.5 h after administration of a 20 mg/kg B/C mixture were also determined (Table 2). Levels in the heart and liver were approximately one-half of those found for the 2-fold higher dose of dexrazoxane. These results also indicated that B and C were metabolized in vivo to ADR-925. ADR-925 levels in brain tissue, similar to those in the dexrazoxane treatment, were also undetectable (<2 µmol/kg wet tissue). ADR-925 was also detected in the heart and liver of animals treated with ADR-925, although at levels that were about 6- to 10-fold lower than those in the dexrazoxane-treated animals. This result is consistent with the polar ADR-925 being much less permeable to these tissues.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) or strongly bind free Fe2+ (Kf 1010.0 M–1) (Huang et al., 1982) or Fe3+ (Kf 1018.2 M–1) (Diop et al., 2000) is likely the basis of the mechanism by which dexrazoxane prevents iron-based doxorubicin-mediated free radical oxidative stress on the heart muscle. Our previous studies on the metabolism of dexrazoxane in humans (Schroeder et al., 2003) and in the rat (Hasinoff and Aoyama, 1999a,b; Schroeder et al., 2002) showed that B, C, and ADR-925 quickly appeared in the plasma after bolus i.v. administration, a result which suggested that dexrazoxane, and B and C were rapidly metabolized to ADR-925. Thus, these results suggested that rats treated with a B/C mixture might also metabolize B and C to ADR-925.
We also previously showed that DHPase, which is present in the liver and the kidney, can efficiently hydrolyze dexrazoxane (Hasinoff et al., 1991, 1994; Hasinoff, 1993, 1994c; Hasinoff and Aoyama, 1999b) and is likely the enzyme that is primarily responsible for the metabolism of dexrazoxane to B and C. We have also shown that DHPase is unable to convert B and C into ADR-925 (Hasinoff et al., 1991). Likewise, we have shown that DHOase is able to enzymatically hydrolyze B and C to ADR-925 but is unable to hydrolyze dexrazoxane (Schroeder and Hasinoff, 2002).
The results of this study showed that the B/C mixture was very rapidly metabolized to ADR-925, a result that suggested enzyme-mediated metabolism. The peak plasma concentration of dexrazoxane of 530 µM seen in our previous rat study (Schroeder and Hasinoff, 2002), after administration of 40 mg/kg dexrazoxane, can be compared with peak plasma concentrations of B and C of 442 and 76 µM, respectively, for a 20 mg/kg dose of the B/C mixture (Table 1). In fact, ADR-925 was maximally present in plasma at 2 min when the first blood sample was taken. The rapid appearance of ADR-925 suggested that the initial pharmacokinetics could be analyzed by a model in which the initial concentrations of the ADR-925 metabolite were largely determined by metabolism of B and C. Kinetic analysis of the data (Fig. 2b) in this formation rate-limited model showed that the initial ADR-925 elimination rate constant of 0.32 min–1 was in fact similar in magnitude to the B and C metabolite formation rate constants kfb and kfc of 0.13 and 0.20 min–1, respectively. This simplified model, which only analyzed the data for B and C and ADR-925 up to 30 min, of necessity, involved several simplifying assumptions. It was assumed that B and C, at least initially, were only eliminated through metabolism to ADR-925. It was also assumed that the initial formation rate-limited elimination of B and C occurred through nonsaturating processes. The rapid metabolism of B and C was also a limitation on obtaining a more accurate formation-limited pharmacokinetic analysis. The elimination of ADR-925, when ADR-925 was administered alone, was biphasic and gave an average -phase distributive phase rate constant of 0.40 min–1 (Fig. 3). This fast distributive phase precluded a valid comparison with km in the formation-limited model (Table 1) because the ADR-925 was produced from metabolism of B and C, and these intermediates would already have undergone their own prior distributive phase. This study did not directly address the question of whether or not doxorubicin administered after dexrazoxane would affect the metabolite profile or clearance of the intermediates of dexrazoxane or its metabolites, since the animals were not treated with doxorubicin. Thus, we cannot rule out the possibility that doxorubicin treatment may affect the metabolism and the pharmacokinetics of dexrazoxane or it metabolites
We previously used the ratio of the initial plasma concentrations of B to C, compared with that produced by purified DHPase of 4:1 (Hasinoff, 1993), after the administration of dexrazoxane to either rats or humans, as evidence that DHPase was involved in the metabolism of dexrazoxane to B and C (Hasinoff and Aoyama, 1999a; Schroeder et al., 2003). We previously showed that purified DHOase, which is present in the liver, kidney, and heart (Kennedy, 1974), is able to hydrolyze B and C to ADR-925 (Schroeder et al., 2002). Thus, if DHOase were the enzyme solely responsible for the metabolism of B and C in vivo, DHOase would be acting on B and C under nonsaturating conditions, given that the Km values for B and C are 4.8 and 11 mM, respectively (Schroeder et al., 2002). Thus, under these conditions, DHOase would hydrolyze B and C at a rate ratio equal to the ratio of their Vmax/Km values of 0.9 (Schroeder et al., 2002). It can be seen from the data of Table 1 that the initial formation rate constant ratio kfb/kfc had a value of 0.68 ± 0.15, a value that compares reasonably well to 0.9 for the pure enzyme. Thus, these results are consistent with DHOase being largely responsible for the in vivo metabolism of the B/C mixture. We also previously reported that plasma, blood, and plasma concentrations of Ca2+ and Mg2+ were able to promote the hydrolysis of B and C 3- to 5-fold over its background rate of hydrolysis (Buss and Hasinoff, 1997; Schroeder et al., 2005). Thus, these metal ions may partially contribute, along with DHOase, to the metabolism of B and C.
We have also shown in this study that rats treated either with dexrazoxane or the B/C mixture yield tissue levels that are relatively high in ADR-925. The values we obtained can be compared with the total of all metabolite forms of 14C-labeled dexrazoxane levels determined at 5 min in heart and liver of 320 and 640 µmol/kg, respectively, for mice administered 100 mg/kg dexrazoxane (Mhatre et al., 1982). Because the liver contains both DHPase and DHOase, which may act sequentially to form ADR-925, it was not unexpected that these tissues contained ADR-925. However, the fact that the heart does not contain DHPase (Hamajima et al., 1996) and the supernatant of a heart homogenate does not have dexrazoxane-hydrolyzing activity (Hasinoff et al., 1991) suggests that the ADR-925 found in the heart was from uptake of circulating B and C or ADR-925. In support of this conclusion, we did show that isolated neonatal myocytes were able to slowly take up B, C, and ADR-925 and displace iron from an intracellular fluorescence-quenched iron-calcein complex (Hasinoff et al., 2003). The much lower levels of ADR-925 in heart and liver when the rats were treated with ADR-925 reflect the low cellular permeability of the highly polar dianionic ADR-925.
The lack of detectable ADR-925 in brain tissues is consistent with the polar dexrazoxane and its three metabolites not being able to cross the blood-brain barrier. Dexrazoxane is currently in clinical trials as an extracerebral rescue agent in cancer patients with brain metastases treated with high-dose etoposide (Holm et al., 1998; Schroeder et al., 2003, 2004). Because neither dexrazoxane nor its metabolites were found in brain tissue, these results suggest that dexrazoxane will not antagonize the topoisomerase II poisoning activity of etoposide (Sehested et al., 1993) in cancerous brain tissue.
This study did not directly address how dexrazoxane exerted its cardioprotective effects in the heart. However, in conclusion, these studies showed that the one-ring metabolites B and C of dexrazoxane were rapidly metabolized in the rat to ADR-925. Their rapid in vivo hydrolysis provides a mechanism by which B and C can be activated to its iron-binding form, ADR-925, which has antioxidant activity. The initial plasma levels of ADR-925 that were observed were consistent with the formation rate-limited enzyme-mediated metabolism of B and C. Some of the metabolism of B and C may take place in the heart, which contains the enzyme, DHOase, that can metabolize B and C to ADR-925.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: ICRF-187, dexrazoxane; ADR-925, N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[(N-(2-amino-2-oxoethyl)]glycine; B, N-(2-amino-2-oxoethyl)-N-[(1S)-2-(3,5-dioxo-1-piperazinyl)-1-methylethyl]glycine; C, N-(2-amino-2-oxoethyl)-N-[(2S)-2-(3,5-dioxo-1-piperazinyl)propyl]glycine; DHPase, dihydropyrimidine amidohydrolase (dihydropyrimidinase); DHOase, dihydroorotase; HPLC, high-pressure liquid chromatography.
Address correspondence to: Brian B. Hasinoff, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada. E-mail: B_Hasinoff{at}UManitoba.ca
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  P. E. Schroeder, D. Patel, and B. B. Hasinoff The Dihydroorotase Inhibitor 5-Aminoorotic Acid Inhibits the Metabolism in the Rat of the Cardioprotective Drug Dexrazoxane and Its One-Ring Open Metabolites Drug Metab. Dispos., September 1, 2008; 36(9): 1780 - 1785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R.J. H. Bird and S. M. Swain Cardiac Toxicity in Breast Cancer Survivors: Review of Potential Cardiac Problems Clin. Cancer Res., January 1, 2008; 14(1): 14 - 24. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
), C (
), and ADR-925 (
) data were calculated from a one-compartment fit model described by eqs. 1, 2, and 3 and gave average half-lives of 4.8, 3.3, and 8.3 min, respectively. The data plotted are averages from three rats except for the B datum at 45 min, which was from two rats. b, average rat plasma concentrations of B, C, and ADR-925, but replotted for times up to 30 min only, for fitting to an ADR-925 formation rate-limited model. The smooth lines (solid lines for B and C and a dashed line for ADR-925) were calculated from simultaneously fitting the whole concatenated data set to eqs. 4, 5, and 6. These equations describe a model in which ADR-925 is formation rate-limited by its rapid in vivo metabolism.