SEARCH

SEARCH BY CITATION

Keywords:

  • doxorubicin prodrug;
  • mitochondria cytochrome oxidase;
  • DNA mitochondrial;
  • reactive oxygen species

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Doxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long-term and explain in part the delayed onset of heart dysfunction. DOXO-EMCH a 6-maleimidocaproyl hydrazone derivative of doxorubicin, is an albumin binding prodrug which has entered clinical trials because of its superior antitumor and toxicological profile. In the present work, we examined the chronic cardiotoxicity of DOXO-EMCH in direct comparison with doxorubicin. Rats (11 weeks of age) were treated with intravenous doxorubicin (0.8 mg/kg weekly for 7 weeks), an equimolar dose of DOXO-EMCH (1.1 mg/kg), or with 3.3 mg/kg of DOXO-EMCH. Controls received saline. Animals were euthanized at 48th week. Rats exposed to doxorubicin had a severe clinical, and histopathological cardiomyopathy with depressed myocardial activity of cytochrome c-oxidase (COX, 26% of controls), reduced expression of the mtDNA-encoded COX II subunit, decreased mtDNA copy numbers (46% of controls), and high levels of malondialdehyde and superoxide (787% of controls). All parameters were highly correlated with myocardial damage. Both DOXO-EMCH groups did not differ from controls with regard to clinical symptomatology, mortality and mitochondrial enzymes, although the myocardia of the high-dose group had slightly increased histopathological abnormalities, depressed mtDNA copies (74% of controls) and elevated superoxide levels (347% of controls). Doxorubicin-exposed hearts and to a lesser extent the myocardia of both DOXO-EMCH groups contained mtDNA-deletions. In summary both DOXO-EMCH doses were superior over doxorubicin with respect to clinical and histopathological evidence of cardiomyopathy, myocardial COX-activity, COX II expression, mtDNA-content, mtDNA mutation loads and superoxide production in rats. © 2006 Wiley-Liss, Inc.

Doxorubicin is an antineoplastic anthracycline that is widely used in the treatment of leukemia and lymphoma, breast and ovarian carcinoma and many other solid tumors. Bone marrow suppression with maximum toxicity after 7–10 days and a rapid recovery thereafter generally limits the escalation of single doses. Cumulative doses of doxorubicin exceeding 500 mg/m2 in contrast, are curtailed by a late-onset and irreversible cardiotoxicity.1

The antitumor potency and toxicological profile of anthracyclines has been the impetus for a diligent search for more effective and less toxic anthracycline analogues, and ∼2,000 derivatives have been developed during the past 20 years.2 Despite these efforts, daunorubicin, epirubicin, idarubicin, pirarubicin, zorubicin, aclarubicin and carminomycin have not satisfactorily improved the therapeutic index of anthracyclines in clinical practice.3

To improve the therapeutic potential of doxorubicin, we have developed a macromolecular prodrug in which doxorubicin is derivatized at its C-13 keto-position with a thiol-binding spacer molecule (i.e., 6-maleimidocaproyl hydrazone of doxorubicin, abbreviated DOXO-EMCH, Fig. 1).4 Because of its maleimide group, DOXO-EMCH binds quantitatively and selectively to the cysteine-34 position of albumin after intravenous administration. The cysteine-34 of albumin carries an accessible thiol group which is a specific target of DOXO-EMCH because thiol groups are not present in the majority of serum proteins and because this group is the most reactive thiol group in human plasma.4 The reaction with albumin follows second-order kinetics and is complete within a few minutes. Albumin accumulates in solid neoplasms due to their high metabolic turnover, angiogenesis, hypervasculature, defective vascular architecture and impaired lymphatic drainage and is therefore able to transport the payload to the tumor.5, 6 In the slightly acidic environment often present in the extracellular environment of neoplasms but also in their intracellular endosomal or lysosomal compartments, the acid-sensitive hydrazone linker allows doxorubicin to be released from its carrier.

thumbnail image

Figure 1. Chemical structure of DOXO-EMCH, a 6-maleimidocaproyl hydrazone derivative of doxorubicin.

Download figure to PowerPoint

DOXO-EMCH unlike its free doxorubicin parent, achieved complete remissions in a murine renal cell carcinoma model and in 2 breast carcinoma xenograft models.4 Moreover, DOXO-EMCH was also superior to doxorubicin with regard to drug toxicity in mice, rats and dogs,7 and exhibited a good safety profile in a recently completed clinical Phase I study.8

We have developed a rat model of chronic anthracycline toxicity in which we showed that quantitative and qualitative alterations in mitochondrial DNA (mtDNA) are somatically acquired during doxorubicin exposure, and are associated with respiratory chain defects as well as increased production of reactive oxygen species.9 Furthermore, the mitochondrial lesions were heart specific and accumulated even in the absence of subsequent doxorubicin applications, thus sufficiently explaining the delayed onset of clinical and histological cardiomyopathy. Very similar mitochondrial damage was recently also observed in the hearts of tumor patients treated with doxorubicin.10

In this work, we investigated the chronic cardiotoxicity of DOXO-EMCH in rats and demonstrate the superiority of the prodrug over free doxorubicin with regard to histological, functional and genetic damage to the myocardium and its mitochondria.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

The investigation was approved by the state animal ethics board and conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health.11 Male Wistar rats were purchased at Charles River (Sulzfeld, Germany), were housed in a normal night–day rhythm under standard conditions of temperature, humidity and fed a normal rat chow (SSniff R/M-H, Spezialdiäten, Germany). At 10 weeks of age, the rats received an intravenous port (Rat-O-Port®, Uno Roestvaststaal, Zevenaar, Netherlands) under anesthesia with Forene™ (Abbott). Based on the mortality in earlier investigations,9 the rats were divided into 4 experimental groups of different size: 9 animals served as controls (Group A) and received intravenous saline (300–700 μl). Group B (n = 15) received equivalent volumes of doxorubicin at a dose of 0.8 mg/kg, freshly dissolved in water from lyophilized powder (Pharmacia, Germany). Group C, the DOXO-EMCH low-dose group (n = 10), received intravenous DOXO-EMCH, synthesized according to a published procedure4 and freshly reconstituted in sterile 10 mM sodium phosphate, 5 % D-(+)-glucose (pH 6.4) and at a dose equivalent to 0.8 mg/kg of free doxorubicin (1.1 mg/kg). Group D (n = 10) was injected with a higher dose of DOXO-EMCH (3.3 mg/kg), a dose that with respect to subacute mortality was equitoxic to 0.8 mg/kg of doxorubicin in a 4-cycle repeat dose study in rats.7 All animals received 7-weekly injections through the port, beginning at 11 weeks of age and were killed by cervical dislocation at 48 weeks of age, immediately before postmortem examination and organ collection. Heart weights were recorded. Left ventricle and apex were snap frozen and cryopreserved in liquid nitrogen until subsequent analysis. Aliquots were fixed in glutaraldehyde (3%) for subsequent electron microscopy.

Cardiomyopathy score and mitochondrial ultrastructure

The severity and extent of myocardial lesions was scored on a qualitative/quantitative morphological grading scale on apical heart sections (4 μm), stained with haematoxilin and eosin (HE).12 The evaluating person was blinded to the treatment status of the animals. Two tissue samples were randomly selected from each group and examined by electron microscopy as described.9

Enzyme activities

The enzyme activity of cytochrome c-oxidase (COX), succinate dehydrogenase (SDH) and citrate synthase (CS) were measured spectrophotometrically in freshly prepared tissue extracts, as described.13 COX is a multisubunit respiratory chain complex which is encoded by both nuclear DNA (nDNA) and mtDNA. SDH is also a respiratory chain enzyme, but encoded entirely by nDNA. CS is a nDNA-encoded component of the Krebs-cycle and located in the mitochondrial matrix.

mtDNA-encoded respiratory chain protein

The mtDNA encoded subunit I of cytochrome c-oxidase (COX I) was quantified by immunoblot. The COX I signal was normalized to the expression of the subunit IV of cytochrome c-oxidase (COX IV), which is encoded by nDNA. Blots were also probed with a third antibody (Research Diagnostics, Flanders, NJ) against glycerol aldehyde phosphate dehydrogenase (GAPDH), an enzyme which is entirely encoded in the nucleus. Further details are described elsewhere.14, 15

mtDNA copy number and frequency of the common mtDNA-deletion

Total DNA was extracted with the QIAamp DNA isolation kit (Qiagen, Hilden, Germany). mtDNA and nDNA copy numbers were determined by quantitative PCR using the ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA). mtDNA was amplified between nucleotide positions 2,469 and 2,542 with the forward primer, 5′-AATGGTTCGTTTGTTCAACGATT-3′and the backward primer 5′-AGAAACCGACCTGGATTGCTC-3′. mtDNA was quantified with a FAM-fluorophore labeled probe (5′-6FAM-AAGTCCTACGTGATCTGAGTT-TAMRA-3′). For the quantification of nDNA copies, we selected the GAPDH gene between nucleotide positions 494 and 671, using the forward primer 5′-TGCACCACCAACTGCTTAG-3′ and the backward primer 5′-GGATGCAGGGATGATGTTC-3′. In this case we used a HEX-fluorophore labeled probe (5′-HEX-CAGAAGACTGTGGATGGCCCCTC-TAMRA-3′).

Each 25 μl reaction contained 20 ng of genomic DNA, 100 nM probe, 200 nM primers and Taq-man Absolute Master Mix® (Abgene, Hamburg, Germany). Triplicate amplifications of mitochondrial and nuclear products were performed separately in optical 96-well plates (Applied Biosystems) under the following conditions: An initial incubation at 50°C for 2 min was followed by 10 min at 95°C and 40 denaturing steps at 95°C (15 sec), alternating with combined annealing/extension at 60°C (1 min). Absolute mtDNA and nDNA copy numbers were calculated using serial dilutions of plasmids with known copy numbers.16

The sequence of normal rat mtDNA contains direct repeats between which base pairs may be deleted by slipped mispairing during replication.17 A 4,834-bp deletion is the most frequent deletion in rats, similar to the age-related “common” 4,977-bp deletion in humans. The common mtDNA-deletion was probed by PCR using extradeletional primers and short extension cycles as described.9 Sequencing (MWG Biotech, Germany) confirmed that the 459-bp PCR product represented the common deletion.

Malondialdehyde and superoxide production

Malondialdehyde (MDA) is 1 of the end products of lipid peroxidation and an indicator of free radical production and oxidative stress. MDA was spectrophotometrically quantified in tissues with an assay for thiobarbituric acid reactive material.18

Superoxide production was measured in situ on transverse tissue sections with the oxidative fluorescent dye dihydroethidium (Sigma, Taufkirchen, Germany).9, 19 The intensity of the fluorescence was quantified using Scion Image™ (Scion Corp).

Statistics

Group means were compared by ANOVA, chi-square, fisher exact, unpaired t-test or Wilcoxon analysis, as appropriate. Regressions were computed by nonlinear exponential regression analysis. All calculations were performed using the Sigma Plot 2000™, version 6.0 (SPSS) statistical package.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Mortality, macroscopic and microscopic pathology

Five of the 15 rats from the doxorubicin Group B died in Weeks 32, 35, 38, 44 and 46, respectively. Postmortem examination revealed pleural effusions and a dilated myocardium in the Group B animals that died at Weeks 32, 38 and 44. One of the 10 animals in the DOXO-EMCH low-dose group died in Week 31, showing no macroscopic pathology. All these rats were excluded from the analysis because we were unable to determine the exact postmortem time (Table 1).

Table I. Mortality and Autopsy Findings Among Experimental Groups
 ControlDoxorubicin (0.8 mg/kg) (B)DOXO-EMCH (1.1 mg/kg) (C)DOXO-EMCH (3.3 mg/kg) (D)p among all groups
  • Significance (p < 0.05) vs.

  • 1

    controls and vs.

  • 2

    doxorubicin. High statistical significance (p < 0.001) vs.

  • 3

    control and vs.

  • 4

    doxorubicin.

Rats treated from Week 11–17 (n)9151010NA
Rat mortality before Week 48 (n)05100.045
Rats available for subsequent investigations at Week 48 (n)910910NA
Mean body weight at Week 46 (g ± SD)639 ± 96487 ± 1341656 ± 582638 ± 7720.002
Mean heart weight (g ± SD)1.25 ± 0.111.83 ± 0.1231.40 ± 0.0941.48 ± 0.114<0.001
Increased respiratory rate at Week 48 (n)0/95/1010/920/1020.001
Rats with pleural effusions (n)0/98/1030/940/104<0.001
Rats with liver enlargement (n)0/910/1030/940/104<0.001
Rats with renal pathology (n)0/910/1030/940/104<0.001

The maximal medium body weight of Group B was reached by Week 46 and at that time point was lower when compared with all other groups (Table 1). From Weeks 46 to 48, Group B rats lost about 6% of body weight, whereas there was a statistically significant weight gain in all the other groups (data not shown). In 5 of the 10 Group B animals who survived until euthanasia, an increased respiratory rate was noted after Week 46, 9 had macroscopic evidence of myocardial dilatation and 8 had pleural effusions (Table 1). All 10 animals had enlarged livers of a dark red, engorged appearance and also macroscopic renal pathology.20 Although the mean heart weight of Group B rats was higher than that of controls (146%, p = 0.004) and of Group C animals (131%, p = 0.001), the heart weight of Group C was statistically similar to controls (p = 0.16). The heart weight of Group D was slightly (118%) increased, compared to controls (p = 0.04).

The degree of myocardial damage, as assessed with the cardiomyopathy score, was substantially elevated in Group B (p < 0.001, compared to controls), but not in both DOXO-EMCH groups (Table 2). Among the DOXO-EMCH treated animals, however, the cardiomyopathy score was slightly elevated in the group receiving the high dose when compared with its low-dose counterpart (p = 0.02)

Table 2. Mitochondrial Effects of Doxorubicin and DOXO-EMCH in Heart
 ControlDoxorubicin (0.8 mg/kg) (B)DOXO-EMCH (1.1 mg/kg) (C)DOXO-EMCH (3.3 mg/kg) (D)p (B vs. control)p (C vs. control)p (D vs. control)p (C vs. doxorubicin)p (D vs. doxorubicin)p (C vs. D)
  • Data are group mean ± SD. NS, not significant; NA, not applicable.

  • 1

    μmol/min/g protein.

  • 2

    percentage of control mean.

  • 3

    copies/cardiomyocyte.

  • 4

    μmol/g tissue.

Cardiomyopathy score1.6 ± 1.17.2 ± 2.51.2 ± 0.82.9 ± 1.9<0.001NSNS<0.001<0.0010.02
COX154 ± 2414 ± 1039 ± 1236 ± 16<0.001NSNS<0.0010.002NS
SDH160 ± 2675 ± 3944 ± 1356 ± 19NSNSNS0.04NSNS
COX/SDH-ratio2100 ± 1826 ± 2297 ± 677 ± 44<0.001NSNS<0.0010.004NS
CS13440 ± 4704346 ± 6943565 ± 6063579 ± 5120.004NSNS0.020.02NS
COX I/COX IV-ratio2100 ± 1564 ± 22105 ± 1984 ± 24<0.001NSNS<0.001NS0.04
COX IV/GAPDH-ratio2100 ± 15100 ± 1495 ± 1494 ± 9NSNSNSNSNSNS
mtDNA copies3676 ± 117314 ± 119608 ± 75497 ± 128<0.001NS0.005<0.0010.0040.03
Animals with detectable mtDNA-deletion (%)01001060<0.001NS0.01<0.0010.04NS
Intensity of the mtDNA-deletion++(+)+NANANANANANA
MDA445.5 ± 17.2153.6 ± 59.649.3 ± 17.966.9 ± 32.3<0.001NSNS<0.001<0.001NS
Superoxide2100 ± 33787 ± 282123 ± 62347 ± 276<0.001NS0.02<0.0010.0020.03

The myocardial ultrastructure of Group B rats was characterized by complete myofibrillar disarray and large clusters of intermyofibrillar mitochondria. The cristal architecture of the mitochondria was lost and there were large deposits of electron-dense material (Fig. 2), whereas only an increased number of slightly enlarged mitochondria was noted in the myocardia of the high-dose DOXO-EMCH group. The DOXO-EMCH low-dose group and the control animals showed no ultrastructural pathology.

thumbnail image

Figure 2. (A) Representative electron micrographs of hearts from untreated control animals, (B) Doxorubicin, (C) DOXO-EMCH low-dose, and (D) High-dose groups. Mitochondria are marked with a star. Scale bar: 0.6 μm.

Download figure to PowerPoint

Respiratory chain function

The mean enzyme activity of COX within Group B animals was reduced (26% of control values), whereas there was no apparent decrease of COX activity in the other groups (Table 2). All anthracyclines did not affect the mean myocardial SDH activity. We also normalized the enzyme activity of COX (which requires intact mtDNA) to the activity of SDH (which is independent of mtDNA) by calculating the COX/SDH-ratio. The mean COX/SDH-ratio was reduced in Group B hearts when compared with controls (p < 0.001), but unchanged in the hearts of both DOXO-EMCH groups. The enzyme activity of CS was increased in the Group B hearts (126% of control values). Among all rats, the histological degree of myocardial damage correlated inversely with the absolute COX-enzymatic activity (r = −0.69, p < 0.001) and the COX/SDH-ratio [r = −0.77, p < 0.001, Fig. 3(A)].

thumbnail image

Figure 3. Correlations between the degree of myocardial damage, parameters of mitochondrial integrity and cardiac superoxide-content.

Download figure to PowerPoint

Thus, both DOXO-EMCH groups showed a superior toxicological profile when compared with free doxorubicin, with respect to the mtDNA-encoded enzyme activities and CS, whereas there were no statistical differences between both DOXO-EMCH groups.

mtDNA-encoded respiratory chain subunits

The mean COX I/COX IV-ratio was reduced in Group B and remained essentially unchanged in the hearts of both DOXO-EMCH groups (Table 2), whereas the COX IV/GAPDH-ratio did not statistically differ between all groups. The COXI/COXIV-ratio was positively correlated with both the absolute COX activity (r = 0.38, p = 0.02) and the COX/SDH-ratio (r = 0.55, p = 0.006) and inversely correlated (r = −0.86, p < 0.001) with the cardiomyopathy score [Fig. 3(B)].

Copy number of wild-type mtDNA

mtDNA copy numbers in hearts exposed to doxorubicin were approximately half of those in controls (Table 2). The DOXO-EMCH high-dose group also had a slight mtDNA depletion (by 26% of control values). Among all groups, the myocardial mtDNA amount was inversely correlated with the cardiomyopathy score [r = −0.81, p < 0.001, Fig. 3(C)], and positively correlated with the absolute COX activity (r = 0.49, p = 0.03), the COX/SDH-ratio (r = 0.55, p = 0.006) and the COX I/COX IV-ratio (r = 0.63, p < 0.001).

mtDNA deletion

A 459-basepair PCR product was amplified from all Group B but none of the control hearts (Fig. 4). Sequencing confirmed that the 459-basepair PCR products represented a mtDNA fragment, in which 4,834 basepairs between 2 direct 16-basepair repeats had been deleted, (e.g., the “common” mtDNA deletion). This PCR product was also detected in 1 animal of Group C and 6 animals of Group D (Table 2), although its intensity was always lower than the lowest intensity of the PCR product from each Group B animal (Fig. 4).

thumbnail image

Figure 4. Representative PCR-products (arrow) on agarose gel electrophoresis as a semiquantitative index of the “common” mtDNA deletion. A 100-basepair DNA standard (Sigma, Taufkirchen, Germany) was added to lanes 1 and 7. Lane 2, PCR products from control; lane 3, Group B myocardia; lane 4, PCR-product from the 1 Group C heart; and lane 5, from 1 of the 6 Group D hearts in which the “common” deletion was detectable. Negative control without DNA template is shown in lane 6.

Download figure to PowerPoint

Lipid peroxidation and superoxide production

In doxorubicin-exposed myocardia, MDA levels as an indirect indicator of ROS formation were increased by a factor of 3.4 compared to controls, and by factors of 3.1 and 2.3 when compared with the DOXO-EMCH low-dose and high-dose groups, respectively (Table 2). Myocardial MDA was inversely correlated with the COX enzyme activity (r = −0.38, p = 0.02), the COX/SDH-ratio (r = −0.58, p = 0.003), the COX I/COX IV-ratio (r = −0.65, p < 0.001) and the mtDNA-content (r = −0.77, p < 0.001) and positively correlated with the cardiomyopathy score (r = 0.78, p < 0.001).

Myocardial superoxide was increased by 787% in Group B compared to controls, and was also higher when compared with both DOXO-EMCH groups (Table 2). Superoxide levels were also elevated in Group D hearts (347% of the control mean). Superoxide levels correlated positively with the cardiomyopathy score [r = 0.74, p < 0.001, Fig. 3(D)] and the MDA level (r = 0.65, p < 0.001) and negatively with the COX activity (r = −0.65, p < 0.001), the COX/SDH-ratio (r = −0.83, p < 0.001), the COXI/COXIV-ratio (r = −0.72, p < 0.001) and the mtDNA/nDNA-ratio (r = −0.71, p < 0.001). Superoxide production, but not MDA content, was increased in the high-dose DOXO-EMCH group, compared to the low-dose.

Skeletal muscle

In contrast to the heart, there were no macroscopic or microscopic abnormalities in skeletal muscle. With the exception of the COX I/COX IV-ratio, which was slightly increased in the DOXO-EMCH low-dose group when compared with the high-dose (p = 0.04), all parameters in skeletal muscle did not differ statistically between all groups (Table 3). PCR did not identify any mtDNA deletion in skeletal muscle (Table 3).

Table 3. Lack of Mitochondrial Effects of Doxorubicin and DOXO-EMCH in Skeletal Muscle
 ControlDoxorubicin (0.8 mg/kg) (B)DOXO-EMCH (1.1 mg/kg) (C)DOXO-EMCH (3.3 mg/kg) (D)p (B vs. control)p (C vs. control)p (D vs. control)p (C vs. doxorubicin)p (D vs. doxorubicin)p (C vs. D)
  • Data are group mean ± SD. NS, not significant; NA, not applicable.

  • 1

    μmol/min/g protein.

  • 2

    percentage of control mean.

  • 3

    copies/cardiomyocyte.

  • 4

    μmol/g tissue.

COX133 ± 1931 ± 1432 ± 1234 ± 9NSNSNSNSNSNS
SDH139 ± 1842 ± 2137 ± 1442 ± 15NSNSNSNSNSNS
COX/SDH-ratio2100 ± 5895 ± 4297 ± 3797 ± 42NSNSNSNSNSNS
CS13581 ± 2853611 ± 2593614 ± 3923685 ± 407NSNSNSNSNSNS
COX I/COX IV-ratio2100 ± 21106 ± 21117 ± 17100 ± 17NSNSNSNSNS0.04
COX IV/GAPDH-ratio2100 ± 2095 ± 25109 ± 2195 ± 16NSNSNSNSNSNS
mtDNA copies3670 ± 155630 ± 108679 ± 160636 ± 148NSNSNSNSNSNS
Animals with detectable mtDNA-deletion (%)0000NSNSNSNSNSNS
MDA487 ± 3383 ± 2086 ± 2080 ± 29NSNSNSNSNSNS
Superoxide2100 ± 1799 ± 991 ± 1392 ± 10NSNSNSNSNSNS

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Over the past 5 years, we have investigated a prodrug concept that exploits the fact that endogenous albumin accumulates in solid tumors and thus can serve as a carrier that passively targets the anthracycline to the malignancy.4, 21, 22 A high degree of protein-binding, especially to albumin, is generally considered a disadvantage because only the free drug can exert its pharmacological effect, the incorporation of an acid-sensitive or enzymatically cleavable bond between the drug and the albumin-binding moiety ensures a specific release of the drug at its site of action. As a result of our preclinical work, DOXO-EMCH emerged as a clinical candidate because of its rapid and selective binding to circulating albumin, high plasma stability and high water-solubility,4 its superior efficacy in 3 murine tumor models, and an ∼3- to 5-fold increase in the MTD in mice, rats and dogs when compared with doxorubicin.7

We have previously established a conclusive model of the pathogenesis of chronic doxorubicin cardiomyopathy by showing that the clinical onset of the cardiomyopathy is associated with heart-specific quantitative and qualitative lesions of mitochondrial DNA (mtDNA), respiratory chain defects and an increased production of reactive oxygen species.9 According to this model, a relatively minor injury is acquired in myocardial mitochondria during acute doxorubicin exposure and then accumulates with time also in the absence of the anthracycline until the bioenergetic capacity of the organelles is severely impaired. This model is based on a vicious circle in which doxorubicin liberates free radicals with consequent quantitative and qualitative defects in mtDNA and secondary impairment of mtDNA-encoded respiratory chain subunits. The dysfunctional respiratory chain then closes the vicious circle by generating free radicals itself. mtDNA replication may also be inhibited directly by doxorubicin through DNA strand cross-linking, DNA adduct formation, inhibition of topoisomerase Type 2 or through the intercalating properties of the anthracycline.23, 24, 25, 26 Recently, genetic, functional and ultrastructural alterations have also been identified in the mitochondria of human hearts exposed to doxorubicin.10

The aim of this study was to examine the chronic cardiotoxicity of DOXO-EMCH in a previously established rat model. DOXO-EMCH was applied in a low dose, which is equimolar to free doxorubicin, and a 3-fold higher dose, a dose which exhibits superior antitumor efficacy. The main finding of our investigations is that both DOXO-EMCH doses unlike the positive control with free doxorubicin exhibited no significant toxicity with respect to rat symptomatology. Although a direct comparison in our model is lacking, the absence of clinical cardiotoxicity with the high dose of DOXO-EMCH suggests an advantage over other conventional anthracyclines such as epirubicin. Epirubicin is 2-fold less cardiotoxic than doxorubicine on a milligram-per-milligram basis, but has similar cardiotoxicity when equi-myelosuppressive doses are administered.3 PK2, a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer prodrug of doxorubicin exhibited an ∼5-fold reduction in cardiotoxicity relative to free doxorubicin.27 When compared with these models however, our study has a much longer time of follow-up and requires lower-cumulative doses and thus is likely to represent the late-onset cardiotoxicity of patients more closely. It would be interesting to now compare DOXO-EMCH with pegylated liposomal doxorubicin, which has shown reduced cardiotoxicity not only in preclinical models, but also clinically.28, 29

It is also important to note that the degree of histopathological abnormalities and the toxicity with respect to some mitochondrial parameters was slightly elevated in the high-dose DOXO-EMCH group, suggesting a dose-dependent quantitative effect, rather than a qualitative superiority of the toxicological profile of DOXO-EMCH. In earlier investigations, the cardiomyopathy appeared to only become clinically and histologically apparent, when the degree of combined respiratory chain and mtDNA-insults exceeded a threshold of less than 30% residual COX/SDH activity.9, 30 This threshold was however not reached by the high-dose DOXO-EMCH group, explaining the lack of clinical manifestations.

The absence of mitochondrial, ultrastructural and clinical cardiotoxicity in the DOXO-EMCH low-dose group and the finding that mtDNA-encoded abnormalities either precede or parallel the histopathological onset of myocardial damage in the high-dose group also support the importance of somatically acquired mitochondrial lesions in the pathogenesis of the cardiomyopathy. These findings also suggest that the analysis of the mitochondrial toxicity in rats may be exploited as a sensitive and cost-effective preclinical screening method for the cardiotoxicity of future anthracyclines.

DOXO-EMCH binding to endogenous albumin prevents the rapid diffusion of doxorubicin into healthy tissues. The following pharmacokinetic parameters were measured for free doxorubicin after intravenous DOXO-EMCH (2.5 mg/kg doxorubicin equivalents) in rats: Cmax 52 μM, AUC 540 hr × μM, volume of distribution 0.3 l/kg, and clearance 7.9 ml/hr/kg. These characteristics differed by 2–3 orders of magnitude with those determined for equimolar doxorubicin: Cmax 0.5 μM, AUC 1.4 hr × μM, volume of distribution 72 l/kg, clearance 2,553 ml/hr/kg. In contrast, the half-lives (25 vs. 19 hr, respectively) were rather similar (Felix Kratz, unpublished results). High myocardial peak concentrations of the free drug in particular appear to be responsible for acute and chronic cardiotoxicity.31 Thus the differences in pharmacokinetic properties between DOXO-EMCH and free doxorubicin offer a plausible explanation for our findings.

Our analysis did not focus on the numerous other mechanisms that may contribute to the delayed onset of chronic doxorubicin cardiomyopathy (excellently reviewed in Ref.2). For example, proapoptotic factors of mitochondrial origin after increased superoxide production have recently been implicated in the execution of cardiomyocyte death.32 We also cannot rule out mutations in nuclear genes necessary for myocardial function, mtDNA maintenance or mtDNA repair.33

In a recently completed Phase I study with DOXO-EMCH, the albumin-binding prodrug showed a good safety profile and antitumor efficacy.8 Forty-one patients with advanced cancer disease were treated with 2–6 intravenous cycles of DOXO-EMCH once every 3 weeks at a dose level of 20–340 mg/m2 doxorubicin equivalents. Treatment with DOXO-EMCH was well tolerated up to 200 mg/m2 without manifestation of drug-related side effects. Myelosuppression (Grade 1–2) and mucositis (Grade 1–2) were the predominant adverse effects at dose levels of 260 mg/m2 and myelosuppression (Grade 1–3) as well as mucositis (Grade 1–3) were dose-limiting at 340 mg/m2. No acute cardiac toxicity was observed. Of 35 evaluable patients, 34% had progressive disease, 51% had disease stabilization, 6% had a minor response, 6% had a partial remission and 3% had a complete remission. The recommended dose for Phase II studies is 260 mg/m2 which is a more than 4-fold increase when compared with standard treatment with doxorubicin (60 mg/m2).

In summary, we show that the chronic mitochondrial and myocardial toxicity of DOXO-EMCH in rats is reduced, compared with free doxorubicin. Our findings and the available efficacy data of DOXO-EMCH suggest that its antitumor effect is dissociated from the cardiac and mitochondrial toxicity. The data support the further clinical development of DOXO-EMCH.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Pharmacia/ Germany for providing doxorubicin and Kerstina Melkaoui for expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med 1998; 339: 9005.
  • 2
    Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004; 56: 185229.
  • 3
    Ewer MS, Benjamin RS, Yeh JT. Cardiac complications. In: KufeD, BastR, HaitW, HongW, PollackR, WeichselbaumR, HollandJ, FreiE, eds. Cancer medicine, 7th edn. Hamilton, Ontario: Decker, 2005. 252541.
  • 4
    Kratz F, Warnecke A, Scheuermann K, Stockmar C, Schwab J, Lazar P, Druckes P, Esser N, Drevs J, Rognan D, Bissantz C, Hinderling C, et al. Probing the cysteine-34 position of endogenous serum albumin with thiol-binding doxorubicin derivatives. Improved efficacy of an acid-sensitive doxorubicin derivative with specific albumin-binding properties compared to that of the parent compound. J Med Chem 2002; 45: 552333.
  • 5
    Kratz F, Warnecke A, Schmid B, Chung DE, Gitzel M. Prodrugs of anthracyclines in cancer chemotherapy. Curr Med Chem 2006; 13: 477523.
  • 6
    Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 2000; 65: 27184.
  • 7
    Kratz F, Kauffmann HM, Ehling G, Unger C. Acute and repeat-dose toxicity studies of the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH), an albumin-binding prodrug of the anticancer agent doxorubicin. Hum Exp Toxicol, in press.
  • 8
    Unger C, Medinger M, Steinbild S, Drevs J, Häring B. Phase I dose-escalation and pharmacokinetic (PK) study of a (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) in patients with advanced cancers. Paper presented at the German Cancer Congress, Berlin, 2006.
  • 9
    Lebrecht D, Setzer B, Ketelsen UP, Haberstroh J, Walker UA. Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy. Circulation 2003; 108: 24239.
  • 10
    Lebrecht D, Kokkori A, Ketelsen UP, Setzer B, Walker UA. Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin. J Pathol 2005; 207: 43644.
  • 11
    NIH office of laboratory animal welfare. Public health service policy on humane care and use of laboratory animals. Available at http://grants.nih.gov 2002.
  • 12
    Della Torre P, Podesta A, Pinciroli G, Iatropoulos MJ, Mazue G. Long-lasting effect of dexrazoxane against anthracycline cardiotoxicity in rats. Toxicol Pathol 1996; 24: 398402.
  • 13
    Silvestri G, Santorelli FM, Shanske S, Whitley CB, Schimmenti LA, Smith SA, DiMauro S. A new mtDNA mutation in the tRNA (Leu(UUR)) gene associated with maternally inherited cardiomyopathy. Hum Mutat 1994; 3: 3743.
  • 14
    Capaldi RA, Marusich MF, Taanman JW. Mammalian cytochrome-c oxidase: characterization of enzyme and immunological detection of subunits in tissue extracts and whole cells. Methods Enzymol 1995; 260: 11732.
  • 15
    Walker UA, Setzer B, Venhoff N. Increased long-term mitochondrial toxicity in combinations of nucleoside analogue reverse-transcriptase inhibitors. AIDS 2002; 16: 216573.
  • 16
    Hammond EL, Sayer D, Nolan D, Walker UA, Ronde A, Montaner JS, Cote HC, Gahan ME, Cherry CL, Wesselingh SL, Reiss P, Mallal S. Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study. J Clin Virol 2003; 27: 97110.
  • 17
    Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S. A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science 1989; 244: 3469.
  • 18
    Tuzgen S, Kaynar MY, Guner A, Gumustas K, Belce A, Etus V, Ozyurt E. The effect of epidural cooling on lipid peroxidation after experimental spinal cord injury. Spinal Cord 1998; 36: 6547.
  • 19
    Miller FJ,Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res 1998; 82: 1298305.
  • 20
    Lebrecht D, Setzer B, Rohrbach R, Walker UA. Mitochondrial DNA and its respiratory chain products are defective in doxorubicin nephrosis. Nephrol Dial Transplant 2004; 19: 32936.
  • 21
    Mansour AM, Drevs J, Esser N, Hamada FM, Badary OA, Unger C, Fichtner I, Kratz F. A new approach for the treatment of malignant melanoma: enhanced antitumor efficacy of an albumin-binding doxorubicin prodrug that is cleaved by matrix metalloproteinase 2. Cancer Res 2003; 63: 40626.
  • 22
    Warnecke A Kratz F. Maleimide-oligo(ethylene glycol) derivatives of camptothecin as albumin-binding prodrugs: synthesis and antitumor efficacy. Bioconjug Chem 2003; 14: 37787.
  • 23
    Cullinane C, Cutts SM, Panousis C, Phillips DR. Interstrand cross-linking by adriamycin in nuclear and mitochondrial DNA of MCF-7 cells. Nucleic Acids Res 2000; 28: 101925.
  • 24
    Keizer HG, Pinedo HM, Schuurhuis GJ, Joenje H. Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity. Pharmacol Ther 1990; 47: 21931.
  • 25
    Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF. Adriamycin induced DNA damage mediated by mammalian DNA topoisomerase II. Science 1984; 226: 4668.
  • 26
    Wiseman A, Attardi G. Reversible tenfold reduction in mitochondria DNA content of human cells treated with ethidium bromide. Mol Gen Genet 1978; 167: 5163.
  • 27
    Hopewel JW, Duncan R, Wilding D, Chakrabarti K. Preclinical evaluation of the cardiotoxicity of PK2: a novel HPMA copolymer-doxorubicin-galactosamine conjugate antitumour agent. Hum Exp Toxicol 2001; 20: 46170.
  • 28
    O'Brien ME, Wigler N, Inbar M, Rosso R, Grischke E, Santoro A, Catane R, Kieback DG, Tomczak P, Ackland SP, Orlandi F, Mellars L, et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol 2004; 15: 4409.
  • 29
    Working PK, Newman MS, Sullivan T, Yarrington J. Reduction of the cardiotoxicity of doxorubicin in rabbits and dogs by encapsulation in long-circulating, pegylated liposomes. J Pharmacol Exp Ther 1999; 289: 112833.
  • 30
    Zeviani M, Gellera C, Antozzi C, Rimoldi M, Moramdi L, Villani F, Tiranti V, DiDonato S. Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNA leu (UUR). Lancet 1991; 338: 1437.
  • 31
    Cummings J, Willmott N, More I, Kerr DJ, Morrison JG, Kaye SB. Comparative cardiotoxicity and antitumour activity of doxorubicin (adriamycin) and 4′-deoxydoxorubicin and the relationship to in vivo disposition and metabolism in the target tissues. Biochem Pharmacol 1987; 36: 15216.
  • 32
    Childs AC, Phaneuf SL, Dirks AJ, Phillips T, Leeuwenburgh C. Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio. Cancer Res 2002; 62: 45928.
  • 33
    Grandjean F, Bremaud L, Robert J, Ratinaud MH. Alterations in the expression of cytochrome c oxidase subunits in doxorubicin-resistant leukemia K562 cells. Biochem Pharmacol 2002; 63: 82331.