The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage
Version of Record online: 27 NOV 2006
Copyright © 2006 Wiley-Liss, Inc.
International Journal of Cancer
Volume 120, Issue 4, pages 927–934, 15 February 2007
How to Cite
Lebrecht, D., Geist, A., Ketelsen, U.-P., Haberstroh, J., Setzer, B., Kratz, F. and Walker, U. A. (2007), The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage. Int. J. Cancer, 120: 927–934. doi: 10.1002/ijc.22409
- Issue online: 27 DEC 2006
- Version of Record online: 27 NOV 2006
- Manuscript Accepted: 29 AUG 2006
- Manuscript Received: 6 JUL 2006
- Deutsche Krebshilfe
- Bonn/ Germany. Grant Number: 106112
- doxorubicin prodrug;
- mitochondria cytochrome oxidase;
- DNA mitochondrial;
- reactive oxygen species
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.
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
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
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
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.
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).
|Control||Doxorubicin (0.8 mg/kg) (B)||DOXO-EMCH (1.1 mg/kg) (C)||DOXO-EMCH (3.3 mg/kg) (D)||p among all groups|
|Rats treated from Week 11–17 (n)||9||15||10||10||NA|
|Rat mortality before Week 48 (n)||0||5||1||0||0.045|
|Rats available for subsequent investigations at Week 48 (n)||9||10||9||10||NA|
|Mean body weight at Week 46 (g ± SD)||639 ± 96||487 ± 1341||656 ± 582||638 ± 772||0.002|
|Mean heart weight (g ± SD)||1.25 ± 0.11||1.83 ± 0.123||1.40 ± 0.094||1.48 ± 0.114||<0.001|
|Increased respiratory rate at Week 48 (n)||0/9||5/101||0/92||0/102||0.001|
|Rats with pleural effusions (n)||0/9||8/103||0/94||0/104||<0.001|
|Rats with liver enlargement (n)||0/9||10/103||0/94||0/104||<0.001|
|Rats with renal pathology (n)||0/9||10/103||0/94||0/104||<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)
|Control||Doxorubicin (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)|
|Cardiomyopathy score||1.6 ± 1.1||7.2 ± 2.5||1.2 ± 0.8||2.9 ± 1.9||<0.001||NS||NS||<0.001||<0.001||0.02|
|COX1||54 ± 24||14 ± 10||39 ± 12||36 ± 16||<0.001||NS||NS||<0.001||0.002||NS|
|SDH1||60 ± 26||75 ± 39||44 ± 13||56 ± 19||NS||NS||NS||0.04||NS||NS|
|COX/SDH-ratio2||100 ± 18||26 ± 22||97 ± 6||77 ± 44||<0.001||NS||NS||<0.001||0.004||NS|
|CS1||3440 ± 470||4346 ± 694||3565 ± 606||3579 ± 512||0.004||NS||NS||0.02||0.02||NS|
|COX I/COX IV-ratio2||100 ± 15||64 ± 22||105 ± 19||84 ± 24||<0.001||NS||NS||<0.001||NS||0.04|
|COX IV/GAPDH-ratio2||100 ± 15||100 ± 14||95 ± 14||94 ± 9||NS||NS||NS||NS||NS||NS|
|mtDNA copies3||676 ± 117||314 ± 119||608 ± 75||497 ± 128||<0.001||NS||0.005||<0.001||0.004||0.03|
|Animals with detectable mtDNA-deletion (%)||0||100||10||60||<0.001||NS||0.01||<0.001||0.04||NS|
|Intensity of the mtDNA-deletion||−||++||(+)||+||NA||NA||NA||NA||NA||NA|
|MDA4||45.5 ± 17.2||153.6 ± 59.6||49.3 ± 17.9||66.9 ± 32.3||<0.001||NS||NS||<0.001||<0.001||NS|
|Superoxide2||100 ± 33||787 ± 282||123 ± 62||347 ± 276||<0.001||NS||0.02||<0.001||0.002||0.03|
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.
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)].
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).
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).
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.
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).
|Control||Doxorubicin (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)|
|COX1||33 ± 19||31 ± 14||32 ± 12||34 ± 9||NS||NS||NS||NS||NS||NS|
|SDH1||39 ± 18||42 ± 21||37 ± 14||42 ± 15||NS||NS||NS||NS||NS||NS|
|COX/SDH-ratio2||100 ± 58||95 ± 42||97 ± 37||97 ± 42||NS||NS||NS||NS||NS||NS|
|CS1||3581 ± 285||3611 ± 259||3614 ± 392||3685 ± 407||NS||NS||NS||NS||NS||NS|
|COX I/COX IV-ratio2||100 ± 21||106 ± 21||117 ± 17||100 ± 17||NS||NS||NS||NS||NS||0.04|
|COX IV/GAPDH-ratio2||100 ± 20||95 ± 25||109 ± 21||95 ± 16||NS||NS||NS||NS||NS||NS|
|mtDNA copies3||670 ± 155||630 ± 108||679 ± 160||636 ± 148||NS||NS||NS||NS||NS||NS|
|Animals with detectable mtDNA-deletion (%)||0||0||0||0||NS||NS||NS||NS||NS||NS|
|MDA4||87 ± 33||83 ± 20||86 ± 20||80 ± 29||NS||NS||NS||NS||NS||NS|
|Superoxide2||100 ± 17||99 ± 9||91 ± 13||92 ± 10||NS||NS||NS||NS||NS||NS|
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.
We thank Pharmacia/ Germany for providing doxorubicin and Kerstina Melkaoui for expert technical assistance.
- 3Cardiac complications. In: KufeD, BastR, HaitW, HongW, PollackR, WeichselbaumR, HollandJ, FreiE, eds. Cancer medicine, 7th edn. Hamilton, Ontario: Decker, 2005. 2525–41., , .
- 4Probing 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: 5523–33., , , , , , , , , , , , et al.
- 8Phase 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., , , , .
- 11NIH office of laboratory animal welfare. Public health service policy on humane care and use of laboratory animals. Available at http://grants.nih.gov 2002.