James H. Doroshow
DAUNORUBICIN (DAUNOMYCIN) AND DOXORUBICIN
The anthracycline antibiotics doxorubicin and daunorubicin, initially discovered over 40 years ago,1, 2 are among the most widely used antineoplastic agents in current clinical practice; their antineoplastic spectrum of action compares favorably with the alkylating agents and the taxanes. Doxorubicin and daunorubicin are especially active against the hematopoietic malignancies such as acute lymphocytic and acute myelogenous leukemia, Hodgkin's and non-Hodgkin's lymphoma, and multiple myeloma, as well as carcinomas of the breast, lung, ovary, stomach, and thyroid, sarcomas of bone and soft tissue origin, and various childhood malignancies. The key features of the two most commonly used anthracyclines, daunorubicin and doxorubicin, are summarized in Table 18.1, and the structures of the anthracyclines now in use are shown in Figure 18.1. Doxorubicin is currently used principally for the treatment of solid tumors, especially breast cancer and lymphoma, while daunorubicin is routinely utilized as part of chemotherapeutic induction programs for acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL). The doxorubicin analog epirubicin is similar to the parent compound with respect to its acute toxicity profile and spectrum of antitumor efficacy but is significantly less potent and only slightly less cardiotoxic. The modestly decreased cardiac toxicity of epirubicin is only a marginal advantage, since other means are currently available to lessen the risk of anthracycline-induced heart damage. Idarubicin, a daunorubicin analog, has significant activity in the treatment of AML but is less active against solid tumors, and thus it is an appropriate alternate anthracycline only in the setting of acute leukemia.
In the clinic, the anthracyclines doxorubicin and daunorubicin have no known antagonistic interactions with any of the other commonly used anticancer agents. Furthermore, these drugs are active over a wide range of doses and in a variety of administration schedules; essentially equivalent antitumor activity is observed whether the anthracycline is given as a single large bolus dose once a month, as a weekly intravenous bolus, or as a prolonged infusion.3, 4 However, changes in drug scheduling do change the pattern of normal tissue injury. The combination of broad antitumor activity, lack of antagonism with other antitumor agents, and flexibility in dose and schedule make doxorubicin and daunorubicin very useful in the design of drug combinations. As a result, anthracycline-containing combination chemotherapy protocols have become standard therapy for cancers of the breast and thyroid; bone and soft tissue sarcomas; essentially all hematologic malignancies; and many childhood solid tumors. Although the acute toxicities associated with anthracycline administration, such as myelosuppression, mucositis, and alopecia, are important in clinical practice, the toxic reaction that causes the greatest concern is the unique cumulative cardiac injury produced by these drugs. Elucidation of the biochemical mechanisms of this cardiac toxicity resulted in the identification of an iron-chelating agent with its own modest antineoplastic activity, dexrazoxane (ICRF-187), which can block the cardiac toxicity of the anthracycline antibiotics in a wide range of animal models. Prospective, randomized clinical trials have shown that this agent is highly effective in reducing the cardiac toxicity of doxorubicin. This development, as well as the demonstration of a steep dose-response curve for doxorubicin in the treatment of solid tumors5 and the feasibility of utilizing colony-stimulating factors with or without peripheral blood progenitor support to ameliorate the bone marrow toxicity of the anthracyclines, has permitted a significant increase in the dose intensity, dose density, and duration of anthracycline therapy and may further increase the clinical utility of this family of drugs.6, 7, 8, 9
TABLE 18.1 KEY FEATURES OF DAUNORUBICIN AND DOXORUBICIN
Figure 18.1 Structures of the four anthracyclines in current clinical use. For epirubicin and idarubicin, arrows point to the sites where these new drugs differ from doxorubicin and daunomycin, respectively.
General Mechanism of Action and Cellular and Molecular Pharmacology
The initial studies of anthracycline cellular pharmacokinetics reported the existence of a carrier-mediated transport system. This was based on the apparent saturation kinetics for uptake, and the κm and Vmax for this carrier were calculated. However, the physical properties of doxorubicin vary over the concentration range at which these studies were done; both doxorubicin and daunorubicin self-associate like other planar molecules by ring stacking, forming polymers.10 This results in progressively less of the added drug being available for uptake into the cell.11 It now seems clear that for doxorubicin and daunorubicin, transmembrane movement is by free diffusion of the un-ionized drug.12 Furthermore, the uptake of the less polar daunorubicin is substantially faster than that of doxorubicin, which is itself substantially faster than the uptake of its polar alcohol metabolite.13
The daunosamine sugar can become protonated within the physiologic pH range with a pKa of 7.6.14, 15 For this reason, both extracellular and intracellular pH can have a significant impact on anthracycline uptake and cytotoxicity.16, 17, 18 For example, intracellular acidosis would result in enhanced drug accumulation because un-ionized drug would enter, would become protonated, and would be unable to diffuse out of the cell. Conversely, relative acidification of the extracellular fluid would result in a shift of drug out of the cell. In this regard, it should be pointed out that tumor masses as small as 1 cm can exhibit extracellular pHs as low as 6 to 6.5.19 More recent in vivo measurements utilizing 31P magnetic resonance spectroscopy have demonstrated that the intracellular pH of tumor cells in xenograft models is most frequently neutral to alkaline while the extracelluar pH is acidic.15 Thus, it is not surprising that simultaneous alkalinization of intra- and extracellular pH has been demonstrated to enhance the uptake and cytotoxicity of doxorubicin in cell culture and in SCID mice carrying the human MCF-7 breast carcinoma.15, 17 Furthermore, the acidification of intracellular organelles, including lysosomes, the Golgi network, and endosomes, in doxorubicin-resistant tumor cells significantly increases drug sequestration in these sites away from targets critical for tumor cell killing; blockade of sodium-proton exchange in acidic organelles produces a redistribution of doxorubicin into the cytoplasm and nucleus that can partially restore doxorubicin sensitivity in cells expressing the multidrug-resistance phenotype.20, 21
The cellular pharmacology of the anthracyclines is also characterized by the ability of essentially all nucleated cells to accumulate these drugs to an extraordinary degree.22 Ratios between the intracellular and extracellular concentration of daunorubicin and doxorubicin are routinely on the order of 30- to 1000-fold both at the end of a short-term in vitro incubation and in leukemic blasts at the end of an anthracycline infusion.23 The accumulation of the anthracyclines is due to DNA binding, rapid association with cell membranes, and storage in several different intracellular compartments; furthermore, there are significant differences in the degree of accumulation based on cell and tissue type.22 This phenomenon is important both for understanding the pharmacokinetics of these drugs and for the therapeutic efficacy of prolonged intravenous infusions.
Recent studies have demonstrated that once the anthracyclines diffuse through the plasma membrane, a substantial portion of the drug is bound to the 20S fraction of the proteasome.24, 25 Following proteasomal binding, anthracyclines are actively transported to the nucleus in an ATP-requiring, nuclear pore–dependent process that utilizes nuclear localization signals found in the 20S proteasomal subunits. The anthracyclines then dissociate from the proteasome due to the higher binding affinity of DNA. This process may facilitate the induction of cellular death programs by the anthracyclines and could provide a rationale for the combination of the anthracyclines with other agents that target proteasomal function.26, 27
In addition to the diffusion of the anthracyclines across the cell membrane, active drug efflux occurs in some cells. The initial demonstration of ATP-dependent drug efflux resulted from the elucidation of the role of the multidrug resistance transporter in acquired anthracycline resistance.28, 29, 30, 31 Cells expressing the MDR1 gene product efflux anthracyclines utilizing the P170 glycoprotein, a membrane protein capable of pumping a wide range of natural products, including the anthracyclines, out of cells. The P170 glycoprotein has adenosine triphosphate binding sites, and drug efflux is dependent on the presence of adequate intracellular ATP pools. Recently, other ATP-dependent drug efflux mechanisms capable of transporting anthracyclines against a concentration gradient have been discovered and may contribute to tumor cell drug resistance and to transport of the anthracycline antibiotics in normal host tissues, including the liver. These efflux mechanisms transport unmodified and/or glutathione-conjugated drug molecules.32, 33, 34, 35, 36, 37, 38, 39, 40
It is apparent from the studies reviewed above that while the general processes by which anthracyclines cross cell membranes have been examined in outline form, the kinetics of this process have not been established definitively, as is the case for methotrexate. The major barrier to such studies is the tendency for these drugs to bind to many intracellular proteins, DNA, phospholipids, and, perhaps, glycosaminoglycans. Because only free drug is presumably available for transport, efflux should be examined as a function of free, not total or bound, drug. However, this has rarely been done in studies of drug efflux utilizing anthracycline-resistant cell lines. Furthermore, the elucidation of several different energy-dependent efflux proteins in both tumor cells and normal tissues complicates the interpretation of prior studies while at the same time providing continuing opportunities for additional evaluations of cellular pharmacokinetic processes.
DNA Intercalation, Topoisomerase II Interactions, and Other Effects on DNA
There remains considerable controversy over the mechanism of action of the anthracyclines and thus over the importance of various intracellular targets. However, there is no disagreement with the observation that the bulk of intracellular drug is in the nucleus and that a portion of the anthracycline in the nucleus is intercalated into the DNA double helix (Fig. 18.2). Detailed studies of daunorubicin affinity for DNA have identified a preference for dGdC-rich regions that are flanked by A:T base pairs.41 In short defined DNA sequences prepared by PCR, daunorubicin binds preferentially to either 5′(A or T)GC or 5′(A or T)CG triplets.42 The consensus sequence for highest doxorubicin affinity is 5′-TCA.43 With respect to DNA interactions, the anthracycline molecule (Fig. 18.1) can be separated by function into three domains: the planar ring, which actually intercalates into DNA; the side chain (and its associated D ring), which provides an important hydrogen-bonding function; and the daunosamine sugar, which binds to the minor groove and plays a critical role in base recognition and sequence specificity.42
Figure 18.2 Three-dimensional view of daunomycin free and intercalated into DNA. This view shows the planar nature of the drug chromophore and how this is critical to DNA intercalation.
It has generally been assumed that all of the drug within the nucleus is intercalated. When tumor cells exposed to doxorubicin or daunorubicin are examined by fluorescence microscopy, an intense nuclear fluorescence is observed, and many investigators have assumed that the intensity of this fluorescence is a measure of drug intercalated into DNA. However, anthracycline fluorescence is quenched significantly upon intercalation, and in fact, this quenching has been used to measure intercalation.44 Thus, the nuclear fluorescence of the anthracyclines is likely to be due to binding of the drug to components of the nucleus by a nonintercalative mechanism. In fact, stable, covalently bound doxorubicin-DNA adducts (containing the daunosamine sugar) have recently been produced under conditions favored by the iron-dependent redox chemistry in which doxorubicin participates.45
Because the anthracyclines concentrate in the nucleus and are good DNA intercalators, DNA intercalation was presumed initially to play an important role in their mechanism of action. Several proposals were advanced to describe how DNA intercalation might lead to tumor cell kill, the best documented of which were studies demonstrating inhibition of RNA and DNA polymerases by the anthracyclines.46, 47 Unfortunately, the drug concentrations required for inhibition of these enzymes are far in excess of that which can be achieved in vivo, which probably explains the lack of correlation between inhibition of RNA and DNA synthesis and the cytotoxicity of doxorubicin.48
Topoisomerase II Interactions
An important advance in our understanding of anthracycline-DNA interactions occurred with the demonstration that anthracyclines cause protein-associated DNA breaks measured by filter elution, which in some cell lines are correlated with cytotoxicity.49, 50, 51 Subsequent investigations have shown that the formation of protein-associated DNA breaks is caused by the formation of a ternary drug-DNA-enzyme “cleavable complex” involving the anthracycline antibiotic and DNA topoisomerase II, an enzyme associated with the nuclear matrix that plays a critical role in releasing torsional strain in DNA as well as in chromosome condensation.52 Anthracyclines inhibit topoisomerase II by trapping DNA strand passage intermediates that can be detected as protein-associated DNA single- and double-strand breaks linked to the enzyme.53, 54 It had previously been presumed that through intercalation the anthracyclines altered the three-dimensional conformation of DNA, which arrested the cycle of topoisomerase II action at the point of DNA cleavage. However, topoisomerase II–associated DNA cleavage can be demonstrated at doxorubicin concentrations (10-8 M) well below the dissociation constant for DNA intercalation,55 as well as with anthracycline analogs that do not intercalate into DNA.56 Thus it is possible that anthracyclines may stimulate topoisomerase II–mediated DNA cleavage by a nonintercalative mechanism. Studies evaluating the interaction of the anthracyclines with topoisomerase II have demonstrated that doxorubicinone actively inhibits the purified enzyme, suggesting that the anthracycline sugar is not required for enzyme inhibition. Since the daunosamine sugar plays an important role in DNA binding, it may be possible to dissociate DNA intercalation further as a mechanism of action.54 It has been demonstrated that the anthracyclines produce topoisomerase-related DNA cleavage in specific regions of the DNA (with an adenine at the 3′ end of one break site); this may provide a clue to gene-specific effects of these drugs, including specific sites of cleavage by anthracycline analogs.57, 58 It is also now clear, as will be described subsequently, that anthracycline resistance may be associated with alterations in the level or function of topoisomerase II, indicating a potentially important role for this enzyme in anthracycline action.59, 60, 61
Recent molecular studies performed both in human cell lines and in yeast model systems have more clearly defined the role of the alpha isoform of topoisomerase II in the production of protein-associated DNA cleavage after doxorubicin exposure.62, 63 Overexpression of the antisense construct of topoisomerase IIα in human U937 monocytic leukemia cells down-regulates topoisomerase IIα mRNA levels by >70%, with a concomitant reduction in the cytotoxicity of daunorubicin. Furthermore, a detailed reexamination of doxorubicin-related single-strand cleavage and topoisomerase-DNA complex formation has confirmed this process to be ATP-dependent and specific for topoisomerase II (not I),64 an observation originally described more than a decade earlier.65
Although anthracycline interactions with DNA topoisomerase II clearly occur in many mammalian cell lines, it is likely that the formation of cleavable complexes alone is only potentially lethal and is not in itself sufficient for tumor cell killing. Although the initial correlations of protein-associated single-strand cleavage and cytotoxicity in L1210 cells, which were performed at clinically relevant drug concentrations, suggested a direct relationship between topoisomerase II–mediated DNA damage and cytotoxicity for doxorubicin,49, 66 subsequent investigations have demonstrated a dissociation between tumor cell killing and the kinetics of DNA break formation and disappearance for doxorubicin and its analogs.67 In some cell lines, only DNA double-strand cleavage can be associated with cytotoxicity,68 and in others, DNA single-strand cleavage is modest and double-strand cleavage essentially undetectable at even supralethal drug concentrations.69, 70 Furthermore, recent evidence from several different model systems does not provide uniform support for a causal relationship between the level of topoisomerase II and the sensitivity of human cell lines to doxorubicin in vitro.71, 72 Finally, correlations between topoisomerase IIα content or activity in primary tumors and clinical outcome for breast cancer patients treated with anthracyclines have not been demonstrable.73, 74
It has been appreciated for some time that in addition to stabilization of the cleavable complex, the anthracyclines and a number of other antineoplastic compounds can inhibit the catalytic activity of the enzyme without trapping the complex.53, 75 This observation, important for the development of new anthracyclines as well as combination regimens, underlies the recent demonstration that certain anthracycline analogs (not doxorubicin) antagonize the cytotoxicity and DNA cleavage of etoposide through inhibition of cleavable complex formation.76, 77, 78 Furthermore, certain anthracycline analogs have recently been found that inhibit both topoisomerase I and II, which may explain their nonoverlapping resistance profiles and altered spectrum of action.79, 80,81 Doxorubicin is also known to exhibit more cytotoxicity than expected per DNA break. This might mean either that doxorubicin-associated breaks are qualitatively different from those produced by other topoisomerase II–active drugs or that other mechanisms of action might be operating in parallel. Thus, questions remain regarding the precise role of cleavable complex formation in the cytotoxicity of the anthracycline antibiotics.
Other Effects on DNA
In addition to the physicochemical effects of the anthracyclines on DNA and their interactions with topoisomerase II, doxorubicin produces other effects on DNA. Among the most important of these is that doxorubicin and daunorubicin form DNA-anthracycline complexes that significantly modify the ability of a specific class of nuclear enzymes, the helicases, to dissociate duplex DNA into DNA single strands in an ATP-dependent fashion; the entire process of strand separation is thus hindered, limiting replication.82 This effect occurs at clinically relevant drug concentrations (<1 µM) and parallels, at least in part, the cytotoxic spectrum of several anthracycline analogs.83 The mechanism of helicase inhibition involves the formation of an irreversible ternary complex between anthracyclines that possess an unblocked daunosamine sugar, DNA, and the helicase.84 Given the diversity of human DNA helicases, differential effects of the anthracyclines could be related to their interactions with this class of nuclear enzymes. It has also been shown that in the absence of significant DNA double-strand cleavage, doxorubicin interferes with DNA unwinding and produces nonoligosomal fragmentation of nascent DNA during continuous exposure to very low drug concentrations.70, 85 This DNA effect is associated with tumor cell differentiation and suppression of c-myc oncogene expression by both doxorubicin and certain of its analogs and suggests yet another potential growth inhibitory pathway for the anthracyclines.86, 87
As will be reviewed subsequently, it has also been demonstrated that doxorubicin can undergo cycles of reduction and oxidation in essentially all intracellular compartments, including the nucleus and the mitochondrion, leading to the formation of reactive oxygen species.88 Doxorubicin redox cycling has been shown to oxidize DNA bases in human chromatin and in intact tumor cells, which may provide a cytotoxic mechanism unrelated to strand cleavage.89, 90, 91Mitochondrial DNA is also susceptible to oxidative stress in vitro and in the rat after doxorubicin administration where the production of 8-hydroxyguanosine, a byproduct of hydroxyl radical attack on DNA, is significantly increased in cardiac and liver mitochondria above the level observed in nuclear DNA from these two organs.92, 93, 94
Evidence of DNA base oxidation has also recently been demonstrated in patients treated with anthracycline antibiotics. Urinary hydroxymethyluracil (an oxidative by-product of thymine) has been observed within 24 hours of drug treatment in patients receiving combination chemotherapy that included an anthracycline.95, 96 Following bolus therapy with the anthracycline analog epirubicin, a wide variety of oxidized DNA bases can be found in chromatin isolated from patient lymphocytes using gas chromatography/mass spectroscopy (GC/MS).97 In patients receiving a 96-hour infusion of doxorubicin, producing steady-state drug levels of 0.1 µM, a 2- to 5-fold increase in 13 different oxidized DNA bases, including thymine glycol, 5-OH-hydantoin, 5-OH-uracil, 4,6-diamino-5-formamindo-pyrimidine, and 5,6-di-OH-uracil, was observed by GC/MS in peripheral blood mononuclear cells beginning 72 hours after the initiation of treatment.98 These data are important because this spectrum of DNA base damage is the same as that produced by ionizing radiation, which is known to be produced by the hydroxyl radical.99, 100 Thus, these studies provide unequivocal evidence of the oxidative metabolism of doxorubicin and its analogs under clinical conditions.
It is important to remember that doxorubicin and daunorubicin are both mutagens and carcinogens;101 only recently have investigators begun to map the base substitutions and deletions produced by doxorubicin that appear to occur adjacent to preferential doxorubicin DNA binding sequences. Furthermore, it is now well established that therapy with doxorubicin (or epirubicin), when used in combination with cyclophosphamide, is associated with a dramatically increased risk of a second malignancy, specifically acute mono- or myelomonocytic leukemia.102, 103, 104
Because of the inhibitory effects of oxidized DNA bases on the action of DNA polymerases and other DNA repair mechanisms, as well as their own intrinsic mutagenic properties, the clinical investigations that demonstrate the production of oxidized DNA bases in hematopoietic cells after treatment with doxorubicin indicate a potential mechanism for anthracycline-related mutagenicity and carcinogenicity, as well as provide insights into the mechanism of tumor cell killing by the anthracyclines and their potentially adverse consequences for the synthesis of genes comprising the mitochondrial respiratory chain.105, 106, 107, 108
In addition to the production of oxidized DNA bases, the intracellular reactive oxygen metabolism of the anthracyclines leads to the iron-dependent production of formaldehyde from a variety of intracellular carbon sources, which can subsequently react to produce a drug-formaldehyde conjugate containing two anthracycline molecules.109, 110, 111 Such conjugates have a unique ability to form novel covalent DNA cross-links that markedly enhance the therapeutic activity of the anthracycline. They also provide the opportunity to produce targeted anthracycline prodrugs that possess a unique spectrum of anticancer activity.112, 113
Drug Activation by One- and Two-Electron Reduction
During DNA intercalation and binding to topoisomerase II, the anthracyclines act as chemically inert compounds that owe their activity to their ability to bind to key macromolecules and distort the three-dimensional geometry of these targets. However, the anthracyclines are chemically reactive and possess an extraordinarily rich chemistry that even now has not been fully documented.114, 115
The one-electron reduction of the anthracyclines was initially described in hepatic microsomal systems116, 117, 118 but was later shown to play a central role in the cardiac toxicity of this class of drugs119, 120, 121 and may be involved in antitumor activity as well.122, 123, 124 All of the clinically active anthracyclines are anthraquinones. As is true of quinones in general,125, 126 the anthracyclines are able to undergo one- and two-electron reduction to reactive compounds that cause widespread damage to intracellular macromolecules, including lipid membranes, DNA bases, and thiol-containing transport proteins (Fig. 18.3).127, 128,129, 130 As outlined in Figure 18.4, the one-electron reduction of doxorubicin or daunorubicin may occur in essentially all intracellular compartments, including the nuclear membrane, and is catalyzed by flavin-centered dehydrogenases or reductases, including cytochrome P-450 reductase, NADH dehydrogenase (complex I of the mitochondrial electron transport chain), xanthine oxidase, and cytochrome β5 reductase.131, 132, 133 In addition, recent studies demonstrate that all three isoforms of nitric oxide synthase (at their flavoprotein domains) are capable of catalyzing the one-electron reduction of doxorubicin, with the subsequent production of superoxide and a decrease in nitric oxide.134, 135 Furthermore, doxorubicin can directly inhibit nitric oxide synthase activity,135, 136which could produce significant alterations in vascular tone both in the heart and in tumors.137, 138 It can also be metabolized by lactoperoxidase and nitrite.139 All of these flavoenzymes are widely distributed in mammalian tissues, and anthracycline-mediated free-radical formation has been demonstrated in a wide range of organs and tumor cell lines. In addition to flavoproteins, doxorubicin can be reduced in the heart by oxymyoglobin, leading to the production of strong oxidant species.140
Figure 18.3 One-electron reduction of doxorubicin. This reduction occurs at the quinone oxygens of the chromophore. The semiquinones react rapidly with oxygen, when it is available, to yield the one-electron reduction product of oxygen, superoxide.
One-electron reduction of the anthracyclines leads to the formation of the corresponding semiquinone free radical. In the presence of oxygen, this free radical rapidly donates its electron to oxygen to generate superoxide anion (O2•-). Although not highly toxic itself, the dismutation of superoxide yields hydrogen peroxide (H2O2). Under biological conditions, the anthracycline semiquinone or reduced metal ions such as iron reductively cleave hydrogen peroxide to produce the hydroxyl radical (OH•) or a higher oxidation state metal with the chemical characteristics of the hydroxyl radical, one of the most reactive and destructive chemical species known.141, 142 It is now commonly accepted that reduced metals are critical components in the formation of toxic free-radical intermediates and may well contribute to the cytotoxicity of the anthracyclines.143 However, it remains an area of active investigation how reduced metal species, including iron, become available for these free-radical reactions.
Because oxygen radical formation occurs as a result of normal metabolic processes (including mitochondrial respiration) and is a common mechanism of action for several naturally occurring toxins, most mammalian cells have elaborate defenses against oxygen radical toxicity.144 Superoxide dismutase, catalase, and glutathione peroxidase act in concert to reduce superoxide, hydrogen peroxide, and lipid hydroperoxides to water or nontoxic lipid alcohols without the formation of the hydroxyl or peroxyl radicals (Fig. 18.4). Glutathione, a sulfur-containing tripeptide, can react with many radicals as well as function as part of the glutathione peroxidase cycle to reduce peroxides to less reactive compounds. There are also specific DNA repair systems to handle oxidative damage to DNA.145, 146, 147 However, antioxidant defenses are not equally distributed in various tissues in the body. For example, glutathione concentration is higher in the liver than in most other tissues and tumors. Catalase activity is lower in the heart than in the liver.120, 148 Likewise, the activity of several flavoproteins capable of activating the anthracyclines differs from tissue to tissue. These variations in drug activation and antioxidant defense provide ample opportunity for tissue specificity in terms of toxicity and antitumor activity. For example, the unique cardiac toxicity of the anthracyclines may result, in part, from the low level of cardiac catalase coupled with the extraordinary cardiac content of mitochondria and myoglobin, which enhance drug activation, as well as the sensitivity of cardiac glutathione peroxidase to free-radical attack,120, 149 which destroys the activity of this critical enzyme at the same time that anthracycline administration stimulates cardiac hydrogen peroxide formation.150 In contrast, while anthracyclines can easily be activated to reactive intermediates by hepatic enzymes, the liver has a more active free-radical defense system and is able to actively efflux anthracyclines and anthracycline metabolites. The importance of hepatic antioxidant systems is shown by the fact that pretreatment in rodents with agents that significantly diminish the level of reduced glutathione, such as carmustine (BCNU) or acetaminophen, dramatically sensitizes hepatocytes to doxorubicin-induced free-radical injury.151, 152
The role of oxygen radical formation in tumor cell killing rather than cardiac toxicity continues to be defined.153, 154, 155, 156 However, several lines of evidence support this hypothesis. First, doxorubicin resistance in tumor cell lines can frequently be reversed by agents that decrease glutathione concentration.157, 158,159, 160, 161, 162 This observation cannot be explained by DNA or topoisomerase interactions. Second, anthracycline-enhanced free-radical formation has been detected in a range of tumor cell lines,122, 163, 164 the best studied of which are human breast cancer cell lines where both intra- and extracellular reactive oxygen species were demonstrated.123, 165 Extracellular as well as intracellular antioxidants have also been demonstrated to decrease the cytotoxicity of the anthracyclines in a wide variety of cells, including human tumor cell lines.123, 124, 143, 166, 167, 168, 169, 170 In addition, some anthracycline-resistant cancer cell lines exhibit increases in various aspects of the oxygen free-radical defense system, including increases in glutathione and the selenoprotein glutathione peroxidase.161, 171, 172, 173, 174 Alterations in glutathione peroxidase activity produced by manipulation of selenium status significantly affect tumor cell killing by doxorubicin.175, 176 Transfection of the human cytosolic glutathione peroxidase in the sense orientation produces doxorubicin resistance177 while antisense expression sensitizes cells to doxorubicin cytotoxicity.178 Finally, 4-fold overexpression of the manganese superoxide dismutase in CHO cells produces 2.5-fold resistance to doxorubicin,179 while inhibition of the copper-zinc superoxide dismutase with 1,25-dihydroxyvitamin D3 significantly enhances doxorubicin-related cytotoxicity.180
Figure 18.4 Anthracycline antibiotic cell death program. Anthracycline antibiotics can be metabolized at the cell surface, at complex I of the mitochondrial electron transport chain, in the cytosol, or at the nuclear envelop by flavin-containing dehydrogenases, leading to the production of reactive oxygen species with the potential to alter intracellular iron stores at multiple intracellular sites. This free-radical cascade can initiate both apoptotic and necrotic death programs associated with mitochondrial membrane injury, DNA base oxidation, altered calcium sequestration, energy loss, and altered proliferative potential. The effects of anthracycline-enhanced reactive oxygen production are modulated by intracellular antioxidant enzymes (glutathione peroxidase, catalase) and antiapoptotic proteins. (Please see color insert.)
Reservations about the role of oxygen radical formation in tumor cell killing arise from several observations. First, most of the studies demonstrating anthracycline-enhanced hydroxyl radical formation have utilized drug concentrations in excess of that which would be clinically relevant. This limitation is in part technical in that it is not possible at present to detect hydroxyl radicals at concentrations much below 10-7. However, recent studies using fluorescent probes to detect hydrogen peroxide production by flow cytometry after doxorubicin exposure in human colon carcinoma cells successfully demonstrated peroxide formation after treatment with 0.4 µM of doxorubicin,164 and enhanced oxidative respiration and reactive oxygen production have been observed in human breast cancer cells using chemiluminescent probes at doxorubicin levels between 0.05 and 0.1 µM.181 Second, many tumors for which doxorubicin has great clinical utility, such as breast cancer, are clearly hypoxic, and thus the applicability of the chemistry outlined in Figures 18.3 and 18.4 might be questioned. However, under low partial pressures of oxygen, iron-mediated lipid peroxidation and DNA damage from the doxorubicin semiquinone are actually enhanced.182 Third, there is no question that glutathione depletion does not sensitize all tumor cell lines to the cytotoxic effect of doxorubicin183 and that many doxorubicin-resistant cells have no alteration in antioxidant defense enzymes.184 Fourth, it has frequently been presumed that oxygen radical–mediated effects on DNA were unlikely because intercalated drug could not be reduced. However, doxorubicin covalently bound to oligonucleotides can still be activated by cytochrome P-450 reductase,185 and daunorubicin intercalated into calf thymus DNA can be reduced by a superoxide-generating system; under these circumstances the semiquinone is accessible to hydrogen peroxide for reaction, and the disproportionation of the semiquinone to the 7-deoxyaglycone may occur by intramolecular electron transfer, with migration of electrons over several base pairs.186, 187, 188 Finally, as outlined previously, recent studies have demonstrated the presence of oxidized DNA bases, by-products of anthracycline redox cycling, in both the urine96 and peripheral blood mononuclear cells of patients receiving anthracycline therapy.97, 189 These reports provide the first, albeit indirect, evidence demonstrating the products of redox cycling in human tissues after anthracycline administration using standard treatment schedules.
Role of Iron
Although free-radical formation was originally proposed as the basis for anthracycline cardiac toxicity in 1977 and various animal model studies suggested that hydroxyl radical scavengers could blunt doxorubicin-related heart damage, free-radical scavengers were initially unsuccessful cardioprotective agents in humans.190 These results suggested that some additional variable was involved. This variable proved to be the interaction of anthracyclines with iron. Because of its reactivity, free-iron concentrations in the body are in the range of approximately 10-13 M, whereas total iron concentrations are between 10-4 and 10-5M. Most of the iron in tissues is stored in ferritin. Doxorubicin has been found to release iron from ferritin in two ways. The drug can slowly abstract iron from the ferritin shell directly;191 a much more rapid release of iron follows conversion of the anthracycline to its semiquinone, which can release iron under hypoxic conditions or through the reducing power of the superoxide anion in air.192, 193 Doxorubicin can also release nonheme, nonferritin iron from microsomes.194, 195, 196, 197 In addition, doxorubicin can increase the uptake of iron by a transferrin receptor–mediated process, enhancing intracellular iron availability.198 The hydroxyquinone structure of doxorubicin and daunorubicin represents a powerful site for chelation of metal ions, especially ferric iron. Iron anthracycline complexes have been shown to possess a wide range in interesting biochemical properties in vitro.199, 200 These complexes can bind DNA by a mechanism distinct from intercalation, cause oxidative destruction of membranes, and oxidize critical sulfhydryl groups. It remains unclear, however, whether these tight-binding iron-anthracycline complexes are stable and produce toxic effects intracellularly201, 202 or whether the “delocalization” of catalytic amounts of protein-bound iron by anthracycline-stimulated free-radical formation is responsible for hydroxyl radical formation in tissues.
These investigations suggested that the most effective way to interfere with the generation of highly reactive oxidants after anthracycline exposure would be to pretreat with a chelating agent that might withdraw iron from free-radical reactions. This hypothesis has been confirmed, and an iron chelator, dexrazoxane (ICRF-187), has been shown to prevent doxorubicin-induced lipid peroxidation203 and cardiac toxicity in a wide range of animal models.204, 205 Randomized, controlled clinical trials in humans have confirmed the ability of this agent to diminish markedly the cardiac toxicity of doxorubicin.206, 207 Dexrazoxane is a highly effective iron chelator in vivo; during phase I studies, it caused a 10-fold increase in urinary iron clearance.208, 209 Dexrazoxane is itself a prodrug in that it must undergo hydrolysis to become an effective iron chelator (Fig. 18.5). This hydrolysis to ICRF-198, the major iron-binding metabolite of dexrazoxane, can occur spontaneously at physiologic pH but is markedly enhanced after uptake into cardiac myocytes, with conversion of the parent drug to ICRF-198 in less than 60 seconds.210 The parent drug is very lipid soluble and enters cells by passive diffusion. ICRF-198 has been demonstrated to efflux iron from iron-loaded myocytes; these studies suggest that the same chelating ability may be available to remove iron that has been released from cardiac iron-storage proteins.210
Figure 18.5 Dexrazoxane and its analogy to EDTA (ethylenediaminetetraacetic acid). Dexrazoxane is much more nonpolar because the carboxylic acid groups have been fused into amide rings. This allows ready entry into the cell. Dexrazoxane can undergo hydrolysis to yield a carboxylamine able to bind iron.
Important additional studies have further amplified our understanding of the role of iron in anthracycline biochemistry and in the mechanism of drug-induced cardiac toxicity.211, 212, 213, 214 The alcohol metabolite of doxorubicin, doxorubicinol, which is produced by the two-electron reduction of the C-13 side chain carbonyl group, has been demonstrated to cause the delocalization of low-molecular-weight Fe(II) species from the iron-sulfur center of aconitase in a redox-dependent fashion. The formation of a doxorubicin-iron complex with aconitase interferes with critical interconversions of cytosolic aconitase with iron regulatory protein-1(IRP-1); IRP-1 plays an essential role in iron homeostasis and, hence, in the regulation of critical intracellular metabolic processes (such as the action of mitochondrial electron transport proteins, myoglobin, and various cytochromes). These effects of doxorubicin metabolites suggest that iron-dependent reactions occur that may not be directly related to the formation of reactive oxygen species. This could help to explain the utility of dexrazoxane, compared to free-radical scavengers that do not chelate iron, in the prevention of both acute and chronic anthracycline cardiotoxicity.197
Doxorubicin is a powerful chelator of other metal ions, including Cu2+ and Al3+. The chelation of aluminum by doxorubicin is effective enough that a doxorubicin solution left in contact with aluminum foil for only 1 hour will change from the orange-red of doxorubicin to the bright cherry red of the aluminum complex. A similar reaction occurs with iron-containing alloys; doxorubicin left within a syringe needle for any significant period of time will also change color by virtue of chelation of metal from the needle. For this reason, every effort should be made in the clinic to keep anthracyclines from prolonged contact with any metal surface.
Two-Electron Reduction of the Anthracyclines
Two-electron reduction of doxorubicin (which may occur by sequential one-electron reductions or directly when strong reducing agents are applied) results in the formation of an unstable quinone methide, which rapidly undergoes a series of reactions leading to the formation of the corresponding deoxyaglycone (Fig. 18.6).114 It is now established that deoxyaglycones are formed in vivo.215, 216 Because the deoxyaglycones exhibit far less cytotoxicity than the parent drug, the current consensus is that this is a pathway for drug inactivation. It is likely that in the absence of oxygen the one-electron reduction product, the semiquinone, reacts with itself to yield parent drug and the two-electron reduction product. The quinone methide intermediate in this pathway has been proposed as a potential monofunctional alkylating agent; however, there is little evidence that this intermediate plays an important cytotoxic role in tumor cells. Finally, two-electron reduction of the anthracyclines using powerful reducing agents to convert doxorubicin to its inactive deoxyaglycone metabolite has been advocated as a means to reduce local tissue injury after anthracycline extravasation.217 Direct enzymatic two-electron reduction is unlikely to occur under physiologic conditions.218
Signal Transduction, Membrane-Related Actions of the Anthracyclines, Apoptosis, and Cellular Senescence
It has been appreciated for more than a decade that the anthracycline antibiotics are membrane-active compounds that produce myriad effects at the cell surface.219 It is only in the more recent past that events occurring at the cell surface have been related more clearly to anthracycline cytotoxicity and DNA damage. Doxorubicin alters the fluidity of both tumor cell plasma membranes220, 221 and cardiac mitochondria;127 it binds avidly to phospholipids, including cardiolipin,222, 223 causes an up-regulation of epidermal growth factor receptor (but not p185HER-2/neu),224, 225 inhibits the transferrin reductase of the plasma membrane,226 induces iron-dependent protein oxidation in erythrocyte plasma membranes in vivo227, and can be actively cytotoxic without entering the cell.228, 229 Furthermore, recent studies suggest that the presence of extracellular doxorubicin is of critical importance for membrane interactions that are intimately related to the evolution of tumor cell kill230 and that doxorubicin cytotoxicity can be manipulated by membrane phospholipid alterations 231, 232, 233 that increase drug uptake but not intracellular distribution. The confluence of these studies suggests that plasma membrane–associated events, modulated by lipid metabolism, could be involved in the mechanism of action of the anthracyclines.
Figure 18.6 Two-electron reduction of anthraquinones. The immediate product is the dihydroquinone, which is not stable. This undergoes rearrangement with loss of the sugar to yield the quinone methide. This structure has activity as an alkylator in pure chemical systems. The most likely fate, however, is progression, via a second arrangement, to yield the 7- deoxyaglycone. This final product is much less active than the parent drug.
Signal Transduction and Anthracyclines
Communication between the cell surface and the nucleus plays a crucial role in growth control; several important signal transduction pathways for mitogenic stimuli can be initiated at the plasma membrane.234, 235, 236, 237, 238 If membrane interactions by the anthracyclines are important for their mechanism of action, it should be possible to show that these compounds interact significantly with known signal transduction programs.239
Several laboratories have provided essential pieces of evidence linking anthracycline action to effects on specific signal transduction pathways, including the protein kinase C system. Although at high concentrations (>100 µM), anthracyclines can inhibit protein kinase C,240, 241 the doxorubicin-iron complex is more active as an inhibitor of diacylglycerol at 10-fold lower concentrations.242 At clinically relevant levels, doxorubicin increases the turnover of phosphoinositides and phosphatidylcholine in sarcoma 180 cells, which leads to the accumulation of diacylglycerol and inositol phosphates and a twofold increase in cytosolic protein kinase C activity.243 Furthermore, activation of the protein kinase C pathway by phorbol esters enhances doxorubicin cytotoxicity and drug-related DNA-protein cross-links, whereas down-regulation of protein kinase C partially prevents cell kill.244 Since protein kinase C can phosphorylate topoisomerase II,245 it is possible that the initiation of membrane signalling by doxorubicin could be involved in the regulation of anthracycline-mediated DNA damage.
The importance of the sphingomyelin pathway in signal transduction has become increasingly clear during the past five years.234 In addition to participating in protein kinase C–related signal transduction, sphingolipid metabolites are involved in transducing signals from a wide variety of cell surface molecules, including interferon-γ, TNF-α, and Fas/APO-1. Recent data suggest that the activation of sphingomyelinases by a variety of cellular stresses, including exposure to the anthracyclines, leads to the release of the critical signalling intermediate ceramide from membrane sphingomyelin.246, 247, 248 Intracellular ceramide accumulation can produce profound effects on cell cycle progression as well as on the effector arm of the cell death program.249, 250, 251 Expression of glucosylceramide synthase (the enzyme that converts ceramide to glucosylceramide) in human MCF-7 cells blocks doxorubicin-induced increases in ceramide after drug exposure, leading to an 11-fold increase in IC50 concentration.252
Signal transduction pathways involving protein kinase C and ceramide also appear to be involved in the regulation of the function of the P170 glycoprotein and the enhanced export of anthracyclines in drug-resistant cells;253, 254, 255 inhibition of protein kinase C has been shown to down-regulate P170 glycoprotein function and enhance the sensitivity of myeloid leukemia cells to daunorubicin, providing a novel strategy for overcoming multidrug resistance.256 Furthermore, certain agents that reverse multidrug resistance, such as cyclosporin A and verapamil, may in part be active through an inhibition of ceramide glycosylation.257
Anthracyclines and Apoptosis
The explosive growth of our understanding of apoptosis (programmed cell death)258, 259, 260 has provided crucial links between many of the pleiotropic effects of the anthracyclines that have been described previously, including anthracycline-related alterations in membrane biochemistry, signal transduction, mitochondrial metabolism, DNA damage, and free-radical formation.
Doxorubicin or daunorubicin exposure can produce the morphological changes associated with apoptosis, such as chromatin condensation, internucleosomal DNA fragmentation, reduced cell volume, and cytoplasmic blebbing in a wide variety of cell lines, including HeLa cells,261 P388 murine leukemia cells,262 M1 myeloid leukemia cells,263 murine small intestinal crypt epithelium,264 thymocytes,265, 266 and others.156, 267 In general, the degree of anthracycline-related apoptosis varies considerably between experimental model systems; in cell culture, the full expression of apoptotic morphology is often not observed until 48 to 120 hours after drug treatment. This variability is due, in part, to wide variation in the expression of both proapoptotic and antiapoptotic molecules in cultured tumor cells, a degree of variability that has also been observed in human tumor samples.38, 268, 269, 270
Intensive investigative efforts have begun to determine the molecular mechanisms of anthracycline-related apoptosis. The picture that is starting to emerge is of a series of biochemical interactions by the anthracycline antibiotics with a wide variety of different initiating death signals ultimately utilizing common effector molecules to produce apoptosis and/or necrotic cell kill. One of the best-described death stimuli is the interaction of the CD95 (APO-1/Fas) surface receptor with its natural ligand CD95L or structurally related antibodies to form a signalling complex that activates proteases of the caspase family to effect the ultimate biochemical reactions resulting in apoptotic morphology. This pathway, which plays a critical role in the regulation of lymphoid cell growth, has been shown to be active in some solid tumors and leukemias. Recent experiments initially suggested that doxorubicin produced apoptosis by inducing CD95L and CD95 receptor formation and that CEM cells, Jurkat T cells, and neuroblastoma cells resistant to anti-CD95 antibody were resistant to doxorubicin-induced apoptosis.271, 272, 273 Although doxorubicin clearly appears to up-regulate CD95L expression after doxorubicin exposure in HeLa cells transfected with a CD95L reporter construct,274 a series of recent studies in different cell lines have determined that acquired resistance to CD95 by clonal selection or by treatment with other anti-CD95 antibodies that inhibit CD95-mediated apoptosis does not concomitantly engender resistance to doxorubicin-related apoptosis,275, 276that CD95 and CD95L are frequently not up-regulated following doxorubicin exposure,277 and that anthracycline-mediated activation of caspase 8, previously supposed to modulate CD95-induced apoptosis specifically, occurs in the absence of signalling through the CD95/CD95L pathway.278 All of these experiments support the hypothesis that doxorubicin and CD95 utilize common downstream effectors of apoptosis but that the initiating death stimulus for either of these molecules may vary.
Apoptosis due to the anthracyclines has also been clearly related to ceramide generation,246, 279 which links the plasma membrane biochemistry of doxorubicin with the induction of the cell death cascade. Only recently, however, has the molecular ordering of anthracycline-induced apoptosis begun to be examined. In these studies, ceramide generation following sphingomyelinase activation leads to important effects on mitochondrial permeability, the activation of proapoptotic caspases, and serine-threonine protein phosphatases.234, 249, 280 Yet it remains to be determined whether ceramide production after anthracycline exposure is associated principally with the activation of cell death signals to the mitochondria or with the effector phase of apoptosis.281
One of the critical steps in translating a wide variety of apoptotic stimuli into either apoptotic or necrotic cell death is the induction of cytochrome c release from the space between the inner and outer mitochondrial membrane.258, 282, 283 Cytochrome c release can lead to caspase activation or to altered mitochondrial electron transport, with subsequent apoptosis or necrosis. Anthracyclines are fully capable of inducing cytochrome c release independent of DNA damage.284, 285 In light of the previously described extensive binding and metabolism of the anthracyclines by complex I of the mitochondrial electron transport chain,132 it is likely that reactive oxygen species, produced by anthracycline-treated mitochondria, can damage mitochondrial membrane integrity and may play an important initiating role in doxorubicin-related apoptosis. Recent studies indicate that various free-radical scavengers inhibit programmed cell death following anthracycline exposure.90, 156, 168, 169, 286 Furthermore, cytokine-mediated induction of ceramide production has been found to be redox-sensitive, and overexpression of antioxidant genes in human tumor cells prevents ceramide production and partially blocks apoptosis.287, 288 These recent observations link many of the known but pleiotropic biochemical effects of the anthracyclines; further studies are likely to define the order of anthracycline-related death signals at the molecular level and the relationship of these signals to intracellular anthracycline metabolism.
Two major endogenous modulators of programmed cell death, Bcl-2 and p53, as well as their associated downstream effectors, play critical roles in regulating anthracycline-related apoptosis. The Bcl-2 protein functions in the outer membranes of mitochondria, nuclei, and the endoplasmic reticulum as an inhibitor of cell death; it has been clearly shown to block apoptosis following anthracycline exposure in many experimental systems.289, 290 Furthermore, in acute myelogenous leukemic blasts and HL-60 cells, the cytotoxicity of daunorubicin is increased by exposure to bcl-2 antisense oligonucleotides.291 The biochemical mechanisms through which Bcl-2 inhibits cell death continue to be elucidated; however, it is currently appreciated that Bcl-2 represses cytochrome c release from mitochondria, interferes with caspase activation, blocks the apoptotic effects of reactive oxygen species, and binds to transcription factors involved in doxorubicin-mediated apoptosis, such as nuclear factor-κB.292, 293,294, 295, 296, 297 Bcl-2 has also been demonstrated to suppress p53-mediated transcriptional activation of several genes involved in the apoptotic process after doxorubicin exposure in MCF-7 cells.292
It appears that programmed cell death resulting from doxorubicin exposure is modulated by the interplay between the expression of bcl-2 and the p53 tumor suppressor gene.298 P53 functions as a transcription factor that causes cell cycle arrest or apoptosis after DNA damage; among the genes activated by p53 are the cyclin-dependent kinase inhibitor p21, cyclin G, and the apoptosis-inducing gene bax. Exposure to anthracyclines leads to elevated steady-state levels of p53 in cells expressing the wild-type gene,267, 299 which produces cell cycle arrest or apoptosis depending on the cell type studied. G1 arrest is due to the induction of p21 in both p53-dependent and p53-independent contexts, which leads to apoptosis or cellular senescence.299, 300, 301, 302 Studies also suggest that p53 induction after doxorubicin treatment produces up-regulation of cyclin G expression, with a consequent increase in the accumulation of cells in the G2/M as well as G1 phase of the cell cycle.303 Finally, mutations in p53, found in almost half of human tumors, lead to diminished apoptosis and doxorubicin resistance in many different tumor cell types.304, 305, 306
Anthracyclines and Cellular Senescence
Recent studies have demonstrated that in addition to necrotic or apoptotic death phenotypes, exposure of mammalian cells to the anthracycline antibiotics may produce prolonged growth arrest that in morphologic and enzymatic terms resembles replicative senescence.307 This response may occur whether or not p53 mutations are present and is characterized by the inability of cells undergoing terminal differentiation to form colonies, while at the same time remaining metabolically active but nonproliferative; furthermore, although not required for the initiation of cellular senescence, both p53 and p21 are positive regulators of the senescence phenotype.308, 309, 310 cDNA microarray analysis has demonstrated that doxorubicin-related senescence is associated with the inhibition of genes associated with cell proliferation and the up-regulation of tumor suppressors.311 The discovery of this novel antiproliferative pathway induced by the anthracycline antibiotics provides additional insight into the pleiotropic nature of their antineoplastic mechanism of action as well as possible new approaches to enhancing their use in oncologic practice.312
Mechanisms of Resistance
Enhanced Drug Efflux
P170 Glycoprotein–Mediated Anthracycline Efflux
A majority of the doxorubicin-resistant cell lines developed in the laboratory exhibit increased expression of the P170 glycoprotein. At present, the role of this protein in enhancing drug efflux and as a mechanism of experimental drug resistance has been conclusively established.28, 313, 314 The evidence supporting this role includes (a) a good correlation between the presence of this protein and a pattern of broad-spectrum drug resistance that includes the anthracyclines, vinca alkaloids, actinomycin D, and the epipodophyllotoxins315; (b) transfer of the cloned MDR1 gene for this protein that demonstrates the full phenotype of multidrug resistance, including resistance to doxorubicin316; and (c) the reversal of anthracycline resistance by a range of compounds, such as verapamil, cyclosporine A, calmodulin inhibitors, and tamoxifen, that block P170-mediated drug efflux by binding to this protein.317, 318, 319, 320, 321, 322 The genetic mechanism behind this increased expression in selected lines in vitro is variable. In some cell lines demonstrating very high levels of anthracycline resistance, the notable finding has been gene amplification, either present in double minutes or integrated within the chromosome as homogeneous staining regions; other lines show only increased messenger RNA coding for the P170 glycoprotein.323, 324 The nature of the resistance that develops after a single prolonged exposure to doxorubicin in a human sarcoma cell line was evaluated using classic fluctuation analysis; induction of MDR1 expression was not demonstrated, but rather resistance to doxorubicin arose from a spontaneous mutation with an apparent rate of approximately 2 × 10-6 per cell generation.325 It is also clear that the expression of the MDR1 gene may, under some circumstances, be transcriptionally modulated by doxorubicin itself as well as inhibitors of protein kinase C and calmodulin.254, 326, 327 For resistance that develops in vivo, the situation is more complex, with most tumors and many normal tissues exhibiting increased expression of a single gene copy.315
Although the physiologic role for this protein has not been established unequivocally, its expression has been documented in a range of normal tissues. Elevated expression is seen in colon mucosa, kidney, adrenal medulla, adrenal cortex, the blood-brain barrier, and many normal bone marrow elements.328,329, 330 In addition, expression in liver is increased after both partial hepatectomy and exposure to carcinogens such as 2-acetylaminofluorene.331 The combination of partial hepatectomy and carcinogen exposure is synergistic, resulting in over a 100-fold increase in expression. Based on this information, it has been postulated that the P170 glycoprotein is part of an integrated system for protecting cells against toxic xenobiotics.332, 333 Other components of this system include the mixed-function oxidases, glutathione transferases, glucuronyl transferases, glutathione, and glutathione peroxidase.
A wide range of human tumors has been examined before and after treatment with anthracyclines and other drugs that participate in the multidrug-resistance phenotype. Increased expression of the P170 glycoprotein is found before treatment in renal, colon, and adrenal carcinomas, some neuroblastomas and soft tissue sarcomas, and occasionally in tumors of lymphoid or myeloid origin. For patients with acute lymphocytic and myelocytic leukemias, expression of P170 glycoprotein carries an adverse prognosis.334, 335 P170 glycoprotein expression is rarely found at significant levels either before or after therapy in small cell carcinoma of the lung, but expression is clearly increased posttreatment in some patients failing primary therapy for leukemia, lymphoma, or myeloma.336, 337Expression in breast cancer is variable.338, 339 Clinical trials evaluating the effect of multidrug-reversal agents on the efficacy of anthracycline-containing chemotherapeutic programs have begun to be available.340, 341, 342 In general, the initial studies of this approach demonstrate that clinical strategies to overcome P170 glycoprotein–mediated drug resistance are most likely to be effective for patients with hematologic malignancies, that better reversing agents able to be administered at the appropriate dose with an acceptable toxicity profile are urgently needed, and that, not unexpectedly, the pharmacokinetics of the anthracyclines may be significantly altered by drugs such as cyclosporine A, which markedly decreases the clearance of doxorubicin and doxorubicinol.343Despite these difficulties, a recent large randomized trial for patients with AML demonstrated significantly improved relapse-free and overall survival when cyclosporine A was added to daunorubicin as part of a standard cytosine arabinoside and daunorubicin induction regimen.344 It remains to be determined whether these results reflect reversal of P-glycoprotein–mediated acquired resistance or are due to the increased levels of daunorubicin and its alcohol metabolite found in patients treated with cyclosporine A.
Multidrug Resistance Protein and Other ATP-Dependent Efflux Mechanisms
In some doxorubicin-resistant cell lines that exhibit decreased drug accumulation, P170 glycoprotein is not overexpressed, and verapamil produces quite variable alterations in resistance.345, 346 These results are explained by a unique, ATP-dependent efflux protein that was discovered in the doxorubicin-selected human small cell cancer line H69/AR. Its mRNA encodes a 190-kd protein that is a member of the ATP-binding cassette transmembrane transporter superfamily.32 The encoded glycoprotein has also been found in doxorubicin-resistant HL-60 leukemia cells and the doxorubicin-resistant HT1080/DR4 human sarcoma line, both of which lack P170 glycoprotein expression.33, 347 A wide variety of other experimental tumor cell systems have also been demonstrated to overexpress the multidrug resistance protein (MRP).348, 349, 350 The overexpression of MRP in drug-sensitive HeLa cells has been shown to produce resistance to doxorubicin, etoposide, and vincristine but not to cisplatin or traditional alkylating agents; thus, MRP expression alone, in the absence of alterations in MDR1 expression or levels of topoisomerase II, can produce anthracycline resistance. Furthermore, doxorubicin export by the MRP gene product is as efficient as that by the P-glycoprotein.351 In contrast to human sarcoma cells, doxorubicin selection of leukemic cells in vitro leads first to the overexpression of MRP at low drug concentrations, with the subsequent expression of the P170 glycoprotein.352 Furthermore, MRP may function coordinately with MDR1 to produce anthracycline resistance in patients with acute leukemia, suggesting that MRP may play an important role in the clinic.353, 354 These studies also suggest that critical tissue specificities may be involved in the evolution of the overexpression of different transport proteins.
MRP expression occurs widely in normal tissues except the liver and small intestine, where expression is limited.355 Studies utilizing MRP knockout models356have suggested a variety of physiological functions for the multidrug resistance protein. These include transport of heavy metals; modulation of ion channels; transport of glutathione conjugates, including leukotriene C4; and the cotransport of GSH with and without other xenobiotics such as the anthracyclines.35, 37Although transport of chemically prepared conjugates of doxorubicin or daunorubicin with glutathione by membrane vesicles from cells overexpressing MRP has been documented,37 the occurrence of such conjugates in vivo has not yet been observed. It is more likely that MRP plays a critical role in the cotransport of GSH with the anthracyclines as part of a series of related gene products rather than as a single GS-X pump.357
When additional multidrug-resistant tumor cell lines were discovered that relied on enhanced drug efflux but lacked either the P170 glycoprotein or overexpression of MRP, studies revealed a novel ATP-binding cassette transporter, initially in breast cancer cells resistant to the combination of doxorubicin and verapamil.358, 359 This transporter was initially named the breast cancer resistance protein (BCRP) because it was cloned from MCF-7 breast cancer cells. Doxorubicin can, at variable levels, be transported by BCRP; however, this depends on the presence of a specific mutation in BCRP at arginine 482 that increases drug efflux.360 BCRP more effectively transports mitoxantrone, camptothecin-related topoisomerase I inhibitors, and quinazoline ErbB1 inhibitors. The role of BCRP expression in the development of anthracycline resistance for patients with acute leukemia is under active investigation.361, 362
Other ATP-dependent doxorubicin efflux pumps have also been characterized.34 Furthermore, a 110-kd lung resistance-related protein has been identified as the major vault protein, a critical component of certain subcellular organelles39; this protein has been associated with an adverse prognosis in patients with AML treated with anthracycline-containing chemotherapy.38, 40
These more recent discoveries, taken together with the finding of an entire family of genes encoding multidrug resistence associated proteins (MRPs),357suggest the possibility that several distinct efflux mechanisms exist for the anthracyclines. This is entirely consistent with the redundant mechanisms present in mammalian cells to resist the toxicity of xenobiotics.
Altered Topoisomerase II Activity
Altered topoisomerase II activity has been implicated as a cause of resistance involving the anthracyclines. The resistance pattern of cells selected for topoisomerase II–mediated drug insensitivity may differ from the classic profile of MDR1 substrates. Nonetheless, it is clear that doxorubicin resistance in P388 and L1210 cells, MCF-7 breast cancer cells, and human small cell lung cancer and melanoma lines can be associated with reduced DNA topoisomerase II activity and drug-induced DNA cleavage.60, 363, 364, 365, 366, 367, 368 When tumor cells are selected for anthracycline resistance in the presence of the cyclosporine A analog PSC-833, which binds the P-glycoprotein, doxorubicin resistance develops in the context of significant reductions in topoisomerase IIα mRNA and protein as well as diminished catalytic activity without overexpression of MDR1, MRP, or the lung resistance-associated protein.369 It is relatively common for tumor cells selected with an anthracycline alone to exhibit both altered topoisomerase II activity and expression of the P170 glycoprotein or MRP.364, 368, 370 The mechanisms of decreased topoisomerase activity that have been described from in vitro studies include the presence of mutations in the topoisomerase IIα gene,63 decreased topoisomerase IIα gene copy number,61 and transcriptional down-regulation of topoisomerase gene expression.371Perhaps the most persuasive evidence that changes in topoisomerase II activity are causally related to doxorubicin resistance has been provided by studies that demonstrate reversal of resistance after transfer of a fully functional topoisomerase II gene into resistant cells.372 The importance of these observations for our understanding of anthracycline resistance at the clinical level remains to be elucidated. However, in a related clinical study, the activity of topoisomerase II in acute myelogenous leukemia cells varied over a more than 20-fold range, with significant cell-to-cell heterogeneity; there was no relationship between enzyme levels and drug sensitivity.373
Altered Free Radical Biochemistry, Sensitivity to Apoptosis, and Other Mechanisms of Anthracycline Resistance
The relationship between changes in intracellular free-radical detoxifying species and doxorubicin resistance has been reviewed above. It is sufficient here to emphasize that there are considerable, and probably tissue-specific, variations in the ability of cells to respond to a drug-induced free-radical challenge through enhanced antioxidant defense.374, 375
In addition to changes in drug export, topoisomerase II activity, or defenses against free radicals, it has recently been appreciated that other resistance mechanisms are at work to prevent doxorubicin-related cell death. Clearly, overexpression of bcl-2 can significantly diminish the toxicity of doxorubicin, as can mutations in p53.289, 304, 306 However, as described above, the varied downstream effectors of anthracycline-mediated programmed cell death may individually play critical roles in drug sensitivity beyond that produced by bcl-2 or p53 per se.257, 376, 377, 378 Furthermore, in light of the broad importance of these mediators of tumor cell killing for essentially all classes of antineoplastic agents,379 it is also likely that alterations in components of the cell death cascade play an important role in resistance to the anthracyclines acquired in the clinic prior to anthracycline administration.
Potent nuclear DNA repair systems also contribute substantially to the ability of tumor cells to withstand the cytotoxic effects of doxorubicin.380 Attention has focused on the loss of DNA mismatch repair genes, such as MLH1, in the production of the doxorubicin-resistant phenotype, although the pathways to cell death that are interfered with remain unclear at present.381, 382, 383, 384 Mutations producing decreased levels of poly(ADP-ribose)polymerase in V79 cells also dramatically decrease the efficacy of doxorubicin.385 Since ADP-ribosylation is a well-known posttranslational modification of topoisomerase II and plays an important role in NAD+ utilization, these results suggest that critical aspects of intermediary metabolism may modify the relationship between DNA cleavage reactions and tumor cell killing.386, 387, 388 Inhibitors of this enzyme are also capable of producing doxorubicin resistance in human tumor cells.389, 390However, resistance patterns apparently mediated by alterations in ADP-ribosylation may, in fact, represent partial blockade of certain downstream effectors of the apoptotic cascade, since poly(ADP-ribose)polymerase is a critical substrate for the caspases.258
Additional mechanisms of DNA repair of relevance to the pharmacology of the anthracycline antibiotics are the activities of the hereditary breast cancer susceptibility genes BRCA1 and 2 that participate in a common DNA damage response pathway.391 BRCA1 and 2 are initially up-regulated, then depleted by exposure to doxorubicin in p53-competent cells.392, 393 It has been suggested that a deficiency in BRCA1 may sensitize cells to killing by doxorubicin; however, this has not been universally observed.394, 395 The level of BRCA1 mRNA expression may also provide predictive information regarding overall therapeutic efficacy in women treated with an anthracycline-containing chemotherapeutic program.396
Very few drug interactions have been documented for the anthracyclines. Heparin, a large polyanion, binds to the aminosugar of doxorubicin and daunorubicin, creating insoluble aggregates. Coadministration of heparin and doxorubicin can lead to an increase in the rate of doxorubicin clearance. In rodents, phenobarbital has been shown to increase, and morphine decrease, doxorubicin disappearance.397, 398
Doxorubicin and daunorubicin can cause radiosensitization of normal tissues and subsequent radiation recall. The most significant aspect of this problem occurs with the heart. A cardiac radiation exposure of 2,000 cGy given in conventional 200-cGy per day fractionation results in a doubling of the rate at which cardiac toxicity develops so that a cumulative doxorubicin dose of 250 mg/m2 is equivalent to a dose of 500 mg/m2 in the absence of radiation. This is not an uncommon problem, in that doxorubicin is used for first-line therapy of breast cancer and Hodgkin's disease, for which mediastinal or chest wall radiation therapy is also often used. Recent radiation techniques have reduced the cardiac radiation dose that occurs during treatment following breast-conserving surgery, which lessens this risk somewhat.
Recently, the disposition of doxorubicin was found to be significantly altered when it was administered immediately after a short i.v. infusion of paclitaxel.399This is due to the presence of high levels of Cremophor EL, the diluent in which paclitaxel is prepared, in the plasma after paclitaxel administration. Cremophor EL is a substrate for the P- glycoprotein and can significantly affect the biliary excretion of doxorubicin.400 This interaction is not observed if paclitaxel is given over 24 hours (which lowers the concentration of the diluent in plasma) or if docetaxel, rather than paclitaxel, is combined with doxorubicin, since the former taxane is not prepared in Cremophor EL.
Dose and Schedule of Administration
Doxorubicin has been successfully administered using a wide range of schedules, and at present there is little evidence that changes in schedule make any significant difference in antitumor activity. As mentioned earlier, the antitumor activity of doxorubicin is proportional to the area under the curve (AUC), not to peak drug levels. Thus, a dose of 60 mg/m2 is approximately equally effective administered as a bolus or infused over 1, 2, or 4 days. Myelosuppression is also proportional to AUC and changes very little over this broad range of schedules. The most common schedule has been 45 to 60 mg/m2 every 18 to 21 days. Because of evidence that peak level correlates with cardiac toxicity, weekly dosing at 20 to 30 mg/m2 has also been evaluated and seems to be both less cardiotoxic and approximately as effective as bolus dosing. This trend has been extended to the administration of a 96-hour infusion that is convincingly less cardiotoxic while preserving antitumor activity.401 As an added benefit, prolonged infusions dramatically lessen the nausea and vomiting associated with bolus administration of doxorubicin. The only major negative aspect of infusional doxorubicin is a tendency for mucositis to increase in intensity as the infusion is prolonged. Daunorubicin is usually administered as a brief intravenous infusion in doses of 30 to 45 mg/m2 daily for 3 days as induction therapy for AML.
Pharmacokinetics and Metabolism
The basic pharmacokinetic constants for doxorubicin and daunorubicin are listed in Table 18.1. The pharmacokinetics of these drugs are dominated by tissue binding. During the early distributive phase, drug levels fall rapidly as the drug gains ready access to all tissues of the body except the brain. During this phase, the bulk of the drug binds to DNA throughout the body, and in general tissue levels of the drug are proportional to their DNA content.402 In addition, plasma protein binding accounts for approximately 75% of the drug in the plasma.403 In spite of this plasma protein binding, tissue-plasma ratios range from 10:1 to 500:1 by virtue of the higher affinity of the drugs for DNA than for plasma. Tumor levels have rarely been measured in humans, but multiple myeloma patients were studied during a 96-hour infusion of doxorubicin. Doxorubicin levels of about 10 µM were documented in myeloma cells by the end of the infusion.404 However, with the extensive binding of these drugs to DNA and proteins, the free-drug pool probably represents a very small fraction of the drug concentrations measured in both plasma and cells. Unfortunately, there have been no detailed studies of the pharmacokinetics of this free-drug pool.
After bolus administration, or after the conclusion of a constant i.v. infusion, an initial doxorubicin half-life of 10 minutes is followed by a secondary half-life of 1 to 3 hours. The terminal half-life of 30 to 50 hours accounts for over 70% of the total drug AUC for doxorubicin. As a result of this prolonged terminal phase, plasma levels of drug remain above 10 nM for the greater part of a week after a single dose of 60 mg/m2 of doxorubicin.403 In tissue culture, levels as low as 1 to 5 nM are cytotoxic for sensitive tumor cells after extended exposures. Even accounting for the difference in protein content between tissue culture media and plasma, these results suggest that drug levels sufficient for tumor cell kill may persist for prolonged periods. Doxorubicinol is the primary metabolite of doxorubicin in human plasma but is present in concentrations far smaller than those of the parent compound. Approximately 50% of the drug is excreted in the bile, both as parent drug and as various metabolites, including glucuronides and sulfates. Less than 10% of the administered drug appears in the urine; however, this is sufficient to cause a reddish-orange discoloration of the urine in many patients. When administered at higher than standard dose levels (>100 mg/m2), peak plasma levels of doxorubicin may reach 6-7 µM. When administered as a continuous i.v. infusion at high dose (150 to 165 mg/m2), steady state doxorubicin concentrations are approximately 0.1 µM.405 The relationship of AUC to dose appears to be linear up to a doxorubicin dose level of 165 mg/m2 whether the drug is administered as a bolus or as a continuous infusion.405, 406
Pharmacokinetic studies of gender and body surface area407, 408 revealed, unexpectedly, that men with normal hepatic function were found to have approximately twice the clearance of doxorubicin (administered as an i.v. bolus) as compared with women; higher drug clearance was associated with an increased conversion rate of doxorubicin to its major alcohol metabolite. The pharmacodynamic implications of these findings are uncertain. The pharmacokinetics of the anthracycline analog epirubicin is independent of body surface area; normalization of epirubicin dose based on surface area has no effect on the drug's observed AUC. Although neutropenia correlated well with epirubicin AUC when the drug was administered as a single agent, the absence of an effect of body surface area normalization on systemic exposure suggests that this procedure neither reduces interpatient variability in anthracycline pharmacokinetics nor variability in hematopoietic toxicity following epirubicin treatment. Drug administration at fixed milligram increments for this drug would appear to be rational, safe, and more efficient than current standard practice.
For daunorubicin, metabolism to daunorubicinol is a major determinant of its plasma pharmacology. The parent drug is cleared rapidly from plasma, with a primary half-life of 40 minutes; the loss of parent drug from plasma correlates with the rapid appearance of C4-O-demethyl daunorubicin, daunorubicinol, their aglycones, and various sulfate and glucuronide metabolites in bile. Within hours after a bolus dose of daunorubicin, the predominant circulating form of the drug is the alcohol metabolite,409 which has a longer half-life (23 to 40 hours) than its parent. The opposite is the case for doxorubicin, where doxorubicinol is typically a minor part of the total AUC.410 The formation of either doxorubicinol or daunorubicinol is a function of the enzymatic conversion of the side-chain carbonyl to an alcohol by one or more enzymes in the hepatic aldoketoreductase family.411, 412, 413, 414, 415
Over the years, there has been considerable controversy as to the importance of the aglycone metabolites of the anthracyclines. There are two families of aglycones to be considered, the 7-deoxy and 7-hydroxy aglycones. As mentioned earlier, the 7-deoxyaglycones are the result of a two-electron reduction of the parent drug to a quinone methide, with subsequent elimination of the sugar. It is important to emphasize that the full two-electron reduction of the anthracyclines to the 7-deoxyaglycone stage occurs stepwise, after initial one-electron reduction by microsomal or mitochondrial flavin-containing enzymes. Thus, the demonstration of 7-deoxy by-products has been argued to be de facto evidence of an anthracycline redox cycle. The 7-hydroxyaglycones result from hydrolysis of the sugar-anthraquinone bond. The latter can arise artifactually during the processing and analysis of the drug. This has especially been a problem with older techniques that depend upon thin-layer chromatography. In the past, many investigators seem to have been confused about the distinction between these two aglycone families, and thus the importance of the 7-deoxyaglycones was dismissed by some. It is now clear that the 7-deoxyaglycones are indeed circulating metabolites of both daunorubicin and doxorubicin. In addition, the formation of the 7-deoxyaglycones exhibits large patient-to-patient variability. 7-Deoxydoxorubicin can range from 1 to 5% of the total drug. In contrast, 7-deoxydoxorubicinol aglycone can range from 0 to 20%.215
Doxorubicinol and daunorubicinol are cytotoxic metabolites but are considerably less active than the parent compound.416 As mentioned earlier, deoxyaglycones are much less active than the corresponding parent drug and are currently viewed as a pathway of drug inactivation. In one study, aglycone levels in patients with acute myelogenous leukemia were statistically significantly higher in nonresponders than in responders.417
Although the bulk of doxorubicin elimination occurs by hepatic metabolism and biliary excretion, the evidence that doses of doxorubicin or daunorubicin must be reduced in patients with compromised hepatic function is somewhat difficult to interpret.418, 419, 420, 421 Altered patterns of metabolism have been observed in individual patients, with prolonged terminal half-life of parent drug, and decreased clearance of doxorubicinol. Patients with abnormal liver function also have a diminished capacity to clear doxorubicin when it is administered as an infusion or a bolus.421, 422 However, no consistent pattern of increased toxicity has emerged in patients with mildly decreased drug clearance. Moderate to severe alterations in hepatic function, however, increase AUC significantly.423Physicians are advised to reduce doses routinely in patients with moderate or severe hepatic dysfunction or when marked replacement of the liver by tumor is present, in which case all forms of chemotherapy carry increased risk.
Table 18.1 provides a summary of the common toxicities of the anthracyclines.
Bone Marrow Suppression and Mucositis
Bone marrow suppression and mucositis are common to other anticancer drugs, and there is nothing unusual about these toxic reactions after anthracycline administration. Both myelosuppression and mucositis follow an acute course, with maximal toxicity within 7 to 10 days of drug administration and rapid recovery thereafter. For daunorubicin, bone marrow suppression is more common than mucositis and is the usual dose-limiting toxicity. Doxorubicin causes these two reactions in more equal severity after bolus dose administration. With weekly dosing or continuous infusion, mucositis frequently becomes the dose-limiting toxicity.
Extravasation of most anthracyclines leads to severe local injury that can continue to progress over weeks to months. The drug has been shown to bind locally to tissues and can still be detected at high levels at the base of a drug-induced ulcer in the soft tissues of the hand or forearm months later.424, 425, 426 These lesions are very difficult to treat. Skin grafting is usually not successful unless preceded by extensive excision of the involved tissue. However, debridement of dead tissue should be undertaken with extreme caution during the initial phases of extravasation injury, and local wound care to prevent infection is most important.427 A wide range of treatments have been used immediately after extravasation in an attempt to lessen the injury. These have included ice, steroids, vitamin E, DMSO, and bicarbonate.428, 429 More recently, the cardioprotectant dexrazoxane has been used to treat acute anthracycline extravasations in combination with subcutaneous granulocyte-macrophage colony-stimulating factor to promote wound healing.430
The cardiac toxicity exhibited by doxorubicin and the other anthracyclines is unique in its pathology and mechanism.431, 432 Although the major limiting factors in the clinical use of anthracyclines in adults are bone marrow suppression, mucositis, and drug resistance on the part of the tumor, for individual patients, most commonly with the use of doxorubicin in breast cancer, cardiac toxicity can develop while the patient's tumor is still responsive to the drug. This is a problem not only for the use of the anthracyclines alone or in combination with other chemotherapeutic agents but also with the monoclonal antibody trastuzumab. Trials have demonstrated synergistic cardiac toxicity for the combination of doxorubicin and trastuzumab,433 an antibody directed against the HER2/neu oncoprotein, which is itself active in the treatment of advanced breast cancer.434 The observed potentiation of anthracycline-induced heart damage by trastuzumab has eliminated its concurrent use with doxorubicin in the population of patients whose tumors exhibit high levels of HER2/neu expression, a group that could benefit most from this combination.435 Finally, children seem to be more sensitive to the cardiac toxicity of this drug, and this has become a significant problem in the use of doxorubicin in pediatric oncology.436, 437, 438, 439 Advances in our understanding of the impact of schedule on the cardiac toxicity of these drugs and the development of successful antidotal agents may reduce the importance of this problem in the future.
Clinical Presentation and Management
The anthracyclines manifest both acute and chronic cardiac toxicity. Acute toxicity is detected most commonly as a range of arrhythmias, including heart block. In its more extreme form, this acute injury can include a pericarditis-myocarditis syndrome with onset of fever, pericarditis, and congestive heart failure.440 This syndrome can occur at low cumulative doses of doxorubicin and can have a fatal outcome. In animal models, doxorubicin administration causes significant increases in circulating catecholamines and histamine, and coadministration of α- and β-adrenergic antagonists along with H1 and H2 blockers has lessened acute and subacute doxorubicin toxicity.441 Clinical trials have not been conducted to see if such treatment might be effective in the management of the acute toxicity of doxorubicin in humans, perhaps because this syndrome is relatively uncommon and idiosyncratic. There has been no clear correlation between the manifestation of arrhythmias and the development of the chronic cardiomyopathy. There is essentially no experience with re-treating patients who have survived the pericarditis-myocarditis syndrome with doxorubicin or daunorubicin.
Cardiac toxicity has been best documented for doxorubicin administered as a bolus dose of 45 to 60 mg/m2 every 21 to 28 days. With this schedule, cardiac toxicity develops as a result of cumulative injury to the myocardium. The pathology of this toxicity, determined after endomyocardial biopsy, has been described in detail; a useful grading system correlates well with the risk of clinical cardiac toxicity442, 443 (Table 18.2). These studies have shown that with each dose of doxorubicin there is progressive injury to the myocardium so that the grade increases steadily with total dose of drug administered. The major changes observed in myocytes are dilation of the sarcoplasmic reticulum and disruption of myofibrils. Early in the development of this toxicity, these changes appear focally in scattered myocytes surrounded by normal-appearing cells. As the toxicity progresses, the frequency of these altered cells increases until a significant proportion of the myocardium is involved. Late in the development of this toxicity, the picture is complicated by the development of diffuse myocardial fibrosis. This pathology is unique to the anthracyclines and allows the pathologist definitely to distinguish this cardiac toxicity from other processes such as viral cardiomyopathy or ischemic heart injury. In addition, Billingham's grading system has allowed correlation of the findings among studies done at a range of institutions and has made possible the advances that have now significantly lessened the risk of this problem.
TABLE 18.2 CRITERIA FOR GRADING ANTHRACYCLINE CARDIOMYOPATHY
The clinical risk of congestive heart failure is low at total doses below 250 to 300 mg/m2 of doxorubicin or 600 to 700 mg/m2 of daunorubicin,444, 445 although cases of fatal congestive cardiomyopathy have been observed after a single dose of doxorubicin. Above these doses, the risk steadily accelerates. The total dose limit at which the risk becomes unacceptable is largely arbitrary, and as discussed above, the risk may in part be dependent on the treatment schedule used. For doxorubicin, the most commonly used total dose limit applied in the past has been 450 to 500 mg/m2, at which the risk of clinically evident cardiac toxicity has generally been believed to be 1 to 10% (Figure 18.7). The corresponding limit applied for daunorubicin has been 900 to 1,000 mg/m2. However, recent large trials that have prospectively evaluated heart function with radionuclide-gated cardiac blood pool scans strongly suggest that subclinical, but not inconsequential, reductions in ejection fraction can be detected routinely after 250 to 300 mg/m2 of doxorubicin.446, 447, 448 Furthermore, changes in ejection fraction may or may not predict the development of heart failure in a specific patient. As mentioned below, certain patients, especially those with breast cancer, may approach this total dose limit with a tumor that is still responsive to doxorubicin when there are few other therapeutic options. It has been established that changing from bolus to 96-hour infusion or the addition of dexrazoxane after a cumulative doxorubicin dose level of 300 mg/m2 will allow a substantial duration of additional anthracycline therapy with a significantly reduced risk of severe cardiac toxicity.3, 449, 450
Figure 18.7 Incidence of congestive heart failure as a function of cumulative dose (in mg/m2) of either daunomycin (▪) or doxorubicin ([dot in square]). Daunomycin is a much less potent cardiotoxin than doxorubicin. (Redrawn from data presented in references 400 and 401.)
It has often been said that doxorubicin-induced cardiac toxicity is difficult to treat and is associated with a high mortality.451 While doxorubicin-induced congestive heart failure may have a fatal outcome, it is eminently treatable with standard measures, and many patients (probably well over half) recover or stabilize at a lower, but clinically acceptable, level of cardiac function.451, 452 It is important to point out, however, that congestive heart failure may occur many months after the discontinuation of doxorubicin and that patients who have been stabilized with adequate medical management are at increased risk during subsequent intercurrent illnesses.451, 453 Children are clearly at risk for developing congestive heart failure many years after discontinuing doxorubicin even if treated long term with an angiotensin-converting enzyme inhibitor.454
There are now a number of techniques available to detect the cardiac toxicity early (Table 18.3). Endomyocardial biopsy provides a definitive assessment of risk. However, this may not be available to the clinician. Ejection fraction measurement by electrocardiogram (ECG)–gated radioisotopic cardiac blood pool scan has been shown to provide an accurate measure of cardiac contractility.455, 456 A significant drop in contractility by this technique is usually seen before the onset of congestive heart failure, but this may not be the case in an individual patient.457 Because of these techniques, clinically significant cardiac toxicity is now detected much earlier, and this has done much to lessen the mortality of this complication. Medical management centers around afterload reduction. With conservative treatment, many patients experience gradual improvement in function, and a few patients can have a rather dramatic return of exercise tolerance. This improvement can, however, take more than 1 year.
Lessening the Risk of Cardiac Toxicity
Fortunately, much can now be done to lessen the risk of cardiac toxicity. First, patient characteristics associated with increased risk have been identified. Hypertension and preexisting cardiac disease predisposing to diastolic dysfunction significantly increase the risk that a patient will develop clinically apparent cardiac abnormalities at a lower cumulative drug dose. Cardiac radiation exposure clearly increases the sensitivity of the heart to anthracyclines; at a radiation dose of 2,000 cGy, the slope of the cardiac biopsy score versus the cumulative doxorubicin dose doubles so that a dose of 250 mg/m2 becomes equal to a dose of 500 mg/m2 in the absence of radiation.458 Modern advances in computer-based radiation treatment planning should be used to minimize cardiac radiation exposure in patients with breast cancer and lymphoma who will receive an anthracycline.
TABLE 18.3 PHYSIOLOGIC TESTS OF CARDIAC FUNCTION
It is now clear that the risk of cardiac toxicity from doxorubicin is a function of peak drug level, not AUC.401 In contrast, both in vitro and in patients, the antitumor activity of doxorubicin is a function of AUC, not peak drug level. Thus, shifting from bolus drug administration to weekly dosing or prolonged infusion results in a significant reduction in the incidence of cardiac toxicity.459 In clinical settings where cardiac toxicity has proved to be a serious problem, such as in breast cancer or in the pediatric malignancies, where the incidence of cardiac dysfunction is higher and the late consequences more profound,460consideration should be given to the use of prolonged intravenous infusions or the cardioprotective agent dexrazoxane. However, in the pediatric cancer patient population, the use of dexrazoxane appears to be more effective than 48-hour doxorubicin infusions in lessening anthracycline cardiotoxicity.461, 462
Dexrazoxane is the first agent that has shown consistent ability to block the development of anthracycline-induced cardiac toxicity in a wide range of animal models. Randomized, controlled clinical trials have proven that this agent dramatically reduces the incidence of cardiac toxicity in patients with breast cancer without significantly altering the antitumor activity of anthracycline-containing combinations.206, 449, 450, 463, 464 In the first such study, 92 patients with advanced breast cancer received either 5-fluorouracil, doxorubicin, and cyclophosphamide or the same regimen plus dexrazoxane. The latter was given in doses of 1,000 mg/m2 by intravenous infusion 30 minutes before the chemotherapeutic drugs. Patients receiving dexrazoxane had an equivalent response rate and duration of time to disease progression as those not receiving dexrazoxane. However, the dexrazoxane-treated patients had significantly smaller decreases in left ventricular ejection fraction at each dose level of doxorubicin, their cardiac biopsies reflected less histologic change, and 11 patients treated with dexrazoxane tolerated doxorubicin doses above 600 mg/m2 while only 1 patient not receiving dexrazoxane remained on study above this dose level. The only negative aspect of this trial was a modest increase in myelosuppression in the arm receiving dexrazoxane. These results have been confirmed in larger trials with both doxorubicin and its analog epirubicin and have led to the approval of dexrazoxane by the US Food and Drug Administration as a cardioprotectant in patients receiving >300 mg/m2 of doxorubicin. This approach also appears to be applicable to pediatric patients with sarcomas receiving doxorubicin.438, 461 To maintain its cardioprotective properties while reducing drug-related granulocytopenia, the currently recommended dose of dexrazoxane is 10 times the doxorubicin dose on a milligram per milligram basis administered no more than 30 minutes before the anthracycline infusion is initiated.
Putative Biochemical Mechanisms of Anthracycline Cardiac Toxicity
In addition to the pathologic picture outlined above, any hypothesis that seeks to explain the cardiac toxicity of the anthracyclines must also account for the alterations in cardiac biochemistry that occur after doxorubicin exposure. The consistent changes observed involve marked alterations in calcium handling in the heart muscle and include loss of high-affinity calcium-binding sites, elevation of cardiac calcium content, and mitochondrial accumulation of calcium.465,466, 467, 468, 469 The other alteration frequently described is a diminished capacity for ATP generation. In terms of muscle physiology, these changes are critical. Calcium plays a central role in linking electrical excitation with contraction; each cycle of muscle contraction is triggered by a rapid rise in free intracellular calcium, and relaxation is dependent on a rapid drop in free calcium. In addition, calcium has been shown to play a major role in regulating the beat-to-beat force of cardiac muscle contraction. The two major sites for the beat-to-beat regulation of calcium are the sarcoplasmic reticulum and mitochondria. Sarcoplasmic reticulum avidly binds calcium that is rapidly released when a wave of electrical depolarization sweeps through the sarcoplasmic membrane. Because extensions of the sarcoplasmic reticulum are in intimate contact with the contractile fibers, sarcoplasmic depolarization leads to rapid onset of muscle contraction. The cardiac mitochondria will accumulate calcium if it is available in preference to making ATP. In general, no mechanism has been offered for a direct interaction of doxorubicin with calcium. Since anthracyclines are good metal chelators, one possibility is that the drugs chelate calcium and as a result alter the distribution of this metal ion. However, doxorubicin does not chelate calcium within the physiologic concentration range for calcium. The pathology of anthracycline cardiac toxicity suggests another, more reasonable hypothesis: the major site of anatomical damage after drug exposure is the sarcoplasmic reticulum, a major site of calcium regulation. Doxorubicin injury to the sarcoplasmic reticulum leads to calcium release.470Calcium is then taken up by the mitochondria, which do that in preference to ATP generation. This sequence would account for the lower ATP levels and the accumulation of calcium within the mitochondria.
TABLE 18.4 MECHANISMS OF ANTHRACYCLINE CARDIAC TOXICITY
How do the anthracyclines trigger damage to the sarcoplasmic reticulum? The hypothesis that best explains the above phenomenon is that the cardiac toxicity of the anthracyclines results from drug-induced free-radical formation (Table 18.4). Within the heart muscle there are several sites where enzyme activity is capable of reducing doxorubicin to the corresponding semiquinone; doxorubicin-stimulated oxygen radical formation by cardiac sarcoplasmic reticulum, cytosol, and mitochondria has been conclusively demonstrated.471, 472 The non-redox-active anthracycline 5-iminodaunorubicin does not generate reactive oxygen species in the sarcoplasmic reticulum or mitochondria and is markedly less cardiotoxic than its parent molecule.473 Doxorubicin can also induce peroxidation of the sarcoplasmic reticulum lipid and oxidant-related sulfhydryl loss; progression of this oxidative damage to the membrane is associated with a drop in both high-affinity calcium binding and the force of contraction.129, 465 Recent studies demonstrate, furthermore, that redox cycling of the doxorubicin quinone selectively inhibits critical hyperreactive sulfhydryl groups on the ryanodine-sensitive calcium channel of the sarcoplasmic reticulum, resulting in enhanced channel activation and subsequent alterations in calcium homeostasis.474 These observations show that doxorubicin is reduced to a semiquinone at the sarcoplasmic reticulum and that this leads to oxidative damage to the sarcoplasmic membrane, with subsequent loss of the capacity of this membrane to bind calcium, thus disrupting the linkage between electrical excitation and contraction. To confirm that oxygen radicals are indeed formed in the heart after doxorubicin exposure, isolated perfused beating rat hearts have been exposed to doxorubicin, and electron spin resonance has been used to detect hydroxyl radicals. In this setting, hydroxyl radicals could easily be detected after exposure of the heart to drug levels (1 µM) attained following bolus dosing at 60 mg/m2 but not at concentrations obtained during a 96-hour infusion (0.04 to 0.1 µM).150 Thus, it seems clear that doxorubicin can trigger the formation of reactive oxygen species in vivo and that this occurs at concentrations that are associated with the development of cardiac toxicity.
As outlined in Table 18.4, the anthracyclines can produce a wide variety of toxic effects in the heart, some of which may contribute to their clinical cardiac toxicity.475, 476 Unfortunately, many of the effects described occur at unrealistically high drug concentrations and do not help to explain the specificity of anthracycline cardiac damage. These considerations do not apply to studies that demonstrate specific down-regulation of cardiac α-actin and troponin I mRNAs477 in a fashion that is not inhibited by free-radical scavengers.478 Taken together with studies that have examined the effect of doxorubicin on the activation of other cardiac-specific genes and regulatory pathways,479, 480 it seems likely that the pathogenesis of anthracycline cardiac toxicity and its morphological expression may be understood more clearly in the future at the transcriptional level.
Why is the heart, and not other tissues, a target for this free-radical damage? Several factors are probably involved. First, cardiac tissue has very low levels of catalase activity; overexpression of catalase in the hearts of transgenic mice reduces the cardiac toxicity of doxorubicin.148 This leaves glutathione peroxidase as the only known pathway for hydrogen peroxide detoxification in the heart. However, doxorubicin administration can produce a rapid drop in glutathione peroxidase activity. Thus, at a time when doxorubicin is stimulating the formation of hydrogen and lipid peroxides, it is also eliminating the major pathway for peroxide removal. This observation suggests that limitations in the ability of the heart enzymatically to detoxify oxygen radicals provide an important basis for it sensitivity to doxorubicin.481 This hypothesis has received support from an unusual experiment. Prolonged exercise causes a marked increase in the activities of superoxide dismutase and glutathione peroxidase in rodents; these mice are then more resistant to the cardiac toxicity of doxorubicin.482 It is also highly likely that the robust affinity of the anthracyclines for mitochondrial lipids483 enhances drug binding in a site-specific manner that markedly increases drug-related cardiac mitochondrial reactive oxygen production.484, 485, 486 Furthermore, the heart is extraordinarily rich in iron proteins that are capable of donating their metal to catalyze strong oxidant formation.
In summary, the free-radical hypothesis has been very effective in accounting for the various characteristics of anthracycline cardiomyopathy. In addition, this hypothesis has led to the identification of an agent that is successful at dramatically reducing the cardiac toxicity of doxorubicin in humans without compromising the antitumor efficacy of this valuable anticancer agent.
A large number of doxorubicin analogs have been brought to clinical trial in the hope of finding a compound with less cardiac toxicity and a broader spectrum of antitumor action.487, 488 The most promising of these analogs are (a) idarubicin, an agent with marked activity in acute nonlymphocytic leukemia and acute lymphocytic leukemia, and (b) epirubicin, which has activity in breast cancer. The important features of these two agents, with relevant references, are given in Table 18.5.80, 127, 346, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506
In the search for analogs of the anthracyclines, a variety of multiringed planar structures with the potential for DNA intercalation have been evaluated for antitumor activity. A promising related class of compounds, the anthracenediones, were synthesized by chemists at American Cyanamid Laboratories507 in the late 1970s and were found to have potent antitumor activity against the P388 and L1210 leukemias. The most active of this series tested was mitoxantrone (dihydroanthracenedione), a planar tetracyclic compound having two symmetrical aminoalkyl side arms but no glycosidic substituent as found in the active anthracyclines (Fig. 18.8). Against P388, it is one of the most active agents tested, yielding a 500% increase in life span and a high percentage of cures.508Subsequent preclinical and clinical evaluation has demonstrated significant differences between this agent and the anthracyclines in terms of mechanism of action, the lesser cardiac toxicity of the anthracenediones, and their diminished potential for extravasation injury and for causing nausea and vomiting or alopecia. Their narrow spectrum of antitumor activity, confined to breast and prostate cancer and the leukemias and lymphomas, has limited the opportunity to replace doxorubicin with mitoxantrone in clinical practice. However, because of the favorable toxicity profile of mitoxantrone, it is an appropriate agent for use in an elderly patient population, such as men with hormone-refractory prostate cancer, where treatment can provide significant palliative benefit.509, 510
TABLE 18.5 KEY FEATURES OF ANTHRACYCLINE ANALOGS
Mechanism of Action
Like the anthracyclines, mitoxantrone binds avidly to nucleic acids and inhibits DNA and RNA synthesis. Its mode of binding to DNA includes intercalation between opposing DNA strands, with preference for GC base pairs.511 Careful studies of the stoichiometry of binding and electron microscopic evaluation of the distortions produced in vitro in plasmid DNA indicate an additional type of binding that produces a compaction of chromatin512 and, with plasmid DNA, lacelike intertwining of the DNA strands. These effects are dependent on the presence of the highly positively charged aminoalkyl side chains and probably represent electrostatic cross-linking of DNA strands. Also found are single- and double-strand breaks in DNA.513 Because the drug has the basic quinone structure found in the anthracyclines, its ability to generate free radicals in a manner similar to that of doxorubicin has been examined. These studies revealed that the drug has a much reduced potential to undergo one-electron reduction, compared to doxorubicin,514, 515 and is less readily reduced enzymatically.516Since some of the single-strand breaks are protein-associated, it appears that these breaks result from the formation of a cleavable complex with topoisomerase II, which occurs in mitoxantrone-treated cells.517 This possibility is heightened by the finding that there is little evidence of lipid peroxidation in cardiac tissue, modest stimulation of oxygen consumption in vitro, and, indeed, inhibition of doxorubicin-induced lipid peroxidation by mitoxantrone;518 all of these findings argue against a free-radical mechanism of tissue injury by mitoxantrone and favor enzyme-mediated DNA cleavage. The reduced potential for free-radical formation may also explain the lesser cardiotoxicity of mitoxantrone, although this drug is able to oxidize critical sulfhydryl groups on the ryanodine receptor of the sarcoplasmic reticulum.475, 476, 519 As is the case for the anthracyclines, mitoxantrone can also readily stimulate apoptosis in a variety of cell lines.520, 521 Ceramide-dependent pathways have been implicated as part of the molecular ordering of mitoxantrone-induced programmed cell death.248
Figure 18.8 Structure of mitoxantrone.
Mechanisms of Drug Resistance
As a planar anthraquinone analog, it is not surprising that mitoxantrone shares cross-resistance with many of the natural products, including the vinca alkaloids and doxorubicin.522, 523, 524 This resistance may be mediated by amplification of the P170 glycoprotein (classic MDR1); however, in some cell lines, decreased intracellular drug accumulation is related to the overexpression of the multidrug resistance protein (MRP).525,526 Alterations in topoisomerase II function have also been well described as a mechanism of mitoxantrone resistance.527 In fact, there are now clear examples in which tumor cells develop pleiotropic resistance based both on enhanced efflux and altered topoisomerase function.528 Recent studies have also clarified a series of prior observations suggesting that mitoxantrone resistance in vitro could occur in the absence of alterations in topoisomerase II or enhanced expression of MDR1 or MRP.359, 523, 529, 530 These investigations have identified a novel member of the ATP-binding cassette superfamily of transporters that encodes a 655–amino acid protein (termed the breast cancer resistance protein) that is capable of enhancing the efflux of mitoxantrone and the anthracyclines from mitoxantrone-selected tumor cell lines. Additional mechanisms of mitoxantrone resistance have been related to altered intracellular pH in tumor cells531 and to modifications in the cellular apoptotic program.
Mitoxantrone is frequently used in combination with arabinosylcytosine in the treatment of acute nonlymphocytic leukemia, and there is evidence for biochemical synergy of the two agents. In studies of leukemic cells taken from patients during therapy, coadministration of mitoxantrone and arabinosylcytosine enhanced the accumulation of araCTP in leukemic blast cells.532 In the same study, mitoxantrone alone produced no detectable single-strand breaks but in combination with arabinosylcytosine induced easily detectable single-strand breaks as determined by alkaline elution of blast cell DNA. The molecular basis for these favorable interactions is not understood. Like doxorubicin, mitoxantrone sensitizes cells to both hyperthermia and ionizing radiation.533
The recommended dosage for bolus intravenous administration of mitoxantrone is 12 mg/m2 per day for 3 days for treatment of AML and 12 to 14 mg/m2 per day once every 3 weeks for patients with solid tumors. The drug has definitely established activity against breast cancer,534 ovarian cancer,535 non-Hodgkin's lymphoma,536 and prostate cancer509 as well as against acute leukemia.537 The drug is administered as a 30-minute infusion and rarely causes extravasation injury if infiltrated. Mitoxantrone should not be administered in solutions containing heparin.
Mitoxantrone can be measured in plasma and urine by HPLC.538, 539 The plasma disappearance of mitoxantrone is characterized by a rapid preliminary phase of clearance, with half-lives of approximately 10 minutes(t1/2α) and 1.1 to 1.6 hours (t1/2β)538, 539 followed by a long terminal half-life of 23 to 42 hours. During this final phase of drug disappearance, the drug concentration in plasma approximates 1 ng/mL (or 2 nM), a level at the margin of cytotoxicity. The pharmacokinetics of mitoxantrone are linear over the dose range from 8 to 14 mg/m2 administered as a short infusion.540 Less than 30% of the drug can be accounted for by the fraction of drug that appears in the urine (less than 10%) or the stool (less than 20%). Like doxorubicin, the drug distributes in high concentrations into tissues (liver > bone marrow > heart > lung > kidney) and remains in these sites for weeks after therapy.538 Although specific guidelines are not available for dose adjustment in patients with hepatic dysfunction, several authors have noted a prolongation of the terminal half-life to >60 hours in patients with liver impairment.539, 541
The specific metabolites of mitoxantrone have not been well characterized.542 The side chains undergo oxidation, yielding the mono- and dicarboxylic acids of anthracenedione, and both have been recovered from urine.543 Neither has antitumor activity.
As an alternative to intravenous infusion, mitoxantrone has been administered by hepatic intra-arterial infusion544 and by intraperitoneal instillation.545, 546These trials were based on the observation that mitoxantrone has a steep dose-response curve in vitro and that optimal concentrations of drug (1 to 10 µg/mL) are achieved only briefly during standard intravenous therapy. Local concentrations much higher than those realized in systemic administration can be achieved by either the intra-arterial or intraperitoneal routes. During intraperitoneal trials, patients with ovarian or colon cancer received 12 to 38 mg/m2 as a single dose every 4 weeks in 2 liters of dialysate. A 1,400-fold advantage was found for intraperitoneal drug concentrations over simultaneous plasma levels. The terminal half-life for disappearance of drug from the intraperitoneal space was 9 hours. Toxicity was primarily leukopenia at the highest doses of drug. Abdominal discomfort and tenderness, as well as catheter dysfunction due to the formation of a fibrous sheath reflecting serositis, are not uncommon with intraperitoneal mitoxantrone.
The primary advantages of mitoxantrone in comparison with doxorubicin are its much reduced incidence of cardiac toxicity, the mild nausea and vomiting that follows intravenous administration, and the minimal alopecia. Early trials of mitoxantrone revealed occasional episodes of cardiac failure,547 primarily in patients who had not been helped by prior doxorubicin. There is no doubt that patients will develop congestive heart failure after treatment with mitoxantrone in the absence of prior anthracycline exposure, although the incidence is less than 5%.533, 548, 549 Our appreciation for the cumulative cardiac toxicity of mitoxantrone has recently been enhanced by reports of heart damage occurring in patients with multiple sclerosis (for which mitoxantrone has been approved by the US Food and Drug Administration).550, 551 In this setting, 5% of patients who receive over 100 mg/m2 of mitoxantrone develop an asymptomatic decrease in left ventricular ejection fraction. The incidence of cardiac toxicity is greatest in patients who have received prior anthracyclines or chest irradiation552 and in those with underlying cardiac disease.533, 549, 552
Other toxicities include a reversible leukopenia, with recovery within 14 days of drug administration; mild thrombocytopenia; nausea and vomiting; and, rarely, abnormal liver enzymes in patients receiving dose levels appropriate for solid tumors.553 One minor, and at times alarming, side effect of mitoxantrone is a bluish discoloration of the sclera, fingernails, and urine.554
In summary, mitoxantrone has not replaced doxorubicin in solid tumor chemotherapy, primarily because of its lesser activity against breast cancer. However, because of its advantageous toxicity profile, it is useful in the palliative therapy of hormone-resistant prostate cancer and is effective in combination therapy for the lymphomas and leukemias.
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