Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

Topoisomerase II Inhibitors: The Epipodophyllotoxins, Acridines, and Ellipticines

Yves Pommier

Fran¸ois Goldwasser

INTRODUCTION

DNA topoisomerase II (Top2) inhibitors have been the subject of considerable biochemical, pharmacological, and clinical investigation. In addition to the epipodophyllotoxins, acridines, ellipticines, and bisdioxopiperazines considered in this chapter, other drugs interact with Top2, including anthracyclines, mitoxantrone, and anthrapyrazoles. However, these other drugs exhibit other mechanisms of action and are described separately (see chapter 18).

The inhibition of cellular topoisomerase by antitumor agents such as adriamycin and ellipticine was first hypothesized by Kohn and coworkers in the late 1970s before eukaryotic topoisomerase II (Top2) had been identified.1 This hypothesis was based on the observations that the DNA breaks induced by adriamycin and ellipticine had unique characteristics in DNA alkaline elution assays: (a) they were only detectable after full deproteinization and therefore were called protein-associated (orprotein-linked)strand breaks, and (b) they were associated with an equal frequency of DNA-protein cross-links. The demonstration that the drug-induced protein-associated strand breaks were mediated through inhibition of Top2 took a few more years. This discovery was facilitated by cellular pharmacology studies of amsacrine, a far more potent inducer of protein-linked DNA breaks than anthracyclines or ellipticines.2, 3 Liu and coworkers, who had isolated mammalian Top2, showed that a number of inducers of protein-linked DNA breaks were acting through Top2.4 Independently, Kohn and coworkers demonstrated that the protein linked to DNA upon m-AMSA treatment is Top2.5 The term cleavage (orcleavable)complex is commonly used to define the enzyme-DNA complex, because cleavage is only detectable after strong protein denaturation by sodium dodecylsulfate (SDS). Our knowledge regarding both the Top2 enzyme and the mechanisms of action of and resistance to Top2 poisons has considerably increased. We better understand the wide variability in the spectrum of antitumoral activity of Top2 poisons as well as some of their specific toxic effects. Interference of these agents with Top2 has led to a model of MLL gene translocations and leukemia in which Top2-mediated chromosomal breakage occasionally is resolved by translocation.6

Extracts from the mayapple or mandrake plant have long been used in folk medicine. The active principle in this plant, podophyllotoxin, acts as an antimitotic agent that binds to tubulin at a site distinct from that occupied by the vinca alkaloids. A number of semisynthetic derivatives of podophyllotoxin have been made. Two glycosidic derivatives, teniposide (VM-26) and etoposide (VP-16) (Fig.19.1), are active against a number of human malignancies. Etoposide was approved by the US Food and Drug Administration (FDA) for marketing by Bristol Laboratories under the trade name VePesid in early 1984. Teniposide (Vumon) has been used in Europe for several years and was approved by the FDA in 1992 for refractory childhood leukemia. More recently, etoposide phosphate (Etopofos; Fig.19.1) was designed as a prodrug of etoposide in order to obtain a water-soluble compound activated specifically with antibody alkaline phosphatase conjugates at the tumor site.7, 8 In fact, etoposide phosphate is almost immediately converted to etoposide in the patient plasma by host endogenous phosphatase. Thus, etoposide phosphate simplifies the formulation of etoposide by being water-soluble and readily converted to etoposide.

Figure 19.1 Chemical structures of the epipodophyllotoxins (VP-16 and VM-26), the acridines (m-AMSA and DACA), and the ellipticines and olivacines (ellipticinium and S16020-2).

It is now well established that etoposide and teniposide exert their antineoplastic effect by inhibiting Top2 and that, in contrast to the parent compound (podophyllotoxin), they are inactive against tubulin. Etoposide and teniposide are very active against malignancies with a high proliferation rate. Etoposide is a key agent in germ-cell malignancies,9, 10 small cell lung cancer, poorly differentiated carcinomas, and poorly differentiated endocrine tumors and is given as first-line therapy in combination with cisplatin. Etoposide is also used in second-line regimens or salvage therapies in non-Hodgkin's lymphomas (NHLs), including HIV-associated NHLs11; high-risk metastatic gestational trophoblastic tumors12; Kaposi's sarcomas; osteosarcomas; Ewing's sarcomas; neuroblastomas; and leukemias. It is also one of the important agents used in preparatory regimens for bone marrow transplantation.13

Teniposide (VM-26) is mainly used in pediatry and neuro-oncology. Teniposide is active in combination in pediatric tumors such as retinoblastoma, neuroblastoma,14 and acute myelocytic leukemias,15, 16, 17 as salvage therapy for initial induction failures in childhood acute lymphoblastic leukemia (ALL), and for non-Hodgkin's lymphoma.18 Teniposide is active against recurrent oligodendroglioma19 and is also incorporated in chemotherapy regimens in primary central nervous system lymphoma.20 Teniposide is known as active in small cell lung cancer and in bladder cancer (by both intravenous and intravesical routes)21, 22 but is infrequently used in these diseases.

Amsacrine, or 4′(9-acridinylamino)-methanesulfon-m-aniside (m-AMSA; Fig. 19.1), is the first rationally synthesized aminoacridine anticancer agent to undergo full clinical development. Amsacrine has substantial efficacy in acute myeloblastic leukemia (AML) and ALL23, 24 but has largely been replaced by newer agents. The acridine derivative, N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA; Fig. 19.1), displays high activity against solid tumors in mice23 and exhibits a dual mode of cytotoxic action involving topoisomerases I and II.24, 25 It has not proven effective in trials thus far.

Representing a third structural class of Top2 inhibitors, ellipticine is an alkaloid derived from the Apocynaceae family, including Ochrosia, Bleekeria vitensis, and Aspidosperma subincanum.26 Despite its promising preclinical activity, severe toxic effects observed in animal studies hampered the progress of ellipticine toward clinical trials. The semisynthetic derivative 2-N-methyl-9-hydroxyellipticinium acetate (NMHE = ellipticinium; Fig.19.1) was briefly tested in breast cancer trials. The drug S16020-2 is a new olivacine derivative structurally related to ellipticinium27 (Fig.19.1). S16020-2 is highly cytotoxic in vitro28 and displays outstanding antitumor activity against various experimental tumors, especially some solid tumor models.29, 30, 31 Its activity is notably higher than that of ellipticinium and comparable to that of doxorubicin HCl, although with a different tumor specificity. S16020-2 is being tested in clinical trials.32

Catalytic inhibitors of Top2 have also been identified (see Table 19.2). One of them, dexrazoxane, a bisdioxopiperazine derivative, has been approved as a cardioprotective agent in association with anthracyclines.33, 34, 35 Among the bis(N-acyloxymethyl) dioxopiperazine derivatives, sobuzoxane (MST-16) has antitumoral activity in non-Hodgkin's lymphoma and T-cell leukemia patients36, 37 and has obtained official approval in Japan.

TABLE 19.1 MAMMALIAN DNA TOPOISOMERASES

 

Topoisomerases I

Topoisomerase IIα

Topoisomerase IIβ

Topoisomerases III

Size of monomer

100 kd (Top1 nuclear)
72 kd (Top1 mt)
Acting as monomer

170 kd Acting as dimer

180 kd Acting as dimer

110 kd

Size of mRNA

4.2 kb (Top1 nuclear)
1.8 kb (Top1 mt)

6.2 kb

6.5 kb

3.8 kb (main transcript; ref. 260)

Chromosome location

20q12–13.2 (Top1 nuclear)
8q24.3 (Top1 mt)

17q21–22

3p24

17p11.2–12 (Top3α)
22q11–12 (Top3β)

Catalytic intermediate

DNA SSB
Covalent linkage to 3′ DNA terminus

DNA DSB
Covalent linkage to 5′ DNA termini

DNA DSB
Covalent linkage to 5′ DNA termini

DNA SSB
Covalent linkage to 5′ DNA termini

ATP dependence

No

Yes

Yes

No

Cell cycle expression

Throughout

G2/M

Throughout

Specific inhibitors
indolocarbazoles

Camptothecins, indenoisoquinolines, (reviewed in ref. 261)

Top2 poisons and catalytic inhibitors, intercalators (see Table 19.2)

Same as topoisomerase IIα(prefermitoxantrone; see ref. 262)

Unknown

SSB, single-strand breaks; DSB, double-strand breaks.
Top1mt: mitochondrial Top1 is the most recently discovered human Top1 gene (ref. 263).

MOLECULAR AND CELLULAR PHARMACOLOGY

DNA Topoisomerase II

Enzymology and Functions

The length of eukaryotic DNA and its anchorage to nuclear matrix attachment regions limit the free rotation of one strand around the other as the two strands of the DNA double helix are separated for DNA metabolism (transcription, replication, recombination, and repair). DNA topoisomerases catalyze the unlinking of the DNA strands by making transient DNA strand breaks and allowing the DNA to rotate around or traverse through these breaks.38 Three families of topoisomerases are known in humans: topoisomerase I (Top1), topoisomerase II (Top2), and topoisomerase III (Top3)38 (Table 19.1). DNA gyrase and topoisomerase IV (topo IV) are the bacterial equivalents of eukaryotic Top2. Quinolones (nalidixic acid, ciprofloxacin, norfloxacin, and derivatives), which are widely used antibiotics, act by inhibiting DNA gyrase and topo IV but have no or very limited effect on the host human Top2.39, 40

Topoisomerase-mediated DNA breaks occur through transesterification reactions in which a DNA phosphoester bond is transferred to a specific enzyme tyrosine residue while the enzyme generates a break in the DNA phosphodiester backbone. Type 1 enzymes (Top1 and Top3) make DNA single-strand breaks, whereas the type 2 enzymes (Top2α and Top2β) make DNA double-strand breaks. In the case of Top1, the enzyme catalytic tyrosine becomes linked to the 3′-terminus of the cleaved DNA, while in the case of Top3 the linkage is to the 5′-DNA terminus of the break. In Top2-mediated reactions, each enzyme molecule of a homodimer becomes linked to the 5′-terminus of each of the cleaved DNA strands (Table 19.1, Fig. 19.4).

Figure 19.2 A: Domain structure of Top2. The three major domains of eukaryotic are illustrated, as well as the site of ATP binding (ATP), the active-site tyrosine (Y805 for Top 2α and Y826 for Top 2β), the nuclear localization sequence(s) (NLS), and the sites of phosphorylation (PO4). The N-terminal domain (homologous to the gyrase B subunit) extends from amino acid 1 to about 660. The catalytic core domain (homologous to the A subunit of gyrase) extends from about residue 660 to 1,200, and the C-terminal domain (no corresponding homology with gyrase) extends from about residue 1,200 to the C-terminus of the enzyme. B: Model for the catalytic cycle of type II topoisomerases (according to reference 41). The unliganded enzyme binds to duplex DNA (labeled G) across the A' domains (step 1). A second duplex DNA strand (labeled T) and ATP bind to the enzyme (steps 2 and 3). Nucleotide binding promotes dimerization of the ATPase domains and closure of the clamp (curved arrows, step 3). Cleavage of the G-strand allows the passage of the T-strand through the cleaved G-strand (step 4 is diagrammed in brackets to indicate that the cleavage complex is a short-lived intermediate in the proposed transportation event). Following G-strand religation, the T-strand is released through the dimer interface in the A' region (step 5). ATP hydrolysis completes the enzyme catalytic cycle.

Both Top1 and Top2 can remove DNA supercoiling by catalyzing DNA relaxation. They can complement each other in this function, at least in yeast, where the absence of Top1 can be compensated for by the presence of the other topoisomerase. However, yeast strains deficient in Top2 are not viable and die at mitosis because Top2 is essential for chromosome condensation and for the proper segregation of mitotic and meiotic chromosomes.38 The reason is that, in addition to its DNA-relaxing activity, Top2 can separate two linked circles of duplex DNA (decatenation). Top2 also catalyzes the reverse reaction (catenation) by allowing one duplex to pass through a double-stranded gap created in the other duplex (strand passage reaction; see next section and Fig. 19.2). Decatenation is essential at the end of DNA replication for the separation of daughter DNA molecules and the segregation of newly replicated chromosomes. The accumulation of Top2 at the end of the S phase and during G2 and its concentration in the chromosome scaffold are consistent with the enzyme's role in separating chromatin loops and condensing DNA at mitosis (see Table 19.1).

Mammalian cells have two Top2 isoenzymes, termed Top2α and Top2β (Table 19.1). They differ in molecular mass, enzymatic properties, chromosome localization, sequence, cell cycle regulation, and cellular and tissue distribution. Although the cellular concentration of Top2β is relatively constant throughout the cell cycle, the Top2α level is tightly linked to the proliferative state of the cell. The concentration of the α isoform increases two- to threefold during G2/M and is, order of magnitude, higher in rapidly proliferating cells than in quiescent populations.

DNA Topoisomerase II Catalytic Cycle

A description of the Top2 catalytic cycle is essential for understanding how Top2 poisons stabilize the cleavage complex of the DNA strand passage reaction (see Fig. 19.2; for more details, see references 41, 42). In contrast to Top1, Top2 enzymes function as a homodimers. Their catalytic activity requires the presence of magnesium as well as ATP as an exogenous energy source. Hence Top2 enzymes act as DNA-dependent ATPases. Recent structural and mechanistic studies reveal a remarkable dynamic behavior of the enzyme structure during its catalytic cycle.41, 42 Top2 catalyzes DNA strand passage according to the two-gate model shown in Figure 19.2.41, 42 The enzyme forms a dimer and initiates its catalytic cycle by binding to its DNA substrate with a preference for DNA crossover regions. Hence, Top2 interactions with DNA are determined both by DNA superstructure (DNA crossovers, bends, etc.) and local DNA sequence. Although Top2 enzymes interact with preferred sequences, they do not have the sequence specificity of restriction endonucleases. This lack of stringency probably allows the enzyme to act at multiple sites of the genome in order to perform its vital functions.

Top2 assumes at least two alternative conformations, open or closed clamp forms in the absence or presence of ATP, respectively.41, 42 The enzyme binds two segments of duplex DNA, referred to as the G and T segments. The G (for g ate) segment is the one cleaved by the enzyme in order to pass the T (for t ransported) segment through the enzyme-DNA complex (Fig. 19.2). Upon ATP binding, Top2 undergoes a conformational change from an open to a closed clamp form (step 3). In the presence of a divalent cation (under physiological conditions Mg++), the tyrosine active residue of each Top2 monomer (tyrosine 805 for human Top2α) attacks a DNA phosphodiester bond four bases apart on the G duplex and becomes covalently linked to the 5′ ends of the broken DNA while the 3′-ends are 3′-hydroxyls. The T segment can then pass through the gap produced in the G segment (step 4). Cleavage of the G duplex is reversible in nature, and under normal conditions the cleavage complex is a short-lived intermediate. After strand passage the T segment is released from the clamp, and the broken ends of the G segment are religated by Top2 (step 5). Upon hydrolysis of ATP by the intrinsic ATPase activity of the enzyme, the Top2-DNA complex is converted back to the open clamp form with release of the G segment. Thus closing and opening of the Top2 clamp are coupled with ATP binding and hydrolysis, respectively. Through its ability to open both strands of a DNA duplex and to catalyze strand passage in concerted reactions, Top2 can perform a variety of DNA topoisomerization reactions. Whereas DNA relaxation is common to Top1, conversion of circular DNA to knotted forms and removal of preexisting knots are specific to Top2. These biochemical reactions are commonly used to assay topoisomerase activities in vitro: relaxation of supercoiled plasmid DNA in the absence of ATP and Mg ++ in the case of Top1 and decatenation of kinetoplast DNA (kDNA) and unknotting of P4 DNA in the case of Top2.43

DNA Topoisomerase II Poisons

Mechanisms of Top2 Inhibition by Anticancer Drugs

The antitumor Top2 inhibitors poison the enzyme by stabilizing the DNA cleavage complexes (step 4 in Fig. 19.2) rather than preventing enzyme catalytic activity. The production of DNA cleavage complexes is due to an inhibition of DNA religation in the case of VP-16, VM-26, and m-AMSA.44, 45, 46 On the other hand, compounds such as quinolones act by inducing the formation of cleavage complexes rather than by inhibiting religation.47 The cleavage complexes can be detected in cells as protein-linked DNA breaks by alkaline elution or by SDS-KCI precipitation assays (for review, see references 1, 43). Cellular topoisomerase-DNA complexes can also be detected using the ICE bioassay (immunocomplex topo assay).48 Inhibition of Top2 catalytic activity without the trapping of cleavage complexes (Table 19.2; Fig. 19.3) was first demonstrated for strong DNA intercalating agents at drug concentrations that saturate the DNA. It is attributed to DNA structural alterations that prevent the enzyme from binding to DNA (steps 1 and 2 in Fig. 19.2) or prevent initiation of the cleavage complex.49, 50 Other DNA binders such as merbarone and the bisdioxopiperazines (ICRF-159, ICRF-187 [= dexrazoxane], and ICRF-193) produce the “closed clamp” type of inhibition, for example, inhibition of Top2 catalytic activity without the trapping of cleavage complexes51, 52 (see Table 19.2). Hence three types of curves that relate drug concentrations to cleavage complexes can be observed for Top2 inhibitors (Fig. 19.3): (a) a monotonal increase of cleavage complexes with drug concentration in the case of non-DNA or weak DNA binders (VP-16, VM-26, m-AMSA, quinolones), (b) a bell-shaped curve (with initial increase in cleavage complexes with increasing drug concentrations, followed by a decrease in cleavage complexes at higher concentrations) in the case of DNA intercalators (ellipticines, anthracyclines, mitoxantrone, anthrapyrazoles), and (c) a monotonal decrease of cleavage complexes in the case of some bulky intercalators (ethidium bromide, ditercalinium, aclarubicin) or non-DNA binders (bisdioxopiperazines) (see Table 19.2) that inhibit catalytic activity without trapping cleavage complexes.

At the biochemical level, Top2 inhibitors exhibit different effects (Table 19.3). The kinetics of cleavage complex formation and reversal in drug-treated cells vary from slow in the case of doxorubicin2 and ellipticine53 to very rapid in the case of VP-16, m-AMSA, and ellipticinium.2, 54 The higher cytotoxicity of doxorubicin versus VP-16 may be explained by the importance of persistent cleavage complexes for cytotoxicity. Most drugs induce not only Top2-mediated DNA double-strand breaks but also Top2-mediated single-strand breaks, the ratio of which varies widely among drugs. Ellipticines produce almost exclusively DNA double-strand breaks, while VP-16 and amsacrine produce 10 to 20 single-strand breaks per double-strand break.2, 44, 54 Anthracyclines produce a mixture of single-strand and double-strand breaks.2 Hence the higher cytotoxicity of anthracyclines compared with amsacrine or VP-16 may be due to the higher frequency of DNA double-strand breaks, which may be more cytotoxic than single-strand breaks.44 Finally, the DNA sequence and genomic localization of Top2 cleavage complexes vary among drugs.55 Drugs that are chemically and structurally related frequently produce closely related patterns of Top2 cleavage, whereas compounds structurally and electronically unrelated produce different patterns both in purified DNA and in drug-treated cells.44, 55, 56

TABLE 19.2 TOPOISOMERASE II INHIBITORS

 

Base-Sequence Preference of Top2 Inhibitors and Drug-Binding Model

The DNA sequencing of drug-induced cleavage sites shows that each class of inhibitor tends to act at Top2 cleavage sites with different base sequence preferences at the 3′- and/or 5′-terminus of the Top2-mediated DNA double-strand break57, 58, 59, 60 (Fig.19.4). These drug-specific preferences for certain bases immediately flanking the cleavage sites suggest that the drugs interact directly with these bases. Since all Top2 inhibitors, whether intercalator or not, have a planar aromatic portion that in some cases mimics a base pair (see Fig. 19.1), the simplest explanation is that the drugs stack inside the cleavage sites at the enzyme-DNA interface. Depending on the drug structure, preferential base stacking would take place either at the 3′- or the 5′-terminus. This hypothesis implies that topoisomerases first cleave the DNA at many sites and that the drugs bind specifically to some sites and prevent DNA religation.61

The base sequence analysis data suggest that stacking at one cleavage site is sufficient for the creation of a DNA double-strand break, a theory consistent with the concerted action of both enzyme monomers during catalysis.57, 58, 59, 60, 61, 62, 63 This type of inhibition, which we refer to as “interfacial inhibition,” is one of nature's paradigms for noncompetitive protein inhibition.64

Determinants of Sensitivity and Resistance to Top2 Inhibitors

Figure 19.5 summarizes the multiple factors that determine the cytotoxicity of Top2 inhibitors. Before topoisomerase inhibitors reach their nuclear target, they have to be taken up by the cells and transported to the nucleus. Reduced drug accumulation and/or altered intracellular drug distribution is a dominant feature of many drug-resistant cell lines. Most clinical antitumor Top2 inhibitors are substrates for the 170-kd transmembrane glycoprotein Pgp, which is a product of the MDR1 gene and responsible for the classical MDR (multidrug-resistance) phenotype.65 MDR-sensitive drugs include doxorubicin and analogs, mitoxantrone, anthrapyrazoles, ellipticines, VP-16, and to a lesser extent m-AMSA analogs. Hence, cells overexpressing Pgp are generally resistant to Top2 inhibitors because the drugs are actively extruded from the cells. The amino group on the daunosamine sugar of anthracyclines is probably involved in the drug recognition by Pgp. This is probably why the deamino derivative hydroxyrubicin is less subject to drug resistance while retaining Top2 inhibitory activity.66

Figure 19.3 Different modes of drug inhibition of Top2. Top2 poisons such as the epipodophyllotoxins (VP-16 and VM-26) and the azatoxins (solid circles) only trap the Top2 cleavage complexes with increasing efficiency as their dose increases. Top2 suppressors such as the bis-dioxopiperazines (open circles) are pure catalytic inhibitors that only inhibit the formation of cleavage complexes. The hatched squares correspond to biphasic inhibitors such as DNA intercalators (anthracyclines, ellipticines, acridines; see Table 19.2), which enhance Top2 cleavage complexes at low concentrations and suppress cleavage complexes at higher concentrations.

Although drug metabolism is not necessary for activity of the Top2 inhibitors described in this chapter, some drugs undergo metabolic modifications that may affect the way they interact with Top2 or/and DNA. Anthracyclines participate in redox reactions that lead to the formation of free radicals that damage cellular lipids or DNA (see Chapter 18). Some adriamycin-resistant and mitoxantrone- resistant cell lines show increased levels of cellular glutathione (GSH) or GSH-conjugating enzymes, which may be a result of GSH-mediated drug inactivation with or without subsequent drug efflux mediated by the putative ATP-dependent glutathione S-conjugate export pump.67 The VP-16 metabolites demethylated on the podophyllotoxin phenyl ring and without the sugar are at least as active as VP-16 against purified Top2 (Fig.19.6).68 These metabolites are more susceptible to oxidation-reduction reactions and may exhibit a shorter half-life and higher reactivity toward other macromolecules than Top2.69 In the case of the ellipticines, similar oxidation-reduction reactions and free-radical formation have been identified and may be responsible for lesions that contribute to the drugs' cytotoxic effects.70, 71

TABLE 19.3 DIFFERENCES AMONG TOPOISOMERASE II INHIBITORS

Other targets besides Top2

Free radicals: anthracyclines, mitoxantrone
DNA intercalation (e.g., anthracyclines, mitoxantrone, ellipticines)

Different effects on Top2

Base sequence preferences and location of DNA cleavage sites (see Fig. 18-3)
Ratio of DNA double- to single-strand breaks: ellipticine > anthracyclines >amsacrine/epipodophyllotoxins
Kinetics of trapping Top2 cleavage complexes (slow for anthracyclines; fast for epipodophyllotoxins/amsacrine)
Inhibition of cleavage complexes at higher concentration (intercalators)
Pure Top2 poisons (epipodophyllotoxins)

Mechanisms of resistance

Substrates for transmembrane transporters: anthracyclines and epipodophyllotoxins more than mitoxantrone, amsacrine, and ellipticines
Specific Top2 mutations affect drug binding differentially (refs. 299,300,301,302,303,304,305,306,307)

See text for references.

The intracellular distribution of Top2 inhibitors has been well characterized for the anthracyclines. Drug-resistant cells tend to exclude doxorubicin from their nuclei. These observations suggest that a nuclear transporter may exist. In the case of m-AMSA, cellular uptake studies suggest that the drug may be retained in some slowly exchangeable intracellular compartment.72 An altered intracellular drug distribution with drug sequestration in cytoplasmic organelles has been described.73

In contrast to other chemotherapeutic agents such as antifolates, the degree of cellular sensitivity to Top2 inhibitors correlates with the abundance of the target enzyme. Indeed, the higher the levels of Top2, the more sensitive is the cell, because more cleavage complexes are formed and more consecutive genotoxic and cytotoxic lesions accumulate (see Fig. 19.5). Cancer cells often have a higher level of Top2α, and the Top2 content in cancer cells is less regulated by growth conditions.74, 75 Top2β appears to be also up-regulated in neoplastic cells compared to normal, quiescent tissues.76 Coamplification of the erbB2, Top2α, and retinoid acid receptor α genes, all localized on chromosome 17q, has been observed in breast cancer cells.77 Top2α expression can also be stimulated by Top1 inhibitors (see “Therapeutic Interaction” below). Treatment of AML patients with topotecan leads on average to a threefold increase in Top2α expression.78 Furthermore, a permanent twofold increase of both Top2α and β was reported for topotecan-resistant human cell lines.79

Figure 19.4 Top2 cleavage complexes in the absence of drug (top) and base sequence preferences for Top2 inhibitors. Top2 is shown as filled circles.

Figure 19.5 Determinants of sensitivity and resistance to Top2 inhibitors. (LRP, lung resistance–related protein; MRP, multidrug resistance protein; Pgp, P-glycoprotein.)

 

Figure 19.6 Metabolism of etoposide. Both etoposide-o-dihydroxy and 4-demethylepipodophyllotoxin aglycone are active against Top2.

Many cell lines resistant to Top2 inhibitors show decreased Top2 protein levels. The two main mechanisms for decreased Top2 transcription are likely to involve DNA modifications (promoter mutations, gene rearrangements, CpG methylation) and transcription factor alterations. Methylation of the Top2 gene has been reported in association with decreased transcription.80 Different stress conditions can decrease Top2α expression. For instance, p53, which itself is activated by DNA damage, down-regulates Top2α.81, 82 Glucose-regulated stress, such as hypoxia and nutrient deprivation, also lead to decreased expression of Top2α protein with associated resistance to VP–16.83, 84 These findings may be relevant for poorly vascularized human tumors. It seems that it is mostly Top2α that is altered in cell lines selected with VP-16 or teniposide.85, 86 Although Top2α is also affected in cell lines selected with DNA intercalators such as mitoxantrone, doxorubicin, and the ellipticines, the most dramatic changes seem to concern the β-isoform, which is strongly down-regulated or even missing.87, 88

Another way of diminishing drug-induced cleavage complexes is by a shift in enzyme localization from the nucleus toward the cytoplasm, as observed for several cell lines resistant to Top2 inhibitors.86, 87, 89 In such cases, the cytoplasmic Top2α is still catalytically active and able to carry out its normal functions during mitosis when the nuclear membrane is disassembled.

Top2 mutations have been observed in cell lines selected in vitro. The Top2 mutations are clustered in two regions, one located near the ATP binding site and the other around the catalytic tyrosine (Fig. 19.2). The presence of mutations near the catalytic tyrosine is consistent with the drug-stacking model at the interface of the enzyme-DNA complex.64 The existence of the second mutation cluster near the putative ATP-binding site suggests that Top2 folding brings this second region near the catalytic domain and that Top2 inhibitors bind at the interface of these two Top2 regions.61, 90, 91 The differential resistance to epipodophyllotoxins, m-AMSA, and quinolones of some of the drug-resistant mutants is consistent with preferential interactions of each class of drugs with certain Top2 amino acids and with the preferred DNA bases around the Top2 cleavage sites.

Changes in Top2 phosphorylation may also contribute to Top2-mediated drug resistance. However, in resistant cell lines, reduced cleavage complexes have been associated with both hyperphosphorylation and hypophosphorylation of Top2. Decreased Top2α protein levels were associated with hyperphosphorylation of the enzyme in VP-16-resistant cell lines.92 Hypophosphorylated Top2α has been reported for VP-16-resistant erythroleukemia93 and HL-60 cells94 and for teniposide-resistant leukemia cell lines.95 Top2 phosphorylation increases in parallel with the cellular need for the enzyme: during the S phase of the cell cycle, with a peak at the G2 phase.96 Casein kinase II is probably the main kinase responsible for Top2 phosphorylation in cells.97, 98, 99 Drug-induced cleavage complexes are reduced to approximately 50% after in vitro phosphorylation of Top2α by casein kinase II and protein kinase C.100 This finding is in contrast with Top1 phosphorylation, which increases camptothecin activity.101

Since both normal and cancer cells express Top2, it is likely that drug-induced cleavage complexes are not sufficient for selective killing of cancer cells. DNA synthesis inhibition provides only partial protection against VP-16.102 The interaction of transcription with cleavage complexes may play a prominent role in the activity of Top2 inhibitors, since VP-16 cytotoxicity is decreased by RNA synthesis inhibitors.103, 104 The dependence of Top1 and Top2 inhibitor cytotoxicity on ongoing replication and transcription probably explains why simultaneous treatment with camptothecin and VP-16 has been found to be antagonistic.103,104, 105 VP-16 may suppress camptothecin effects by inhibiting replication, and camptothecin may suppress the effects of VP-16 by inhibiting transcription.

Poly(adenosine diphosphoribose) synthesis also may be important for cell killing, since poly(adenosine diphosphoribose polymerase)-deficient Chinese hamster cells are resistant to VP-16 and hypersensitive to camptothecin.106 These observations indicate that events downstream from the cleavage complexes are critical to cytotoxicity. Such events may involve the accumulation of genetic alterations, such as sister chromatid exchanges (SCE),107 illegitimate recombinations,108, 109 and apoptosis.110

DNA repair must intervene to correct drug-induced and topoisomerase-mediated DNA damage. Yeast cells are usually resistant to topoisomerase inhibitors unless they are RAD52 mutants, for example, deficient in DNA double-strand break repair.111 The ubiquitin proteolysis pathway has been reported to be responsible for ubiquitination and degradation of Top1 and Top2 cleavage complexes.112 Abnormal cell cycle control (checkpoints) has recently emerged as a key element in possibly explaining the differential responses of normal versus neoplastic cells to DNA damage. Therefore, alterations of cell cycle control may play a critical role in the cytotoxicity of topoisomerase inhibitors. Lack of arrest in G1, as in cells with mutated or absent p53 genes, may not provide the cell with the time required to repair damage and may lead to an accumulation of further damage. Hence deregulation of cyclins, cell cycle–regulated kinases and phosphatases, and p53 mutations may sensitize cells to topoisomerase inhibitors.113, 114, 115, 116 Furthermore, pharmacological abrogation of drug induced S-phase and G2-phase checkpoints may provide a novel effective strategy for enhancing the chemotherapeutic activity of topoisomerase inhibitors.117

Another determinant of sensitivity to topoisomerase inhibitors is the predisposition of the cell to undergo apoptosis.110 Some cells, such as human leukemia HL-60 cells, are known to be hypersensitive to a variety of injuries, including DNA damage by topoisomerase inhibitors. The underlying mechanism for this hypersensitivity may be the facile induction of apoptosis.118 Overexpression of the c-myc proto-oncogene and down-regulation of the bcl-2 gene have been involved in committing the cells to an apoptosis-prone phenotype. p53 could play a key role in the case of VP-16 and mediate apoptosis in response to DNA damage without regulating apoptosis induced by glucocorticoids. Apoptosis also may play a role in drug-induced side effects such as hematopoietic or intestinal toxicity. Indeed, hematopoietic progenitors may be prone to apoptosis. Hence studies on the pharmacologic regulation of apoptosis may prove useful. Several classes of agents can suppress topoisomerase inhibitor–induced apoptosis,118 and bcl-2 overexpression renders cells resistant to VP-16.119

In summary, cellular response to Top2 inhibitors is complex, and several mechanisms are commonly associated in laboratory cell lines: Pgp and/or MRP overexpression, reduction of Top2 protein levels, changes in subcellular localization, Top2 phosphorylation, and Top2 mutations. This multifactorial resistance is likely to be applicable to human cancers, which underlines the importance of a multiparametric approach for the evaluation of clinical response to Top2 inhibitors.

CLINICAL PHARMACOLOGY

Epipodophyllotoxins

Two epipodophyllotoxins derivatives are presently used in cancer chemotherapy, etoposide (VP-16) and teniposide (VM-26). Etoposide phosphate is an improved formulation of etoposide. Etoposide remains one of the most active anticancer agents, and it is used as a key component for chemotherapy, especially for testicular cancers; as an adjuvant (EP protocols)9; for first-line metastatic (BEP regimen) and second-line salvage therapies (VIP regimen); and in intensification regimens. Teniposide is a more potent Top2 inhibitor (about 10-fold with purified Top2), and it is mainly used in pediatric tumors and in neuro-oncology.

Drug Assays

Quantitation of epipodophyllotoxins and their metabolites involves either HPLC or immuno-based assays. Two immuno-based assays have been developed more recently: radioimmunoassays (RIAs) and ELISA methods. An ELISA method with a sensitivity of 0.5 ng/mL and with no cross-reactivity with the aglycone metabolite has been developed.120

Pharmacokinetics

Intravenous Etoposide

For both epipodophyllotoxins, there is significant interpatient variability in pharmacokinetic parameters.121, 122 Following an i.v. dose, etoposide decay in plasma follows a two-compartment pharmacokinetic model with a terminal half-life of 6 to 8 hours in patients with normal renal function (Table 19.4). Interpatient and intrapatient variability in pharmacokinetic parameters following i.v. administration is substantial and can exceed 35%.123, 124 The volume of distribution123 averages 4 to 10 L/m2. The peak plasma concentration and the area under the curve (AUC) are proportional to the administered dose125, 126 up to doses of 800 mg/m2, and the elimination half-life is independent of dose. However, protein binding averages 96% but is nonlinear and influenced by the individual concentration of drug and albumin,127, 128 resulting in elevated free-drug concentrations in patients with low serum albumin. Other conditions, including elevated serum bilirubin concentrations, that compete for albumin binding also increase the concentration of the free, or biologically active, drug, resulting in greater toxicity.129, 130 Thus, the toxicity of etoposide correlates best with the pharmacokinetics of the unbound fraction of the drug. Etoposide penetrates the CSF poorly, with CSF concentrations less than 5% of simultaneously measured plasma levels.121, 123, 131 Pleural fluid penetration and ascitic fluid penetration of etoposide are poor.121, 131

TABLE 19.4 KEY FEATURES OF EPIPODOPHYLLOTOXIN DERIVATIVES

 

Etoposide (VP-16)

Teniposide (VM-26)

Mechanism of action

Inhibition of Top2, Nonintercalator

Same but ≈ 10-fold more potent

Pharmacokinetics

Terminal half-life = 6–8 hr

Terminal half-life = 9.5–21 hr

Elimination

Hepatic metabolism, Renal excretion 35–40%

Probable hepatic metabolism

Toxicities

Neutropenia, Thrombocytopenia, (mild) Alopecia, Hypersensitivity, Mucositis (high doses)

Same as etoposide

Precautions

Reduced dose proportionate to creatinine clearance

Possible increased toxicity in hepatic failure

Etoposide is eliminated by both renal and nonrenal mechanisms.132 Approximately 40% of administered etoposide is cleared through the kidney unchanged.123, 124, 126, 129, 133 Etoposide dosage should be reduced in proportion to reductions in creatinine clearance.129, 130 Hemodialysis membranes are not permeable to etoposide, and the pharmacokinetics of etoposide are not affected by the interval between chemotherapy and hemodialysis.134

Several metabolites of etoposide have been identified in humans121, 130, 135 (Figure 19.6). The main metabolite is etoposide-glucuronide, which is eliminated in the urine. A catechol metabolite with significant cytotoxic activity is formed following etoposide O-demethylation in the liver.136 Cytochrome P450 3A metabolizes etoposide to a catechol metabolite, which is further oxidized to a quinone. The etoposide-o-dihydroxy also can be converted to the o-quinone derivative. Both of these, as well as the 4-demethylepipodophyllotoxin, remain active against Top2. Ortho-quinone and semi-quinone free radicals of etoposide may covalently bind to DNA and induce DNA strand breakage.137, 138 Biliary excretion is a minor route of elimination. Minor alterations in liver function, such as transaminase elevations, do not require dose reduction if renal function remains normal,139 but elevated bilirubin may decrease the clearance of unbound etoposide127 and increases the unbound fraction of drug, leading to a greater hematologic toxicity. Therefore, etoposide dose should be reduced by 50% in patients with total bilirubin levels of 1.5 to 3.0 mg/dL. No etoposide should be given in patients with more than 5.0 mg/dL bilirubin.140 Age-related reduction in etoposide clearance has been suggested.141

Dose-Escalation Strategies with Etoposide

·     High-dose etoposide (VP-16) with bone marrow transplantation. Etoposide has been used in most high-dose protocols because its nonhematologic toxicity is only moderate.142 At very high doses up to 1,500 mg/m2, bone marrow rescue is necessary, and the dose-limiting toxicities becomes mucositis and hepatotoxicity. The maximal tolerated dose (MTD) of etoposide administered as a single agent is up to 2.5 g/m2. Between 2.5 and 3.5 g/m2, the CSF concentration of etoposide becomes significant.

·     Loco-regional infusions of etoposide. Based on the previous demonstration of a high peritoneal-plasma ratio of drug exposure for intraperitoneal etoposide, intraperitoneal administrations of etoposide have been done in ovarian cancer patients with peritoneal carcinomatosis.143, 144, 145 The doses of etoposide143, 144, 146, 147 ranged from 100 to 600 mg/m2. The calculated peritoneal-plasma ratio of unbound etoposide was 35. Etoposide can be combined intraperitoneally with cisplatin.147

The administration of intrapleural etoposide has been proposed as a way to prevent the recurrence of neoplastic pleural effusions.148 Incomplete and slow systemic absorption from intrapleurally administered drug has been documented.149 The intraventricular administration of etoposide is feasible.150 CSF peak levels exceed more than 100-fold those achieved with intravenous infusion. The half-life in CSF based on the pharmacokinetic data analysis of four patients was 7.4 ± 1.2 hours. Intrathecal injections of etoposide have been reported using a 0.5-mg dose daily for 5 days, followed by a second course with two injections per day of the same dose.151 No toxic effect was reported. The intra-arterial route (using carotid or vertebral arteries) has been used to increase etoposide uptake in brain tumors.152 Intra-arterial infusion of etoposide has been used also for the treatment of liver metastases of testicular tumors.153

Oral Etoposide

Prolonged administration of etoposide aims for extended Top2 inhibition, thus preventing tumor cells from repairing DNA breaks. Oral administration of etoposide represents the most feasible and economic strategy to maintain effective concentrations of drug for extended times. Nevertheless, the efficacy of oral etoposide therapy is contingent on circumventing pharmacokinetic limitations, mainly low and variable bioavailability.154 Inhibition of small-bowel and hepatic metabolism of etoposide with specific cytochrome P450 inhibitors or inhibition of the intestinal P-glycoprotein efflux pump has been attempted to increase the bioavailability of oral etoposide, but the best results were obtained with daily oral administration of low etoposide doses (50 to 100 mg/day for 14 to 21 days). Saturable absorption of etoposide was reported for doses greater than 200 mg/day, whereas lower doses were associated with increased bioavailability, although they were characterized by high interpatient and intrapatient variability. Pharmacokinetic parameters such as plasma trough concentration between two oral administrations [C(24, trough)], drug exposure time above a threshold value, and area under the plasma concentration-time curve have been correlated with the pharmacodynamic effect of oral etoposide. Pharmacokinetic-pharmacodynamic relationships indicate that severe toxicity is avoided when peak plasma concentrations do not exceed 3 to 5 mg/L and C(24, trough) is under the threshold limit of 0.3 mg/L.

Oral etoposide is formulated in a hydrophilic gelatin capsule. Peak plasma levels are obtained 0.5 to 4 hours after administration.155 The mean bioavailability is 50%,126, 155, 156, 157, 158, 159 with substantial interpatient and intrapatient variability (range, 19 to 100%).123, 126, 160, 161, 162 The bioavailability of doses lower than 100 mg (50 mg/m2) approaches 75%,159 whereas the bioavailability of doses above 100 mg/m2 decreases below 50%. Etoposide has been given orally over a prolonged 21-day schedule at a dose of 50 mg/m2 per day with dose-limiting myelosuppression. The 50 mg/m2 dose has a higher bioavailability (91% to 96%) than that generally reported for higher doses of etoposide, and many patients maintained plasma concentrations of 1 µg/mL or greater for the entire period of treatment. Bioavailability is not linear and decreases with doses greater than 200 mg,161 possibly implying a saturable absorptive mechanism in the gastrointestinal (GI) tract. In addition to saturation of uptake, the very low aqueous solubility and the low stability at acid pH likely contribute to the erratic etoposide bioavailability.157, 161, 163 The intestinal P-glycoprotein mediates the efflux of etoposide, and the use of P-glycoprotein–inhibiting agents such as quinidine might increase the intestinal absorption of etoposide.164 Oral bioavailability of etoposide is not affected by food or by concurrent i.v. chemotherapy.160 A pharmacodynamic model was prospectively tested for the therapeutic monitoring of 21-day oral etoposide165 (see below).

Oral etoposide is well tolerated in the elderly166 and active in NHL and AL patients. However, a study compared the effects of oral etoposide and intravenous etoposide in advanced small cell lung cancer patients on survival and quality of life167 and indicated that the oral route had a lower antitumoral activity and should not be used as first-line treatment for this disease.167

In conclusion, the development of oral etoposide was emphasized both because it might allow prolonged exposure to active concentrations of etoposide and because it appeared as an interesting alternative to improve quality of life in patients treated with palliative chemotherapy. However, both the high interindividual variation in bioavailability and the results of the only study that compared oral and intravenous etoposide suggest restrictions for its use as first-line therapy.

Etoposide Phosphate (Etopophos)

Etoposide phosphate (Etopophos) is an improved formulation of etoposide. It is a water-soluble prodrug of etoposide that is rapidly and completely converted to etoposide after intravenous dosing, regardless of the duration of administration, the dose used, or the treatment schedule.168, 169, 170 Therefore, etoposide phosphate does not expose the bloodstream to detergents or oils, as does the formulation of the lipid-soluble etoposide and teniposide. Etoposide can be administered as an i.v. infusion in saline solution over 5 minutes without signs of hypotension or acute effects.168 Since it is not formulated with polyethylene glycol, polysorbate 80, or ethanol, etoposide phosphate does not cause acidosis, even when given at high doses. When given as a continuous infusion, etoposide phosphate was stable in pumps for at least 7 days.

The molecular weights of etoposide and etoposide phosphate are 558.57 and 568.55, respectively. To avoid confusion, doses of etoposide phosphate may be expressed as molar equivalent doses of etoposide. The toxicity of etoposide and etoposide phosphate are similar.171 The MTD was 100 mg/m2 per day of etoposide equivalent for 5 days or 150 mg/m2 per day for dosing on days 1, 3, and 5. Etoposide phosphate may exhibit better intestinal absorption than etoposide, with a mean bioavailability of 68%. Etoposide phosphate was proposed as a means to overcome the intersubject and intrasubject variability in absorption observed with oral etoposide.172 However, the same large interindividual variability of etoposide phosphate and etoposide AUC was observed, with 42.3% and 48.4%, as the coefficients of variation.173

In conclusion, etoposide phosphate is an improved formulation of etoposide and is better suited for bolus administration, high-dose treatment, and continuous infusions, provided equivalent antitumor activity is demonstrated.

Teniposide (VM-26)

Like etoposide, teniposide demonstrates biexponential disposition following intravenous administration. Only 10 to 20% of administered teniposide is found in the urine as metabolites. The large volume of distribution is consistent with the high degree of protein binding (>99%). Its nonrenal clearance is similar to that of etoposide, as is the volume of distribution. An inverse relationship between serum γ-glutamyl-transpeptidase and teniposide plasma clearance has been reported, suggesting an important hepatic component to clearance. A linear relationship exists between dose and AUC.

In mice, teniposide's highest concentrations are in the liver, kidneys, and small intestine. The lowest concentrations are found in the brain. In humans, CSF levels of teniposide are barely detectable, although teniposide is considered clinically more effective than etoposide in the treatment of malignant gliomas. Teniposide slowly diffuses into and is then slowly eliminated from third-space compartments such as ascites.

Teniposide is available only for intravenous administration, but the i.v. formulation of teniposide can be used orally. The bioavailability of oral teniposide appears similar to that of oral etoposide in that absorption is decreased at higher doses and is increased with smaller consecutive daily doses. The mean bioavailability is 41%.162 The MTD for oral teniposide given over 21 days is 100 mg/day, and the dose-limiting toxicity is myelosupression.162, 174

Pharmacodynamics

Etoposide activity shows marked schedule dependency123 and is both concentration-dependent and time-dependent.123 Etoposide clearance is not correlated with body surface area.175 Dosing according to body surface area is a poor predictor of the peak or steady-state etoposide concentration or AUC.176Hematologic toxicity correlates better to the AUC of unbound etoposide than to the AUC of total etoposide.177 Steady-state etoposide plasma levels of more than 1 µg/mL are tumoricidal but are associated with severe myelosuppression when the etoposide plasma levels are continuously higher than 3 µg/mL. The maintenance of a minimum plasma concentration between 2 and 3 µg/mL appears critical for efficacy. Intrapatient pharmacokinetic variability is small (12 to 15%).176 In the absence of measured plasma concentrations, simplifying VP-16 dosing to a fixed dose independent of body surface area—260 mg instead of 150 mg/m2—has been suggested.176 However, several studies indicated that patients with impaired renal or liver function or elderly patients are at risk for increased hematological toxicity, and hence it was proposed that the etoposide dose be reduced and individualized in these patients.178

Various limited-sampling methods have been proposed to estimate etoposide AUC from one or two plasma concentration measurements.179, 180

In patients receiving 21-day oral VP-16, etoposide plasma concentrations are correlated with the neutrophil count at the nadir.165

Toxicity

Hematotoxicity

Myelosupression is the dose-limiting toxicity of etoposide. Although the single MTD is 300 mg/m2, doses of 150 mg/m2 for 3 days and 45 mg/m2 for 7 days have been studied. Bone marrow toxicity is not cumulative. When given orally over 21 days, etoposide is generally well tolerated at a MTD of 50 mg/m2 per day. Myelosuppression is the dose-limiting toxicity, with leukocyte nadirs occurring between days 22 and 29.

Nonhematological Toxicities

Common nonhematological toxicities include nausea and vomiting in 10 to 20% of patients and alopecia in 10 to 30% of patients, depending on dose and schedule. Rare cases of anaphylaxis and chemical phlebitis have been reported. Hypotension, fever, bronchospasm, diarrhea, and mucositis are uncommon.181Hypotension has been noted in 5 to 8% of patients receiving etoposide but is rare when the drug is given over 1 hour and is probably related to the vehicle (polysorbate 80 plus polyethylene glycol).181

Etoposide-Induced Secondary Leukemia

The increased frequency of illegitimate recombination events induced by Top2-reactive agents may account for their leukemogenicity.107. Etoposide was demonstrated to be mutagenic in patients.182 Acute myelogenous leukemia (AML) cases related to prior treatment with epipodophyllotoxins (etoposide and teniposide) have been identified.6, 183, 184, 185, 186, 187, 188 In contrast to alkylating agent–associated secondary AML, epipodophyllotoxin-associated AML exhibits a shorter latency period, with a median of 24 to 30 months.6, 186, 189 The epipodophyllotoxin phenotype is most often monocytic (FAB M4 or M5). Acute promyelocytic leukemia following treatment with epipodophyllotoxin occurs infrequently.187, 188 Secondary myelodysplastic syndrome and chronic myelogenous leukemia have been described.6 Therapy-related leukemia and myelodysplasia were also reported following prolonged administration of oral etoposide in breast cancer patients and NHL patients.190, 191 In one review of 37 patients, 21 of 30 patients had M-4 or M-5 AML, with 14 of 28 patients having an 11q23 abnormality. The mean latency period was 33 months. Other studies suggest a shorter latency of 2 years.6, 185, 186 Weekly or biweekly schedules of etoposide might be associated with increased risk of secondary leukemia.6 The administration of L-asparaginase prior to etoposide might also increase the risk of leukemia.6, 192

The follow-up by the National Cancer Institute Cancer Therapy Evaluation Program of patients treated with epipodophyllotoxins did not show evidence of significant variations in the incidence of secondary leukemias in patients who had received low (.1.5 g/m2), moderate (1.5 to 2.99 g/m2), or high cumulative doses (>3 g/m2).186 The calculated 6-year rate for development of leukemia was between 0.7% and 3.2%, and the highest rate was observed in the group of patients who received the lowest doses.186 Most other studies found a correlation between the cumulative dose of etoposide and the risk of secondary leukemias. In the Indiana University experience, among the patients with germ cell malignancies treated with cis- platinum, etoposide, and bleomycin (PEB) at conventional doses, 2 of 538 patients developed AML after 22 months to 3 years.193 The median follow-up was 4.9 years. The etoposide dose was an important factor for the occurrence of AML following etoposide and cisplatin combination chemotherapy for advanced non–small cell lung cancer.194 The median etoposide dose was 6.8 g/m2 in 4 out of 114 patients who developed AML after 13, 19, 28, and 35 months after the beginning of the treatment, compared with a median etoposide dose of 3.0 g/m2 in the nonleukemic patients. In another report of 212 patients treated with PEB for germ cell tumors, 5 patients developed acute nonlymphocytic leukemia (ANLL) for a mean cumulative risk of 4.7%.195 All these patients had cumulative etoposide doses above 2,000 mg/m2, whereas none of the 130 patients with cumulative dose below 2,000 mg/m2 developed leukemia. In a series of 734 children treated with epipodophyllotoxins, 21 developed secondary AML. The overall risk of developing a secondary leukemia was 3.8%. Subgroup analysis revealed that the risk was substantially greater when the drug was given on a weekly or biweekly schedule (12%). The cumulative risk was substantially less (1.6%) in the children not treated with etoposide or treated with etoposide only during remission induction or every 2 weeks during continuation treatment.186, 196 In a case-control study of the French Society of Pediatric Oncology, 61 patients with secondary leukemia were matched with 196 controls. In multivariate analysis, the risk of leukemia correlated with the type of primary tumor (excess risk in case of Hodgkin's disease and osteosarcoma) and with the cumulative dose of etoposide.197 The risk of leukemia in patients who received more than 6g/m2 was 200-fold higher. The risk of leukemia was not increased by exposure to alkylating agents or radiotherapy. Not only etoposide but also its catechol and quinone metabolites can induce in vitro Top2 cleavage complexes near the translocation breakpoints and are likely to also play a role in the creation of Top2-mediated chromosomal breakage.198

These leukemias frequently involved the long arm of chromosome 11, with translocation of the MLL (myeloid-lymphoid leukemia or mixed-lineage leukemia) gene, which resides at chromosome band 11q23, but other less common abnormalities involved chromosomes 3, 8, 15, 16, 17, 21, or 22.100, 199, 200, 201, 202Most of the breakpoints in the MLL gene occur in a 9-kilobase region that includes exons 5 to 11. This genomic region includes DNA sequences potentially involved in illegitimate recombinations, such as Alu sequences, VDJ recombinase recognition sites, and Top2 consensus-binding sequences. The MLL gene appears to play a role in the regulation of the differentiation of hematopoietic stem cells. Its overall sequence composition is AT-rich. AT-rich sequences often correspond to nuclear matrix attachment regions (MARs), where Top2 cleavage complexes are preferentially formed.203 More than 20 different translocations involving chromosome 11q23 have been described. DNA topoisomerase II cleavage assays have shown a correspondence between Top2 cleavage sites and the translocation breakpoints. The mechanism of the translocation might be a chromosomal breakage by Top2 followed by the recombination of DNA free ends during DNA repair.

Clinical Resistance

Several studies have found an association between Top2α expression and tumor cell proliferation.204, 205 Consistently higher Top2α mRNA levels were observed in high- grade than in low-grade non-Hodgkin's lymphomas206 and in small cell than in non–small cell lung cancer.207 Increased Top2α was correlated with a poor prognosis in breast cancer patients.208 In contrast to Top2α, Top2β is expressed in both proliferating and quiescent cells.76 Several studies have investigated the relationship between Top2 levels in leukemia cells and the response to Top2 inhibitor–containing chemotherapy. No correlation was seen between Top2α or Top2β expression, as assessed by Western blot, and the response to Top2 poisons in adult acute leukemias.209, 210 Both negative estrogene receptor status and c-erbB2 overexpression are associated with high Top2α expression in breast cancer.211 The overexpression of Top2α does not correlate with the antitumoral effect of anthracyclines.212 However, the clinical response to etoposide phosphate infusion and cisplatin in heavily pretreated metastatic breast cancer patients correlated with tumor Top2α expression.213

Acridines

Amsacrine Pharmacokinetics

The key pharmacologic features of amsacrine are given in Table 19.5.

Amsacrine is metabolized via two major routes (Fig. 19.7), both of which result in the formation of conjugated thiols. The major route of metabolism and elimination of amsacrine in the mouse and the rat is cytochrome P-450–dependent. In the liver, the major metabolite is the amsacrine-glutathion-5′-conjugate. Amsacrine and to a greater degree its metabolites are also excreted in urine. The cellular pharmacokinetics suggest that the cellular uptake occurs by a rapid and passive diffusion. Neither amsacrine nor its metabolites penetrate the blood-brain barrier, and amsacrine does not achieve appreciable levels in the CSF.214

TABLE 19.5 KEY FEATURES OF AMSACRINE

Mechanism of action

Inhibition of DNA topoisomerase II, with possible selectivity for Top2β
Weak intercalator

Pharmacokinetics

Plasma disappearance:
Initial half-life = 30 min
Terminal half-life = 7–9 hr
Protein binding: 50% after 2 hr

Metabolism

Conjugation with glutathione

Elimination

Biliary route (65%, as metabolites)
Urinary route (35–50%, as metabolites and parent drug)

Toxicities

Myelosuppression
Mucositis
Vomiting
Hepatotoxicity
Ventricular arrhythmias

Precautions

Increased toxicity if renal or liver failure; reduce dose by 30%.
Monitoring of serum electrolytes; avoid hypokalemia.

Toxicity of Amsacrine

Hematotoxicity of Amsacrine

A number of single-dose and multiple-dose schedules have been tested in phase I trials in adult patients.215 The oral route is not used because of large and unpredictable interindividual variability in absorption. The optimal schedule appears to be 150 mg/m2 per day for 5 days for adult patients with leukemia. Myelosuppression is the most important and dose-limiting toxicity.

Nonhematological Toxicities of Amsacrine

Amsacrine causes phlebitis. Consequently, it is recommended to dilute amsacrine in 500 mL of 5% dextrose and to use a central line for continuous infusion or repeated treatments to avoid reactions at the injection site. Hearing loss and allergic reactions (anaphylaxis, urticaria and rashes, allergic edema) are relatively rare.

Nausea and vomiting are common with amsacrine.216 Diarrhea is less common and occurs in 5 to 17% of patients. In a trial incorporating dose escalation, the stomatitis was dose-related and relatively infrequent at doses under 120 mg/m2. Stomatitis becomes dose limiting for treatments with very high doses in association with bone marrow rescue.217

The incidence of hepatotoxicity may reach 35%.218 Elevation of bilirubin, the most frequent abnormality,219 is usually dose-related and reversible.216Increased liver transaminases are also dose-dependent and reversible.220 Increased alkaline phosphatases are rare.221 However, two cases of fatal hepatotoxicity have been reported, both in heavily pretreated patients.218, 220 Since amsacrine is conjugated in the liver and is excreted in large part via the biliary system, at least 30% dose reduction is generally recommended in patients with impaired hepatic function (elevated bilirubin).

Cardiotoxicity has been reported in children and adults with both high- and low-dose treatments. Although its incidence is low (<5% in most large series), patients may develop arrhythmia, conducting disturbances, congestive heart failure during and after amsacrine administration, and sudden death.216, 222, 223,224 More commonly, the heart rate is decreased by about 10%.225 Because hypokalemia may exacerbate arrhythmias, it has been recommended that serum potassium levels be maintained at or above 4 mEq/L at the time of drug administration.226 Normally, this value can be obtained readily with an infusion of potassium chloride at a rate of 10 mEq/hour administered over 10 hours before amsacrine treatment. In most patients, amsacrine produces a significant prolongation (0.05 to 0.064 seconds) of the corrected QT (QTc) interval.222, 227 The amsacrine-associated QTc prolongation may persist for up to 90 minutes. Tachyarrhythmias in the setting of QTc prolongation usually arise by triggered automaticity and may be precipitated by adrenergic hyperactivity. A prospective study suggested that this effect was present in most patients and occurred with each dose. Amsacrine also may reduce significantly the serum sodium and magnesium concentrations 20 minutes after the start of the infusion. The effect is transient and reverses by 24 hours after the infusion.228 The decrease in magnesium levels may contribute to the amsacrine-induced cardiac arrhythmias.225, 229 Nevertheless, amsacrine has been administered safely to patients with myocardial dysfunction.226

Figure 19.7 Metabolism of amsacrine (m-AMSA).

The impact of amsacrine treatments on fertility has been reported by Da Cunha and coworkers,230 who found that amsacrine has temporary and reversible effects on sperm count and motility.230

In summary, the optimal schedule of administration for amsacrine appears to be a single daily dose. It seems that little advantage is gained by continuous infusion schedules. Patients with normal hepatic function or mild liver dysfunction should tolerate full drug doses. Patients with significant liver dysfunction manifested by serum bilirubin greater than 2 mg/dL should have an initial 30% dose reduction. Subsequent dose escalation may be possible based on clinical tolerance. Patients with moderate renal dysfunction (serum creatinine in the range of 1.2 to 2 mg/dL) should receive full-dose therapy; however, oliguric patients or those with more serious renal disease (serum creatinine greater than 2 mg/dL) should have an initial 30% dose reduction.

DACA

The acridinecarboxamide N-[2-(dimethylamino)ethyl] acridine-4-carboxamide (DACA; Fig. 19.1) is a lipophilic mono-intercalator that entered phase I and II clinical trials on the basis of its mixed inhibition of both topoisomerases I and II24, 25 and its activity in experimental solid tumors.23 However, 120-hour continuous infusions failed to produce clinical antitumoral activity in patients with ovarian cancer231 or non–small cell lung cancer.232

Ellipticine and Olivacine Derivatives

Ellipticines and olivacines have in common the absence of dose-limiting myelosuppression and the absence of alopecia. Elliptinium is presently only available in Europe, and its use is severely limited by its propensity to cause severe intravascular hemolysis and by its modest antitumor activity. One olivacine derivative (S16020-S; Fig. 19.1) is presently in phase I-II clinical trials.

Therapeutic Interactions with the Top2 Inhibitors

Platinum Derivatives and Radiotherapy

Therapeutic synergy between etoposide and cis-platinum has been demonstrated, and this combination is very effective in the treatment of germ cell tumors and small cell lung cancers.233 The synergism between etoposide and platinum derivatives has also been found in experimental systems234 but its mechanisms remain poorly understood. Top2 inhibition might interfere with the repair of the platinum cross-links or produce complex DNA damage that might be inefficiently repaired in tumor cells. In a model of SCLC xenografts, the combination of ifosfamide and etoposide tended to be more potentiating than the standard combination of cisplatin and etoposide.235

In a randomized crossover study, the potential interaction between the platinum agents cisplatin and carboplatin and the metabolism of etoposide was explored.236 Etoposide was given over three days. At first cycle, etoposide was administered with a platinum drug on day 2, and the alternate platinum was administered on the second course. Neither cisplatin nor carboplatin coadministration affected the pharmacokinetics of etoposide during cycle 1. The AUC of etoposide was increased (28%) on day 3 of cycle 2 when cisplatin was given on day 2. These results are consistent with a previous report of reduction of the total clearance of etoposide during concomitant treatment with cisplatin.237 However, given the pharmacokinetic variability seen with etoposide, the clinical impact of these variations is small at conventional doses. The clearance of etoposide is substantially lower in patients receiving concomitant high doses of carboplatin prior to undergoing autologous bone marrow transplantation.238

Etoposide is an excellent radiosensitizer and is widely used in therapeutic protocols combining chemotherapy and concurrent radiotherapy, especially in lung cancer patients.239

Topoisomerase I Inhibitors: The Camptothecins

The interactions between inhibitors of Top1 and Top2 are complex. Tumor heterogeneity might contribute to the efficacy of concurrent administrations of Top2 and Top1 inhibitors. S-phase cells might be most sensitive to camptothecins, while the cytotoxicity of Top2 inhibitors is less cell cycle–dependent. Local pharmacokinetics might also a contributing factor. In cell culture experiments, simultaneous exposure to etoposide and to the topoisomerase I inhibitor camptothecin is antagonistic unless the two drugs are administered with a 4- to 6-hour interval.105 This in vitro antagonism has been confirmed in mouse xenograft systems in vivo for the association of etoposide with either of the two camptothecin derivatives CPT-11 (irinotecan)240 or topotecan.241 Cell cycle responses probably play a key role in the antagonism observed in these simultaneous exposure schedules. Top2 inhibitors deplete the S-phase cells by producing a G2 arrest, whereas Top1 inhibitors are most cytotoxic in S-phase because of a requirement of active replication for the generation of irreversible DNA lesions.105, 242 In the sequential schedules, compensatory enzyme changes might contribute to the enhanced activity. The combination of a Top1 inhibitor with a Top2 inhibitor is associated with a depletion of the target topoisomerase mRNA and protein and with reciprocal increases in the alternate topoisomerase mRNA and, to a lesser extent, protein.240 For these reasons, considerable clinical research has focused on the sequential administration of Top1 and Top2 poisons. A compensatory increase in Top2α and increased sensitivity to etoposide have been reported following treatment with topotecan.241 The Top2α levels declined 5 days after the last dose of topotecan and resulted in restoration of the original response of xenografts to etoposide.241 Hence, schedule dependency plays a crucial role in optimizing the effectiveness of combination chemotherapy with Top1 and Top2 inhibitors.

Clinical trials to date have explored the following:

·     Simultaneous administration of either CPT-11 (irinotecan) or topotecan with etoposide. A phase I combination study was conducted starting with 30 mg/m2 per day of CPT-11 and 40 mg/m2 per day of VP-16 given simultaneously from day 1 to 3. Cytolysis and hyperbilirubinemia were observed as dose-limiting toxicities resulting from this first dose level. The SN-38 enterohepatic circulation and drug-drug interactions have been suggested as playing a role.243 Another phase I combination study tested 60 to 90 mg/m2 per day of CPT-11 on days 1, 8, and 15 with 80 mg/m2 per day of VP-16 on days 1 to 3 every 3 weeks, with G-CSF support.244 Dose-limiting toxicities were diarrhea and leukopenia.244 The concurrent administration of CPT-11 and etoposide for 3 days has also been evaluated.245 The main dose-limiting toxicity was diarrhea. Granulocytopenia was also severe. The recommended doses were 60 mg/m2 per day in combination with 60 mg/m2 per day of CPT-11, and these doses required prophylactic administration of G-CSF.245 The concurrent administration of 5-day etoposide and CPT-11 led to granulocytopenia as a dose-limiting toxicity.246 Several phase II clinical trials have combined etoposide with either CPT-11247 or topotecan.248

·     Sequential administration of topotecan (72 hours continuous infusion on days 1 to 3) and etoposide (75 or 100 mg/m2 per day as a 2-hour infusion on days 8 to 10) has been studied in patients with solid tumors.249 In two of the six patients, Top1 levels in their tumors were successively measured and showed either modest or substantial decrements following topotecan treatment, while in one of these six patients, tumor Top2 levels increased.249Sequential administration of intravenous topotecan and oral etoposide was also evaluated but not at optimum intervals.250

The pharmacologically guided addition of etoposide to weekly chemotherapy consisting of cisplatin and irinotecan has been investigated.251 The Top2 poison was given 2 days after each dose of irinotecan. The dose-limiting toxicities included diarrhea and neutropenia. The quantitation of Top2α protein in peripheral blood mononuclear cells revealed an increase of Top2α at the time of etoposide administration in two patients. Evidence of antitumoral activity was seen in previously treated patients with mesothelioma and gastric, breast, and ovarian cancer.252

Alternatively, sequential administration starting with the Top2 poison has also been tested using the combination of doxorubicin and topotecan.253 The MTD was 25 mg/m2 of doxorubicin followed 2 days later by 1.75 mg/m2 per day of topotecan for 3 days. The treatment appeared feasible and active, encouraging phase II trials.

Another phase I-II study evaluated two sequences—Top1 inhibition followed by Top2 inhibition, or the reverse—using infusions of topotecan and oral etoposide in patients with small cell lung cancer. The dose-limiting toxicity was neutropenia. The combination was feasible and effective.254.

These studies support the continued development of sequential Top1 and Top2 targeting in the treatment of solid tumors.

Association of Topoisomerase II Inhibitors

Topoisomerase II inhibitors may be combined together. Etoposide is commonly given with adriamycin in various combination protocols for the treatment of Hodgkin's lymphomas. Mitoxantrone and VP-16 or amsacrine and VP-16 have been combined in the treatment of acute myelogenous leukemia.255 Such associations may be justified because anthracyclines and mitoxantrone exhibit marked differences in the genomic location of cleavage complexes and therefore may selectively target different portions of the genome55 (see “Base-Sequence Preference of Top2 Inhibitors and Drug-Binding Model” discussed earlier in the chapter).

The pharmacokinetics of etoposide were found unaffected by the dexrazoxane rescue used to reduce the extracerebral toxicity of high-dose etoposide.256

Other Agents

·     The human colony-stimulating factors (CSFs) accelerate hematopoietic recovery from etoposide-induced myelosuppression. In addition, CSFs may limit etoposide-induced myelosuppression by inhibiting apoptosis in the normal bone marrow cells. CSFs can also modulate the growth and drug response of tumor cells with functional receptors for these cytokines. Granulocyte colony-stimulating factor (G-CSF) enhances etoposide-containing standard chemotherapy when given until 48 hours before the next chemotherapy course.257

·     Anticonvulsivant therapy, when used concomitantly with etoposide, may induce hepatic enzyme induction, resulting in a higher etoposide clearance.238, 258

·     Warfarin. Etoposide might displace warfarin from its protein-binding sites, resulting in early elevation in prothrombin time.259 A close monitoring of the INR is recommended to adjust the dosage of warfarin.

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