Edwin W. Willems
Over several decades, microbial fermentation has yielded many valuable compounds, such as the anthracyclines, the bleomycins, and various unusual nucleosides, which are discussed in separate chapters. In this chapter we review two relatively long-standing antibiotics of diverse structure as well as one of the marine-originated ecteinascidins, namely ecteinascidin-743 (ET-743). Dactinomycin (actinomycin D; DACT) is still one of the most valuable drugs in pediatric oncology, whereas mitomycin C (MMC), although largely replaced by newer classes of agents, is still occasionally used in treating tumors of the gastrointestinal and respiratory tracts and for intravesicular treatment of bladder cancer. ET-743 is a promising anticancer agent with unique mechanisms of action.
DACT, a product of the Streptomyces yeast species, was discovered in 19401 and has since been identified as an active anticancer antibiotic for gestational choriocarcinoma, Wilms tumor, neuroblastoma, childhood rhabdomyosarcoma, and Ewing's sarcoma.2, 3 Numerous analogs have been isolated from various sources, but none has demonstrated superiority over DACT. Its key pharmacological features are given in Table 16.1.
MECHANISM OF ACTION AND CELLULAR PHARMACOLOGY
The structure of DACT4 is shown in Figure 16.1. It is a chromopeptide consisting of a phenoxazinone planar chromophore with two pentapeptide rings attached.3 Naturally occurring actinomycins differ in the peptide chains but not in the phenoxazone ring. DACT is known to be a strong DNA-binding drug and a potent inhibitor of RNA and protein synthesis. Actual binding to DNA was shown to be intercalative: the chromophore inserts in between the DNA guanine-cytidine base pairs, while the two chains of the pentapeptide rest in the minor groove.3 DACT can bind to both non-GpC and GpC-containing sequences. Interaction between GpC sequences leads to formation of two hydrogen bonds between each guanine and a pentapeptide.3 Besides binding to double-strand DNA, DACT is also known to bind to single-strand DNA (ssDNA).5 The overall association rate between DACT and DNA does not depend on polynucleotide sequence or length but probably reflects the summation of multiple sites of interaction with DNA.6 When bound to the ssDNA in the open complex formed by the polymerase, DACT prevents reannealing of ssDNA. Results suggest that stabilization of unusual ssDNA hairpins by DACT may be an important aspect of its potent transcription inhibition activity.7, 8
Since DACT is taken up in tissues by passive diffusion, its cytotoxic response depends on the ability of the cell to accumulate and retain the drug.9 It may be noted that increased temperature or alterations in the membrane lipid bilayer markedly enhance transport of DACT in different cells. DACT likely causes cell death by apoptosis, as demonstrated in a variety of cells both in vitro and in vivo.10 Although high doses of DACT inhibited growth in a human embryonal rhabdomyosarcoma cell line and induced cytotoxicity, at low doses the drug induced morphologic and phenotypic differentiation.8 Apparently, low-dose DACT releases these cells from their differentiation blockade, allowing them to recover normal myogenic development. This suggests a potential role for differentiation therapy in the treatment of rhabdomyosarcomas.
MECHANISM OF RESISTANCE
Resistance to DACT is related to increased efflux.11 For example, Chinese hamster ovary cells were found to be cross-resistant to DACT and to other drugs such as vinca alkaloids, anthracyclines, and epipodophyllotoxins.12, 13 In several instances, drug resistance could be overcome with verapamil hydrochloride.14 Human tumor cell lines made resistant to DACT in vitro were found to amplify the P-glycoprotein–encoding MDR gene.15 Resistance is reversed by drugs that inhibit P-glycoprotein function.16
TABLE 16.1 KEY FEATURES OF DACTINOMYCIN
No pharmacokinetic interactions between DACT and other drugs are known.
The pharmacokinetics of DACT have been studied in rat, monkey, and dog.17 In these species, serum levels of DACT declined rapidly after administration, with concomitant accumulation of drug in the tissues. The mean drug half-life in tissues was 47 hours, and metabolites have not been identified. Urinary excretion varies from 6 to 31%, and bile excretion varies from 5 to 11%. A very limited study in humans yielded similar results,18 with a very short half-life of distribution and a long plasma elimination half-life (36 hours). Urinary excretion and fecal excretion were 20% and 14%, respectively, and only 3.3% of the urinary excretion consisted of metabolites. Clearly, more extensive and detailed pharmacokinetic studies are required in humans.
By adsorbing DACT onto polybutylcyanoacrylate nanoparticles,19 one can achieve a significant increase of drug concentration in muscle, spleen, and liver in Wistar rats, whereas urinary excretion is diminished.20 Similar results were obtained by liposome entrapment of DACT; however, a slow-release system has not yet resulted in a higher efficacy.19 It has been demonstrated in humans that single-dose intermittent schedules or daily administration of DACT for 5 days produce similar antitumor activity without increased toxicity.21, 22, 23
Figure 16.1 Structure of dactinomycin. D-Val, d-valine; L-N-Meval, methylvaline; L-Thr, l-threonine; L-Pro, l-proline; Sar, sarcosine.
At the usual clinical dosages of 10 to 15 mg/kg per day for 5 days, DACT causes nausea, vomiting, diarrhea, mucositis, and hair loss. The major and dose-limiting side effect is myelosuppression, with a white blood cell and platelet nadir occurring 8 to 14 days after drug administration.23 Drug extravasation results in soft tissue necrosis.23 In rare cases, DACT treatment leads to severe hepatotoxicity with features of veno-occlusive disease, as in children treated for Wilms tumor.24 DACT can act as a radiosensitizer and may cause radiation recall phenomena, in which patients receiving DACT experience inflammatory reactions in previously irradiated sites.25 The clinical consequences of such reactions may be serious, especially with the involvement of lung. Corticosteroids may ameliorate these reactions.
Mitomycin C (Mutamycin; MMC) was isolated from Streptomyces caespitosus in 1958.26 The initial clinical studies used daily low-dose schedules, which resulted in unacceptably severe, cumulative myelosuppression. Later, an intermittent dosing schedule was introduced, using bolus injections every 4 to 8 weeks, which resulted in more manageable hematological toxicity. With the latter schedule, MMC was found to be active against a wide variety of solid tumors, including breast cancer, non–small cell lung cancer, gastric cancer, pancreatic cancer, gallbladder cancer, colorectal cancer, cervical cancer, prostatic cancer, and superficial bladder cancer. In addition, MMC is used as a radiosensitizer for the treatment of epidermoid anal cancer.27 Its key pharmacological features are given in Table 16.2.
MECHANISM OF ACTION AND CELLULAR PHARMACOLOGY
MMC (Figure 16.2) and other mitomycins have unique chemical structures in which quinone, aziridine, and carbamate functions are arranged around a pyrrolo [l,2- a]indole nucleus.28 They are the only known naturally occurring compounds containing an aziridine ring. MMC is soluble in both aqueous and organic solvents. However, because of its chemical instability in solution, the clinical formulation of MMC is a lyophilized form containing mannitol (Mutamycin) or sodium chloride (Mitomycin Kyowa) as excipients. After dissolution in water, MMC is administered intravenously. The stability of the reconstituted solutions is limited. Storage of MMC at room temperature in unbuffered solutions (pH 7.0) for 5 days seems justified.29
TABLE 16.2 KEY FEATURES OF MITOMYCIN C
Formation of DNA Adducts
MMC cross-links complementary strands of DNA but also induces monofunctional alkylation, with attachment to a single DNA strand.30 It primarily acts as a DNA replication inhibitor, and although monofunctional alkylation is by far the most frequently observed interaction, DNA interstrand cross-linking is considered to be the most lethal type. DNA cross-linking and alkylation require chemical or enzymatic reduction of the quinone function. The primary mechanism of this process involves the C-1 aziridine and the C-10 carbamate groups, although several additional reactive electrophiles, such as a quinone methide and the oxidized forms of aziridinomitosene and leuco-aziridinomitosene, may alkylate DNA as well.31, 32 Several MMC-induced DNA cross-links have been identified,33 including 2,7-diaminomitosene, which specifically alkylates guanines in (G)n tracts of DNA. Selective removal of the aziridine function of MMC results in a switch from minor to major groove alkylation of DNA.34 Acidic activation of MMC is the second mechanism by which DNA alkylation can be produced.
MMC is considered the prototypical bioreductive alkylating agent. Two mechanisms exist through which reductive metabolism mediates the cytotoxic effects of MMC.34, 35, 36, 37 First, under anaerobic conditions, one- or two-electron reduction followed by spontaneous loss of methanol leads to the formation of reactive unstable intermediates.
The suggested mechanism included initial formation of a hydroquinone and its rearrangement to yield a quinone-methide followed by a nucleophilic attack of DNA leading to a monoalkylated product. Intramolecular displacement of the carbamate group would then result in a second reactive site that produces a cross-linked adduct. Although the cross-linking of MMC to DNA in viable cells and cell extracts. In vitro reactions depend on the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) that are capable of activating MMC have been prepared, reproducing the process in models in vitro has been difficult. Although MMC reduction can be easily accomplished, covalent binding to DNA has been difficult to reproduce in cell-free systems. The addition of a reducing agent increases the binding to DNA by creating conditions favorable for the maintenance of the semiquinone radical, the intermediate that is formed by the first electron uptake of MMC. For this reason the semiquinone is believed to bind initially to DNA. It appears that one-electron reduction is sufficient to activate both the C-1 and C-10 electrophilic centers.38
Figure 16.2 Structure of mitomycin C.
Under aerobic conditions a second mechanism comes into play through which MMC develops its cytotoxic effect. Reductive metabolism again leads to the formation of reduced MMC; however, its aerobic fate is different. Molecular oxygen reacts with either the short-lived semiquinone radical or the hydroquinone form to generate the superoxide radical anion, hydroxyl radicals, or hydrogen peroxide.39 Formation of these highly reactive species may lead to cytotoxic effects such as lipid peroxidation or nucleic acid damage and can be prevented by free radical scavengers such as mannitol as well as by protective enzymes such as superoxide dismutase or catalase. Whether the reactive intermediate of MMC is formed through the radical semiquinone or the dianion (hydroquinone form) depends on the half-life of the radical anion. In an aprotic environment, the radical anion may have a considerable lifetime; in protic media, however, it exists only a few milliseconds, with rapid uptake of a second electron. Furthermore, oxygen definitely plays an important role, as it is a specific inhibitor of the two-electron pathway because of interaction with and inactivation of the semiquinone species by oxygen.
Several enzyme systems capable of activating MMC include NADPH-cytochrome P-450 reductase, xanthine oxidase, and xanthine dehydrogenase.35, 39 However, a controversial aspect of the bioreductive activation of MMC concerns the role of an enzyme called DT-diaphorase (DTD).31, 34, 35, 36, 37 DTD is an obligate two-electron reductase that is characterized by its ability to use both the reduced form of nicotinamide adenine dinucleotide (NADH) and NADPH as electron donors and by its inhibition by dicumarol.39 Both MMC-induced cytotoxicity and induction of DNA interstrand cross-links were found to be DTD-dependent and could be inhibited by pretreatment of HT-29 colon carcinoma cell lines with dicumarol.40 The ability of DTD to metabolize MMC to a reactive cytotoxic species suggests that the level of DTD may be an important determinant of the antitumor activity of MMC.
The NADPH–cytochrome P-450 reductase is a flavoprotein containing 1 mole each of flavin mononucleotide and flavin adenine dinucleotide, and its function is to transfer electrons from NADPH to the various forms of cytochrome P-450. The enzyme was shown to be involved in MMC activation to toxic species, with greater cytotoxicity occurring under hypoxic conditions.41, 42 However, similar effects were also shown to occur independent of NADPH–cytochrome P-450.43These and other data may suggest that the enzymes involved in the reduction of MMC under hypoxic conditions may not be the same as those observed under aerobic conditions or that the products of reduction, and hence those responsible for alkylation, may differ.
Analysis of DNA Adducts
Several studies have been published on covalent interactions between MMC and DNA or DNA fragments.33, 44 The actual binding site of MMC in DNA is the N-6 position of adenine residues or either the N-2 or N-7 position of guanine residues. Acid-activated MMC was found to alkylate preferentially the guanine N-7 position, in contrast to reductively activated MMC, which preferentially alkylates the guanine N-2 position, possibly because of the different electronic structures of acid-activated and reduction-activated MMC. The activation mechanism of MMC can presumably now be evaluated from analysis of the DNA adducts formed in vivo.
Induction of Apoptosis
Most anticancer drugs kill cells by apoptosis. The intrinsic threshold of a particular cell for induction of apoptosis may determine its sensitivity to the killing effects of drugs and may constitute an alternative mechanism of drug resistance. A number of (proto) oncogenes and tumor suppressor genes influence the apoptotic pathway, including most prominently p53 and its downstream effector p21.45
Most anticancer drugs are mutagenic, and the alkylating agents in particular have been subject to research on mutagenicity. Although the majority of DNA damage is repaired by the different cellular DNA repair systems, persisting DNA lesions may lead to enhanced mutagenesis owing to the occurrence of errors on replication of a damaged template. Monofunctionally activated MMC has been shown to cause a substantial increase in the mutation frequency in human Ad293 cells transfected with a shuttle vector plasmid pSP189.46 The observed bias of mutations of G:C and the formation of guanine monoadducts suggest that monoadducts may be responsible for the mutations. In Saccharomyces cerevisiae strains, increased mutagenic activity of MMC in terms of frequencies of reversion of mitotic gene conversion and reversion was observed at applied elevated cytochrome P-450 and glutathione contents,47 which confirmed the relevance of metabolizing enzymes and mitochondrial function in MMC's mechanism of action.34[IS1]
MECHANISM OF RESISTANCE
The mechanisms of resistance to MMC are incompletely understood but probably involve changes in drug accumulation, bioactivation, inactivation of the alkylating species, and DNA excision repair. In a series of Chinese hamster ovary cell mutants selected for MMC resistance, a progressive loss of MMC activation capacity and increased capacity for excision repair of DNA was found as cells became more drug resistant.48 The specific bioactivation enzyme system deficient in the resistant cells was not identified in these studies, although the primary activation mechanism in the sensitive parent was sensitive to dicumarol and, therefore, probably DTD. In some resistant cell lines, MMC shares in the MDR phenotype that encompasses doxorubicin, vincristine, and other natural products and that is mediated by overexpression of the drug efflux protein P-170.49 On the other hand, several drugs known to reverse MDR induced by other drugs were not capable of reversing MMC-induced MDR, which suggests other pathways contributing to the process.50 One subline of the HCT 116 human colon carcinoma cell line resistant to MMC had an increased expression of a 148,000–molecular-weight membrane protein, the level of which correlated with the degree of MMC resistance.51 The investigators observed no in vitro cross-resistance to other natural product–type cytotoxic agents. The increased expression of this cell surface protein in drug-resistant phenotypes may be a useful marker for MMC resistance.
MMC has a biexponential decline of the plasma concentration time curves, which corresponds to a two-compartment model with linear pharmacokinetics up to doses as high as 60 mg/m2.52, 53 After a rapid distribution half-life (2 to 10 minutes), the elimination half-life is 25 to 90 minutes (mean, 54 minutes). A remarkable observation in two studies was an unexplained increase in total-body clearance and a decrease in the area under the plasma concentration time curve of MMC after combination chemotherapy that included 5-fluorouracil and doxorubicin.52, 54 No correlations have been found between pharmacokinetic data for MMC and a wide variety of clinical parameters. Most important, impaired liver or renal function does not seem to change the pharmacokinetic behavior of MMC, and therefore neither impairment requires dosage reduction. Urinary recovery after intravenous administration ranged from 1 to 20%, which cannot explain the rapid plasma clearance. Therefore, the suggestion has been made that MMC is rapidly cleared from plasma by metabolism. The liver is thought to be the major organ of biotransformation,55 but the spleen, kidney, brain, and heart may also be involved in this process. The presence of oxygen markedly reduced the rate of metabolism of MMC in liver homogenates, compared with metabolism in a similar but anaerobic system. As biotransformation is required for activity, this supports the theory of a more pronounced metabolic activation under anaerobic conditions.
MMC is erratically absorbed after oral administration. Intravesical MMC therapy to treat superficial bladder cancer results in extremely low plasma levels, with virtually no systemic side effects and a significant exposure at the target site (bladder).56, 57 MMC uptake in bladder tissues is linearly related to drug concentration in urine.58 MMC administered intraperitoneally is rapidly absorbed through the serosal surface into plasma, and hence effective control of local lesions through attainment of high drug levels in the peritoneal cavity is infeasible.59
The most significant and frequent side effect of MMC is a delayed myelosuppression, which seems to be directly related to schedule and total dose.60 Below a total dose of 50 mg/m2, hematological toxicity is rare. At higher doses thrombocytopenia is more frequent than leukocytopenia and anemia. Other toxic reactions usually include mild and infrequent anorexia, nausea, vomiting, and diarrhea. Alopecia, stomatitis, and rashes also occur infrequently. Extravasation results in tissue necrosis, with very disabling ulcers that may require plastic surgery. High doses of MMC may result in lethal veno-occlusive liver disease.61Other more frequent and potentially lethal side effects include hemolytic uremic syndrome (HUS), interstitial pneumonitis, and cardiac failure. The incidence of MMC-induced HUS seems to be less than 10% and is dose dependent, mainly occurring at cumulative doses >50 mg/m2.62, 63 No consistently effective treatment for this syndrome is available. It may be noted that red blood cell transfusion should be avoided.
Pulmonary toxicity of MMC consists of an interstitial pneumonitis.63 Discontinuation of MMC administration may occasionally lead to recovery, and corticosteroid treatment may be helpful in preventing progression of pulmonary dysfunction. The incidence of pulmonary toxicity is approximately 7% of the treated population. Cardiac failure secondary to MMC occurs in a similar percentage, and the incidence rises with cumulative doses >30 mg/m2.64
In the late 1960s, extracts of the Caribbean marine tunicate Ecteinascidia turbinata were found to be active as inhibitors of cell proliferation. However, it was not until the last decade that the active compound, ecteinascinedin-743 (ET-743; Yondelis, trabectedin; NSC648766) was isolated, purified, and synthesized;65, 66, 67 see Figure 16.3 for its chemical structure. ET-743 belongs to the class of the tetrahydro-isoquinoline compounds, which also comprise antibiotic agents such as saframycins, safracins, and naphthyridinomycins. Most likely, ET-743 is produced by the marine tunicate as a defense mechanism to survive in its natural environment.65, 68
ET-743 produces 50% cell death at low picomolar or nanomolar concentrations in a wide variety of in vitro models.65, 67, 69 ET-743 also displayed in vivo activity in preclinical tumor models of human ovarian, breast, non–small cell lung, melanoma, sarcoma, and renal cancer.67, 69, 70 Antitumor effects of ET-743 were observed in clinical phase I trials.67, 71 Currently, phase II/III clinical trials with ET-743 are ongoing, and these suggest activity in soft tissue sarcomas, ovarian and breast cancer, and osteosarcoma.66, 72, 73 Its key pharmacological features are given in Table 16.3.
MECHANISMS OF ACTION AND CELLULAR PHARMACOLOGY
The exact mechanism by which ET-743 exerts its antitumor activity is not completely understood, but DNA appears to be the primary target.
DNA Minor Groove Binding
It seems quite clear that the cytotoxic effects of ET-743 result from the selective alkylation (i.e., causing DNA adducts) of the N2 amino group of guanine in GC-rich regions of the minor groove of DNA (5′-PuGC-3′ or 5′-PyrGG-3′), similar to what was previously reported for the structurally related antitumor antibiotics saframycin, quinocarmycin, and naphthyridinomycin.69, 74, 75 This type of alkylation depends on dehydration of the carbinolamine functional group of ET-743 leading to the formation of an iminium intermediate that subsequently reacts with and causes binding to DNA.75 However, the DNA sequence selectivity of ET-743 differs from other alkylating agents, such as CC1065 or tallimustine (FCE 24517), that covalently bind to N3 of adenine in AT-rich regions.76, 77, 78 Two subunits of ET-743 (A and B) are responsible for DNA recognition and bonding, while the third subunit (C) does not have contact with DNA and protrudes out of the minor groove;75, 78, 79 the C-unit may directly interact with transcription factors.78 Differences in DNA alkylating sequence selectivity and DNA bending direction, as well as the fact that the DNA bond is reversible upon denaturation, also distinguish ET-743 from other DNA alkylating agents;66, 72 these differences may partly explain the observed differences in antitumor activity and toxicity. Moreover, the reported structural changes induced by ET-743 could be important in affecting the recognition and binding of transcription factors or DNA-binding proteins.
Figure 16.3 Structure of ET-743.
TABLE 16.3 KEY FEATURES OF ET-743
Recognition of Transcription Factors and DNA-Binding Proteins
Many DNA-binding drugs interfere with important cellular functions, such as DNA repair, replication, and transcription.68, 80 Therefore, an altered transcription of genes that mediate drug action and apoptosis can negatively impact on therapeutic intervention, including in cancer. As a consequence, impairment of the complex interactions between activators (i.e., promoters and enhancer elements) and their DNA targets (i.e., gene-specific DNA-binding proteins, generally transcription factors) could alter the pattern of gene expression.
Several studies have elucidated the inhibitory effect of ET-743 on DNA binding of transcription factors, such as oncogene products (e.g., MYC, c-MYB, and Maf), transcriptional activators regulated during the cell cycle (E2F and SRF), and general transcription factors (e.g., Sp1, TATA-binding protein (TBP), and NF-Y).65, 75 Inhibition of DNA binding for TBP, E2F, SRF, and NF-Y (and SCR, an important activator of the c-fosgene) was observed at higher ET-743 concentrations (>50 µM) than those required for cytotoxicity (<10 nM). This finding is interesting since NF-Y activates the CCAAT element that is present in 25% of eukaryotic promoters,68 including promoters that regulate genes in the cell cycle.68, 75 In addition, activation of the multidrug resistance gene (MDR1) and heat shock protein 70 (HSP70) promoters, both of which contain the CCAAT box, were significantly inhibited by ET-743, while leaving constitutive gene expression relatively unaffected.81, 82 Therefore, ET-743 is the first pharmacological agent that prevents the activation of MDR1 transcription by multiple stress inducers targeting induced NF-Y-mediated and HSP70-mediated transcription and is distinct from other DNA-interacting anticancer drugs.82 Interestingly, Martinez and colleagues (2001) have shown that nanomolar concentrations of ET-743 and its synthetic analog phthalascidin (Pt-650) both up-regulate and down-regulate the expression of a variety of genes, including those involved in DNA damage response, transcription, and signal transduction (e.g., protein tyrosine phosphatase, CCAAT displacement protein, and p21/waf1/cip1) in intestinal carcinoma HCT116 and breast MDA-MB-435 cell lines.82
The orphan nuclear receptor (SXR) regulates drug metabolism through induction of transcription of the gene for metabolizing cytochrome P-450 enzymes (e.g., CYP2C8 and CYP3A4) and the MDR1 gene that encodes P-glycoprotein (P-gp); P-gp regulates the efflux of a wide range of compounds, including anticancer drugs. ET-743 was found to inhibit SXR and, subsequently, suppress MDR1 gene expression.65, 83 These observations may be relevant in relation to the reported cytotoxicity of ET-743, either alone or in combination with other drugs (see below), since ET-743 could modulate the activity of other anticancer drugs. For example, ET-743 has been shown to inhibit paclitaxel-induced SXR activation and to repress MDR1 transcription through inhibition of SXR at concentrations similar to those required for the above-mentioned inhibition of trichostatin-induced MDR1 transcription.65
Interference with DNA Repair Pathways
The cytotoxicity of ET-743 has been investigated in cell lines with specific defects in DNA repair mechanisms.75, 76, 84, 85, 86 Cells deficient in DNA mismatch repair were as sensitive to ET-743 (but resistant to cisplatin) as proficient (control) cells. Thus, the loss of mismatch repair does not affect the cytotoxicity of ET-743. DNA-dependent protein kinase (DNA-PK) is involved in the DNA double-strand–break repair pathway, which is activated by ionizing radiation and alkylating drugs (chlorambucil). Cells lacking functional DNA-PK (or inhibited by wortmannin) were severalfold more sensitive to ET-743, although double-strand breaks could not be observed in these cells. Thus, loss of DNA-PK activity enhances ET-743′s toxicity.86 Nucleotide excision repair (NER) represents a major DNA repair system able to handle a broad variety of DNA damages, such as ultraviolet lesions and bulky chemical adducts (as seen with cisplatin and mitomycin treatments). NER-deficient cells are less sensitive to ET-743 than NER-proficient (control) cells, in contrast to the reported sensitivities of several other DNA alkylators.85 This result was totally unexpected, since it was thought that NER-deficient cells would be unable to recognize and process ET-743-induced DNA damage and would therefore be unable to survive. NER-deficient cells became sensitive to ET-743 when the mutant repair protein (and any one of the several potential mutations) was restored by transfection. Thus, an intact transcription-coupled NER pathway is essential to ET-743 activity and defects in that it confers resistance to ET-743, as shown for cisplatin in mismatch repair–deficient cells.76, 87 In fact, cisplatin-resistant ovarian carcinoma cells (with increased NER) are sensitive to ET-743.66, 72 The unusual pattern of ET-743′s sensitivity in NER-deficient cells emphasizes that ET-743 represents a new class of anticancer agents able to interact with DNA in a different way than other alkylating agents, which may explain its remarkable activity against tumors that are not sensitive to other DNA-interacting anticancer agents.
Cell Cycle Perturbation, Topoisomerase I, and the Microtubule Network
ET-743 causes perturbation of the cell cycle at specific phases. In vitro studies have demonstrated that ET-743 decreases the rate of progression of tumor cells through the S phase and causes prolonged p53-independent blockade in G2/M phase,77 giving rise to a strong apoptotic response. Interestingly, cells in G1 are more sensitive to ET-743 than cells in S phase or G2/M phase.65, 70, 85 ET-743 does not seem to mediate topoisomerase I–related effects, and its effect on tubulin is not likely related to an antitumor effect.65, 75
MECHANISMS OF RESISTANCE
Multidrug resistance (MDR) is a phenomenon displayed by many tumors and is characterized by various molecular adaptations in cancer cells to allow increasingly high doses of cytotoxic drugs.88 Although studies addressing mechanistic aspects of MDR and aberrant apoptosis in cancer cells have yielded valuable insights, only limited knowledge is available about underlying mechanisms involved in the resistance to ET-743. The relationship between ET-743 and MDR1 seems inconclusive.65, 83 ET-743 inhibits activation of expression of MDR1 (by trichostatin) in tumor cells but not activation of constitutive expression in normal cells. In some tumor cell lines, resistance was mediated through MDR, in others it was not.65 Defects in transcription-coupled NER confer resistance to ET-743, whereas the drug resistance by these cells could be reversed by transfection of the appropriate repair enzyme. In addition, so far, limited evidence is available that ET-743 is a substrate for any of the known ABC transporters (P-gp, MRP1, and BCRP). Moreover, ET-743 shows low or no cross-resistance with several standard chemotherapeutic agents.73
Since it has been demonstrated that ET-743, at least in vitro, is a substrate for CYP3A4 enzymes (see below), it might be expected that the well-known CYP3A4 enzyme inducer dexamethasone increases ET-743′s metabolism. However, a combination of both compounds in B16 tumor cells and osteosarcoma xenografts showed increased activity compared to ET-743 alone.65 This finding may be explained by ET-743–reduced CYP3A4 enzyme expression, repressed CYP3A4 SXR activation, or the presence of cytotoxic metabolites of ET-743 (e.g., N-desmethylyondelis; ET-729). Interestingly, Donald and colleagues (2003) demonstrated the protective effects of single high-dose dexamethasone on ET-743–induced hepatotoxicity in female rats89; this protective effect of dexamethasone could, however, not be mimicked in in vitro experiments using liver cells in culture.90
Due to its limited aqueous solubility, ET-743 has been formulated as a lyophilized pharmaceutical product containing 250 µg ET-743 per dosage unit, 250 mg mannitol (bulking agent), and 0.05 M phosphate buffer (pH 4.0) for stabilization.65 This formulation is light sensitive and stable at room temperature only for a few hours.
ET-743 is administered to cancer patients in µg/m2 dosages, resulting in relatively low plasma levels (pg/mL to ng/mL). Several quantitative bioanalytic methods have been developed that are accurate and sensitive, with the lower limit of quantitation in the therapeutic range, including high-performance liquid chromatography (HPLC) and HPLC combined with mass spectrometry.69, 91, 92
Even though metabolism is the most important route for ET-743′s elimination (<2% is excreted in the urine as unchanged drug),69 information on the metabolism of ET-743 is yet sparse and inconclusive. One possible explanation for this apparent discrepancy is that, owing to its relative high potency, metabolite concentrations in vivo are too low to detect with currently available bioanalytic assays. Even though data supporting the role of CYP3A4 enzymes in the disposition and/or antitumor activity of ET-743 in vivo are still lacking, in vitro studies have demonstrated that ET-743 is a substrate for CYP3A4 enzymes. When incubated with human or rat microsomes that express CYP3A4, ET-743 is metabolized into three species, N-desmethylyondelis (ET-729) and two oxidative degradation products. Rat and human microsomal metabolism of ET-743 was reduced in the presence of chemical CYP3A4 inhibitors or antirat CYP3A2 antiserum and to a much lower extent by CYP2E and CYP2A inhibitors.93 In human liver panel studies, ET-743 disappearance was highly correlated with CYP3A4 activities. Glucuronidation does not seem to be an important detoxification route for ET-743.94
Preclinical studies have demonstrated that exposure time represents an important parameter for the chemosensitivity of cancer cells to ET-743. The drug proved to have a more favorable in vitro therapeutic index when the duration of exposure was extended from 1 hour to periods ranging from 24 hours to 3 days. Many clinical phase I studies with ET-743 have been performed with different infusion schemes: 1-hour, 3-hour, 24-hour, daily times 5, and 72-hour infusion.65, 95 In phase I studies, common pharmacokinetic (PK) parameters, such as clearance, terminal elimination constant (half-life; t1/2), area under the concentration time curve (AUC), maximal concentration (Cmax), and apparent volume of distribution at steady state (Vss), range from 21 to 86 L/h, 26 to 89 h-1, 36 to 55 h•ng/mL, 0.32 to 17 ng/mL, and 808 to 3900 L, respectively. It may be noted from these values that ET-743 is extensively distributed in the body (high Vss), which is reflected by a slow redistribution and elimination (long t1/2), although high interpatient variability was observed. Primary dose-limiting toxicity included thrombocytopenia and neutropenia, hepatic toxicity (with 72-hour infusion), and fatigue. Hematological toxicity, which was reversible and dose-dependent, seems to be related to ET-743′s Cmax, while the AUC may be related to the hepatic toxicity observed with longer ET-743 infusions (e.g., 72-hour infusion). No correlations were observed87 between the grades of nausea, vomiting, or fatigue with either AUC or Cmax. Except for an observed decrease in clearance with increasing dose (1-hour infusion) and a disproportionate increase of AUC beyond a dose of 1050 µg/m2 (72-hour infusion), all phase I studies have shown dose-independent kinetics. Preliminary population PK modeling of the phase II study data (24-hour infusion) showed that ET-743′s PK profile was best described by a three-compartment model, with an comparable clearance (44 L/hour) as observed in phase I studies. In addition, a correlation between total plasma clearance and age was suggested. A dose of 1,500 µg/m2 was evaluated in different clinical phase II studies, and activity has been reported for soft tissue sarcoma, ovarian cancer, and breast cancer. Combination studies and phase III studies are currently ongoing.
With short ET-743 infusions, dose-limiting toxicity was hematological (e.g., thrombocytopenia, neutropenia), but with longer infusions, hepatic toxicity became dose-limiting. The latter consists of acute and reversible transaminitis and cholangitis, which is characterized by elevation of alkaline phosphatases, aspartate aminotransferase, and bilirubin (with peak levels on day 3) and a decrease of hepatic cytochrome P-450 (CYP) enzymes (CYP3A2, CYP1A1/2, and CYP2E1). Pharmacokinetic-pharmacodynamic analyses from clinical studies showed that increasing levels of alkaline phosphatases and aspartate aminotransferase correlated with increasing ET-743 doses and AUC, while neutropenia correlated with Cmax and AUC.96 Liver toxicity was also a consistent feature in studies with mice, rats, and monkeys.70 Donald et al. (2002, 2003) demonstrated that a high dose of dexamethasone protects against ET-743–induced hepatic toxicity in the female rat.25, 90, 97 Clinical studies carefully evaluating this finding in cancer patients are ongoing.
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