Kenneth D. Tew
O. Michael Colvin
Roy B. Jones
The alkylating agents are antitumor drugs that act through the covalent binding of alkyl groups to cellular molecules. This binding is mediated by reactive intermediates formed from a more stable parent alkylating compound. Historically, the alkylating agents have played an important role in the development of cancer chemotherapy. The nitrogen mustards mechlorethamine (HN2, “nitrogen mustard”) and tris(β-chloroethyl)amine (HN3) were the first non-hormonal agents to show significant antitumor activity in humans.1, 2, 3 The clinical trials of nitrogen mustards in patients with lymphomas evolved from the observation that lymphoid atrophy, in addition to lung and mucous membrane irritation was produced by sulfur mustard during World War I. Antitumor evaluation4 showed that the related but less reactive nitrogen mustards, the bischloroethylamines (Fig. 12.1), were less toxic and cause regressions of lymphoid tumors in mice. The first clinical studies produced dramatic tumor regressions in some patients with lymphoma, and the antitumor effects were confirmed by an organized multi-institution study.1, 2, 3 This demonstration of efficacy encouraged further efforts to find chemical agents with antitumor activity, leading to the wide variety of antitumor agents in use today. At present, alkylating agents occupy a central position in cancer chemotherapy, both in conventional combination regimens and in high-dose protocols with hematopoietic cell transplantation (HCT). Because of their linear dose-response relationship in cell culture experiments,5 these drugs have become primary tools used in HCT for a variety of diseases.
Mechanisms of Alkylating Reactions
Traditionally, the pharmacokinetics of alkylating reactions have been described as either a first-order process in which the rate of alkylating agent conversion to reactive intermediate determines the rate of reaction with cellular constituents, or as a second-order process in which tissue constituents must react directly with the intact alkylating agent, resulting in an unstable transition-state molecule (a reactive intermediate composed of the alkylating agent and cellular molecule) that decomposes to form the alkylated cellular constituent. Because alkylating agents are designed to produce reactive intermediates, the parent compounds typically have short elimination half-lives of less than 5 hours.
As a class, the alkylating agents share a common target (DNA) and are cytotoxic, mutagenic, and carcinogenic. The activity of most alkylating agents is enhanced by radiation, hyperthermia, nitroimidazoles, and by glutathione depletion. They differ greatly, however, in their toxicity profiles and antitumor activity. These differences are undoubtedly the result of differences in pharmacokinetic features, lipid solubility, ability to penetrate the central nervous system (CNS), membrane transport properties, detoxification reactions, and specific enzymatic reactions capable of repairing alkylation sites on DNA.6, 7 For example, the nitrosoureas produce a specific site of alkylation on the O-6 position of guanine; resistance to this group of agents is correlated with the presence of a guanine-O6-alkyl transferase.8 Application of techniques such as magnetic resonance imaging and mass spectrometry to the study of the alkylation mechanism and the chemical nature of the intermediates involved are making possible a detailed understanding of these reactions.9, 10 Such approaches, coupled with improved techniques of localizing and studying cellular damage11, 12 and determining sites and mechanisms of detoxification,13, 14, 15 should eventually make it possible to predict the sites of alkylation of an agent and to understand and modify the biologic consequences of such alkylations.
Figure 12.1 Structures of bischloroethylsulfide and bischloroethylamine. A. Bischloroethylsulfide (sulfur mustard). B. Bischloroethylamine (nitrogen mustard general structure).
Types of Alkylating Agents Used Clinically
The important pharmacologic properties of the selected clinically useful alkylating agents are summarized in Table 12.1.
The prototypic alkylating agents have been the bischloroethylamines or nitrogen mustards. The first nitrogen mustard to be used extensively in the clinic was mechlorethamine (Fig. 12.1), sometimes referred to by its original code name HN2 or by the term nitrogen mustard. The mechanism of alkylation by the nitrogen mustards is shown in Figure 12.2. In the initial step, chlorine is lost and the b-carbon reacts with the nucleophilic nitrogen atom to form the cyclic, positively charged, and very reactive aziridinium moiety. Reaction of the aziridinium ring with a nucleophile (electron-rich atom) yields the initial alkylated product. Formation of a second aziridinium by the remaining chloroethyl group allows for a second alkylation, which produces a cross-link between the two alkylated nucleophiles.
After introduction of mechlorethamine, a great many analogs were synthesized in which the methyl group was replaced by a variety of chemical groups. Most of these compounds proved to have less antitumor activity than mechlorethamine, but four derivatives seem to have a higher therapeutic index, a broader range of clinical activity, and can be administered both orally and intravenously. These drugs, which for the most part have replaced mechlorethamine in clinical use, are melphalan (L-phenylalanine mustard), chlorambucil, cyclophosphamide, and ifosfamide (Fig. 12.3). The latter two agents are unique in that they require metabolic activation and undergo a complex series of activation and degradation reactions (to be described in detail later in this chapter).
As can be seen from the structures, these derivatives have electron-withdrawing groups substituted on the nitrogen atom. This alteration reduces the nucleophilicity of the nitrogen and renders the molecules less reactive. Melphalan and chlorambucil retain alkylating activity and seem to be more tumor-selective than nitrogen mustard. Cyclophosphamide and ifosfamide, on the other hand, possess no alkylating activity and must be metabolized to produce alkylating compounds. Cyclophosphamide has been the most widely used alkylating agent and has activity against a variety of tumors.16 In 1972, ifosfamide,17an isomeric analog of cyclophosphamide, was introduced into clinical use. It has greater activity against testicular cancer and soft tissue sarcomas.18, 19Melphalan has been widely used in the treatment of ovarian cancer,20 multiple myeloma,21 and carcinoma of the breast.22 Chlorambucil has been most widely used in the treatment of chronic lymphocytic leukemia,23, 24 lymphomas,23, 25 and ovarian carcinoma26 but it is unavailable in intravenous form and its use has declined sharply in recent years with the development of more effective treatments for each of the listed diseases. Both intravenous melphalan and cyclophosphamide are now heavily used in high-dose regimens combined with HCT.
The aziridines are analogs of the putative ring-closed intermediates of the nitrogen mustards but are less reactive chemically. Compounds bearing two or more aziridine groups, such as thiotepa (Figure 12.3 [thiotepa, triethylenethiophosphoramide]),27, 28, have shown clinical activity against human tumors. Thiotepa is also used primarily as a component of HCT regimens.
This aziridine compound was originally tested for antitumor activity because the nitrogen mustards alkylate through an aziridine intermediate. Both thiotepa and its primary desulfurated metabolite TEPA (triethylenephosphoramide) have cytotoxic activity in vitro. Although the mechanism of action of these compounds has not been explored thoroughly, they presumably alkylate through opening of the aziridine rings, as shown for the nitrogen mustards. The reactivity of the aziridine groups is increased by protonation and thus is enhanced at the low pH more characteristic of tumors than normal tissues.
TABLE 12.1 KEY FEATURES OF SELECTED ALKYLATING AGENTS
Alkyl Alkane Sulfonates
The major clinical representative of the alkyl alkane sulfonates is busulfan (Fig. 12.4), which is widely used in HCT regimens for the treatment of acute and chronic myelogenous leukemia.29 Its alkylation mechanism is shown in Figure 12.5. Compounds with one to eight methylene units between the sulfonate groups have antitumor activity, but maximal activity is shown by the compound with four methylene units.30, 31
Busulfan exhibits second-order alkylation kinetics. The compound reacts more extensively with thiol groups of amino acids and proteins32 than do the nitrogen mustards, and these findings have prompted the suggestion that the alkyl alkane sulfonates may exert their cytotoxic activities through such thiol reactions along with interactions with DNA.32, 33 Brookes and Lawley34 were able to demonstrate the reaction of busulfan with the N-7 position of guanine, and Iwanoto et al.35 have suggested that adenine-to- guanine cross-linking is correlated with the cytotoxic potential of busulfan. Busulfan is markedly cytotoxic to hematopoietic stem cells. This effect is seen clinically in the prolonged aplasia that may follow busulfan administration 36 and can be shown experimentally in stem cell cloning systems.37 The pharmacologic basis for this property of busulfan is not understood, but has stimulated the use of busulfan in HCT protocols.38
Figure 12.2 Alkylation mechanism of nitrogen mustards. (From Colvin M. Molecular pharmacology of alkylating agents. In: Cooke ST, Prestayko AW. Cancer and Chemotherapy, vol 3. New York: Academic Press, 1981:291.)
The nitrosourea antitumor agents in current use were developed after screening of methylnitrosoguanidine and methylnitrosourea at the Wisconsin Alumni Research Foundation (WARF) and demonstrated modest antitumor activity in experimental animal tumor models.39 Careful structure-function studies demonstrated that chloroethyl derivatives such as chloroethylnitrosourea and BCNU (Fig. 12.6) possess greater antitumor activity than methylnitrosourea or other alkyl derivatives, and that the nitrosourea derivatives are more active than the nitrosoguanidines.39, 40, 41 In addition to chloroethyl alkylating activity, the available nitrosoureas can also carbamoylate nucleophiles.42
Figure 12.3 Structures of four analogs of mechlorethamine and thiotepa.
Figure 12.4 Structure of busulfan.
These chloroethylnitrosoureas eradicated intracranially inoculated tumors41 because of their lipophilic character and ability to cross the blood-brain barrier. In its initial trials, BCNU showed significant activity against brain tumors, colon cancer, and the lymphomas.43, 44 Subsequently, cyclohexylchloroethylnitrosourea (CCNU, lomustine) and methylcyclohexylchloroethylnitrosourea (methyl-CCNU, semustine) (Fig. 12.6) demonstrated greater activity against solid tumors in experimental animals.45
The nitrosoureas show partial cross-resistance with other alkylating agents.41 A number of studies have confirmed that these drugs are indeed alkylating agents, and the mechanism of the alkylation reaction has been established (Fig. 12.7). BCNU is well recognized to cross-link DNA after the formation of monoadducts, particularly at the N7 position of guanine. As shown in Figure 12.7, the diazonium hydroxide intermediate formed during BCNU hydrolysis decomposes to form a 2-chloroethyl carbonium ion (or equivalent) capable of rapid alkylation. In a subsequent step occurring over hours, the chloride is displaced by electron-rich nitrogen on the complementary DNA strand base to form a cross-link. DNA-protein cross-links are also possible.46, 47
Isocyanates resulting from the spontaneous breakdown of many of the methyl- and chloroethylnitrosoureas are also shown in Figure 12.7. The role of isocyanate-mediated carbamoylation in antitumor effects is incompletely understood, but this activity may be responsible for many of the toxicities associated with nitrosourea therapy.48, 49, 50
Figure 12.5 Alkylation mechanism of alkane sulfonates. (From Colvin M. Molecular pharmacology of alkylating agents. In: Cooke ST, Prestayko AW. Cancer and Chemotherapy, vol 3. New York: Academic Press, 1981:291.)
Streptozotocin is a unique methylnitrosourea with methylating activity but without carbamoylating activity because the molecule autocarbamoylates through internal cyclization. It is active against islet cell carcinoma.51, 52 The dose-limiting toxicities in humans have been gastrointestinal and renal, and the drug has considerably less hematopoietic toxicity than the other nitrosoureas.
Currently, BCNU is used with HCT regimens for hematopoietic diseases.53 As predicted from the animal studies, the nitrosoureas have shown significant activity against brain tumors.54 When used as an adjuvant to radiation therapy, they enhance survival modestly in patients with grade III and IV astrocytomas.55 The severe hematopoietic depression (especially thrombocytopenia) and pulmonary toxicity produced by these agents are significant limiting factors in their use. BCNU-impregnated wafers implanted directly in brain tumors have been used for regional therapy.
Figure 12.6 Structures of nitrosoureas. BCNU, bischloroethylnitrosourea;CCNU, cyclohexylchloroethylnitrosourea.
Alkylating Agent–Steroid Conjugates
From the rationale that steroid receptors may serve to localize and concentrate appended drug species in hormone-responsive cancers, a number of synthetic conjugates of mustards and steroids have been developed. Of these drugs, two have made the transition into clinical application. Prednimustine is an ester-linked conjugate of chlorambucil and prednisolone. It appears to function as a prodrug for chlorambucil, releasing the alkylating agent after cleavage by serum esterases.56, 57 Estramustine is a carbamate ester-linked conjugate of nornitrogen mustard and estradiol. Serum esterases are prevalent and readily cleave the ester link of prednimustine with the ultimate release of the hormone and the active alkylating drug.
Prednimustine produces altered alkylating agent pharmacokinetics compared with unconjugated chlorambucil because the half-life is prolonged as a consequence of slow hydrolysis of the ester bond.56 In addition, the elimination phase of chlorambucil in patient plasma is significantly longer after administration of prednimustine than after chlorambucil.57 Thus, prednimustine acts as a prodrug, delivering alkylating components over a prolonged period.
Figure 12.7 Alkylation of nucleoside by bischloroethylnitrosourea (BCNU).
The pharmacology of estramustine is governed by the presence of the carbamate group in the steroid-mustard linkage. The altered alkylating structure appears to eliminate alkylating activity of the molecule.58, 59 Steroid hormone60 or other mitotic inhibiting activities61 may explain the activity of estramustine Neither prednimustine or estramustine is heavily used, but estramustine is being explored for treatment of prostate cancer in conjunction with radiotherapy.
Prodrugs of Alkylating Agents
Therapy with alkylating agents is compromised by their high level of toxicity to normal tissues and their lack of tumor selectivity. Cyclophosphamide and ifosfamide were prodrugs synthesized in the hope that high levels of phosphamidases in epithelial tumors would selectively activate the drugs.62 A variety of strategies for more selective delivery of alkylating agent to tumor have been explored including cleavable tumor-directed antibody-alkylating agent conjugates,63, 64 alkylating agent-glutathione conjugates, which might be selectively cleaved by glutathione transferase P1 expressed in high levels in tumor cells,64 or using viral vectors to deliver activating enzymes to tumor cells.65 The glutathione conjugate is being explored in randomized clinical trials.
Sites of Alkylation
Any alkylating agent producing reactive intermediates binds to a variety of cellular constituents66 including nucleic acids, proteins, amino acids, and nucleotides. As an example, the active alkylating species from a nitrogen mustard demonstrate selectivity for nucleophiles in the following order: (a) oxygens of phosphates, (b) oxygens of bases, (c) amino groups of purines, (d) amino groups of proteins, (e) sulfur atoms of methionine, and (f) thiol groups of cysteinyl residues of glutathione.67 This ranking, however, assumes there are no steric or hydrophilic/hydrophobic barriers to the tissue nucleophile, and this is seldom the case. In addition, glutathione conjugation is often favored in the presence of glutathione transferases, which offer catalysis. Thus, generalizations about alkylating agent targets are fraught with difficulty.13, 14, 15 In addition, it seems likely that a matrix of biochemical targets of alkylating agents may contribute to cytotoxicity, though DNA is generally favored as the primary target. Proof of this hypothesis may be emerging from three areas of research where cytotoxicity is correlated to: (a) activity of DNA repair enzymes, perhaps best shown for BCNU and repair by alkylguaninealkyltransferase (AGT),68 (b) changes in a matrix of genetic and epigenetic events measured and analyzed using gene expression arrays,69 and (c) direct measurement of specific DNA adducts using PCR or mass spectrometric analysis.70 The stringency of such analyses requires that alternative toxic pathways not involving DNA be excluded, a difficult requirement to meet. For this reason, mechanistic understanding of alkylating agent activity must be considered incomplete.71, 72, 73,74, 75, 76, 77
In the DNA molecule, the phosphoryl oxygens of the sugar phosphate backbone are obvious electron-rich targets for alkylation. A number of studies have shown that alkylation of the phosphate groups does occur78, 79 and can result in strand breakage from hydrolysis of the resulting phosphotriesters. Although the biologic significance of the strand breakage caused by phosphate alkylation remains uncertain, the process is so slow that it seems unlikely that it is a major determinant of cytotoxicity, even for monofunctional agents.80
Extensive studies with carcinogenic alkylating agents such as methyl methane sulfonate have shown that virtually all the oxygen and nitrogen atoms of the purine and pyrimidine bases of DNA can be alkylated to varying degrees. The relative significance to carcinogenesis or cytotoxicity of alkylation of each of these sites remains uncertain. Various reports have indicated that alkylation of the O-6 atom and of the extracyclic nitrogen of guanine may be of particular importance for carcinogenesis.81, 82, 83
Studies of the base specificity of alkylation by the chemotherapeutic alkylating agents have been much less extensive. Busulfan and mechlorethamine alkylate the N-7 position of guanine. Guanine cross-links (two guanine molecules abridged at the N-7 position by an alkylating agent) have been isolated from acid hydrolysates of the reaction mixtures.34
Reaction of the nitrogen mustard with native DNA, however, produces alkylation of the N-1 position of adenine in addition to N-7–alkylated guanine. The reason for the enhanced alkylation of the N-7 position of guanine is uncertain, but it may be the result of base stacking and charge transfer that enhance the nucleophilic character of the N-7 position.84 Melphalan preferentially alkylates guanine N-7 or adenine N-3.70
Base sequence influences the alkylating reaction. The N-7 position of guanine is most electronegative and, therefore, most vulnerable to attack by the aziridinium cation intermediate of the nitrogen mustards when the base is flanked by guanines on its 3′ and 5′ sides. The key site of DNA attack for the nitrosoureas as well as nonclassic methylating agents such as procarbazine and dacarbazine seems to be the O-6 methyl group of guanine.8 Enhanced repair of this site is associated with drug resistance.85 Thus, the preferred sites for alkylation vary by alkylating agent and chemical environment around the DNA base in question.
On the basis of their isolation of the guanines linked at N-7 by alkylating agents, Brookes and Lawley84, 86 postulated that the bifunctional alkylating agents such as the nitrogen mustards produced interstrand and intrastrand DNA-DNA cross- links and that these cross-links were responsible for the inactivation of the DNA and cytotoxicity. On the basis of the Watson-Crick DNA model, these authors suggested that appropriate spatial relationships for cross-linking by nitrogen mustards or sulfur mustard occurred between the N-7 positions of guanine residues in complementary DNA strands (Fig. 12.8).
The importance of cross-linking is supported by the fact that the bifunctional alkylating agents, with few exceptions, are much more effective antitumor agents than the analogous monofunctional agents, as originally described by Loveless and Ross.87 Furthermore, increasing the number of alkylating units on the molecule beyond two does not usually increase the antitumor activity of the compound.
Direct evidence that DNA cross-linking occurs as the result of treatment of DNA or cells with bifunctional alkylating agents was provided initially by relatively insensitive physical techniques, including sedimentation velocity studies and denaturation-renaturation studies.46, 87, 88, 89, 90, 91 These techniques, however, could not detect DNA interstrand cross-linking in mammalian cells exposed to therapeutic levels of alkylating agents in vitro or in tissues after in vivo drug administration. In 1976, a more sensitive assay for DNA interstrand cross-linking in cells, the alkaline elution method,92 was reported and had the necessary sensitivity to detect DNA cross-linking in cells and tumor-bearing animals exposed to minimal cytotoxic levels of alkylating agents.11, 93, 94 These studies, and others using ethidium bromide fluorescence to detect cross-links, have shown that DNA cross-linking by bifunctional alkylating agents correlates with cytotoxicity and that DNA in drug-resistant cells has lower levels of cross-linkage.95, 96 The alkaline elution technique also has detected DNA-protein as well as DNA-DNA cross-links,97 which supports data from previous investigators.98, 99, 100 The work of Ewig and Kohn97 suggests that DNA-protein cross-links do not play a major role in cytotoxicity.
Figure 12.8 Cross-linking of DNA by nitrogen mustard. (Modified and reproduced with permission from Brookes P, Lawley PD. The reaction of mono- and di-functional alkylating agents with nucleic acids. Biochem J 1961;80:486.)
In addition to these target effect-response studies, inactivation of the AGT promoter in gliomas is correlated with improved antitumor activity and survival in patients treated with BCNU.101 Becuause AGT repairs guanine alkylation products produced by BCNU, decreased enzyme activity would be expected to increase DNA alkylation, implying that DNA is a critical target for BCNU effects. Thus, evidence increasingly supports the hypothesis that DNA interstrand cross-linking is the major mechanism of alkylating agent cytotoxicity.
Work by Ludlum et al.47 and Kohn et al.46 suggests that the chloroethylnitrosoureas cross-link via a unique mechanism. The spontaneous decomposition of the chloroethylnitrosoureas generates a chloroethyldiazonium hydroxide entity48 that can alkylate DNA bases to produce an alkylating chloroethylamine group on the nucleotide in the DNA strand. This group could then alkylate an adjacent nucleotide on the complementary DNA strand in a slower step, producing an interstrand cross-link. The mechanism of alkylation by thiophosphates such as thiotepa likely begins with protonation of the aziridine N, which leads to ring opening. Cross-linking can proceed by one of several mechanisms, either activation of the free chloroethyl carbon or activation of a second aziridine ring on the original molecule. Although interstrand cross-links are important mediators of the cytotoxic effects of alkylating agents, the monofunctional DNA alkylations exceed cross-links in number and are potentially cytotoxic. This hypothesis is supported by the fact that certain clinically effective agents, such as procarbazine and dacarbazine, are monofunctional alkylating compounds and do not produce cross-links in experimental systems. The basis of the cytotoxic effects of monofunctional alkylation may be single-strand DNA breaks. Although apurinic sites in the DNA lead to spontaneous hydrolysis of an adjacent phosphodiester bond, this process is probably too slow to be of biologic significance.80 Endonucleases produce single-strand breaks at apurinic sites,102, 103however, and may be responsible for the toxic and therapeutic effects of the monofunctional agents. The presence of apurinic sites may produce cross-links, but the low frequency of these cross-links makes it unlikely that they are responsible for the antitumor activity of the monofunctional agents.104
Limited data suggest that alkylation is nonuniform along the DNA strand and may be concentrated in specific regions. One determinant of regional specificity of DNA alkylations may be chromatin structure12, 105; areas of active transcription seem to be most vulnerable. The impact of alkylation of specific DNA regions on cytotoxicity requires further study.
In summary, the preponderance of evidence supports the hypothesis that the major factor in the cytotoxicity of most of the clinically effective alkylating agents is interstrand DNA cross-linking, which results in inactivation of the DNA template, cessation of DNA synthesis, and, ultimately cell death. Cell checkpoint proteins, including most prominently p53, are responsible for the recognition of DNA alkylation and strand breaks. Recognition of DNA damage leads to a halt in cell-cycle progression and initiation of programmed cell death. Cells containing mutated p53 have greater resistance to alkylating agents.106
An increased knowledge of alkylation mechanisms and targets may make it possible to improve the therapeutic index of these agents. For example, the therapeutic index of alkylating agents should improve if the alkylation of tumor cells were increased without a simultaneous increase in normal tissue alkylation. This might be accomplished by coadministration of the relatively tumor-specific inhibitors of GST P1 which are now under study.107 As previously noted, GST inhibition increases the antitumor effectiveness of alkylating agents.
The uptake of alkylating agents into cells is a critical determinant of cellular specificity. The cellular uptake of only a few alkylating agents has been examined, however. Wolpert and Ruddon108 and Goldenberg and Vanstone109 demonstrated that the uptake of mechlorethamine by Ehrlich ascites tumor cells and by L5178Y lymphoblasts is by active transport systems. Melphalan is transported into several cell types by at least two active transport systems, which also carry leucine and other neutral amino acids across the cell membrane.110, 111, 112 High levels of leucine in the medium protect cells from the cytotoxic effects of melphalan by competing with melphalan for transport into the target cells.113 In contrast to mechlorethamine and melphalan, which are carried by active transport systems, the highly lipid-soluble nitrosoureas BCNU and CCNU enter cells by passive diffusion.114
Studies of cellular uptake of alkylating agents that require metabolic activation (such as cyclophosphamide or ifosfamide) are hampered by uncertainty about whether parent drug, metabolites, or a combination of both are the most critical moiety for transport.
The emergence of alkylating agent–resistant tumor cells is a major problem that limits the clinical effectiveness of these drugs. One mechanism for drug resistance is that of decreased drug entry into the cell. Numerous studies have shown that L5178Y lymphoblast cells resistant to mechlorethamine may have decreased uptake of the drug.100, 108, 115 The extracellular domain of the leucine-melphalan transporter expresses CD98.116 Reduced expression of CD98 on human myeloma cells is associated with melphalan resistance,117 suggesting that transport alteration may be an important resistance mechanism.
Among other mechanisms of resistance to alkylating agents, changes in sulfhydryl content have been implicated in experimental tumors. For example, the increased nonprotein sulfhydryl content of Yoshida sarcoma cells appears to be responsible for resistance to mechlorethamine.118 Calcutt and Connors119found that tumor cells resistant to alkylating agents often possessed a higher ratio of protein-free to protein-bound thiol compounds and suggested that the increased thiol content might function with and inactivate the alkylating agent intracellularly. Glutathione (GSH) is the major intracellular nonprotein sulfhydryl compound. Increased GSH content of melphalan-resistant ovarian carcinoma cells has been reported,120 which has led to the experimental use of buthionine sulfoximine, an inhibitor of glutathione synthesis, to reverse resistance to cyclophosphamide, melphalan, and the nitrosoureas in experimental tumors.121 This reversing agent has been studied clinically but may have limited usefulness as it depletes glutathione from both tumors and normal tissues. Although increased intracellular glutathione content may be found in resistant cells, additional enzymatic detoxification mechanisms that conjugate alkylating intermediates or metabolize them to inactive derivatives at an increased rate have been identified in drug-resistant mutants. Examples of such mechanisms include elevated glutathione transferase levels in mechlorethamine-resistant cells122 and increased aldehyde dehydrogenase activity, which converts aldophosphamide to its inactive carboxyphosphamide, in cells resistant to cyclophosphamide.123, 124, 125
Another potential mechanism to explain resistance of cells to alkylating agents is the enhanced repair of the lesions generated by alkylation. Because DNA appears to be the most critical target for the alkylating agents, the repair of DNA has been a major focus of study.11 Enhanced excision of alkylated nucleotides from DNA appears responsible for the resistance of bacteria to alkylating agents.46, 89, 90 Mammalian cells are capable of such excision repair of sulfur mustard–alkylated nucleotides.126
Repair of DNA alkylation products and cross-links often involves complex systems involving multiple enzymes. AGT (guanine-O6-alkyl transferase) repairs single-strand BCNU methylation products in nitrosourea-resistant cells and is an unusual example of a single enzyme repair process. Nucleotide excision repair (NER) is a primary mechanism for excising single-strand alkylation products and may involve up to 25 factors and steps.127 Interstrand cross-link repair is even more complex and involves some elements of the NER pathway, a variety of less well-understood activities to cleave the second strand, insertion of new bases, and homologous recombination. The complexity of these pathways has limited adequate analysis in this area. In addition, exposure to alkylating agents leads to induction of a series of complementary defensive responses, including decrease in drug-activating enzymes (the P-450 system), increase in glutathione transferase, and increase in DNA repair capacity. This pattern of changes has been most clearly demonstrated in preneoplastic liver nodules after exposure to alkylating carcinogens.128
As mentioned previously, apoptotic cell death after DNA damage is mediated through p53, which blocks cell-cycle progression, initiates attempts to repair damage, and ultimately activates apoptotic pathways. Defects in damage recognition or apoptotic signaling may lead to relative resistance.129 Multiple mechanisms of cellular resistance often occur in a given tumor cell population and are responsible for the drug resistance seen clinically. Goldenberg130 found that L5178Y lymphoma cells resistant to mechlorethamine are 18.5-fold more resistant to mechlorethamine than the wild-type sensitive cells but are uniformly only twofold to threefold resistant to a variety of other alkylating agents. On this basis, Goldenberg suggested that specific resistance to mechlorethamine occurred because of decreased transport into the cell, whereas the general cross-resistance was the result of other mechanisms, such as enhanced repair capacity. This hypothesis is consistent with the observation of Schabel et al.131 in experimental animal tumors and with clinical experience. In both situations, varying degrees of cross-resistance between alkylating agents are seen, but a tumor that is resistant to one alkylating agent may remain significantly responsive to another. This finding forms the rational basis for the use of combinations of alkylating agents in high-dose chemotherapy regimens with HCT.132
Reversal of Resistance
Because of the pivotal importance of GSH to alkylating agent detoxification, four separate approaches to modulation have been adopted: (a) precursors of GSH have been given to replete GSH in normal tissues, thus reducing the host toxicity; (b) specific inhibitors of GSH biosynthetic enzymes have been administered to decrease intracellular GSH; (c) inhibitors of detoxifying enzymes such as GSTs have been given to decrease the tumor cell's ability to protect itself against alkylating metabolites; and (d) other precursor thiols have been administered to protect normal tissues.
Because GSH cannot readily cross cell membranes, early efforts to increase intracellular GSH relied on administration of the constituent amino acids, especially cysteine. More recently, a number of monoesters of GSH have been synthesized that are able to traverse the cell membrane and enhance intracellular GSH.133 In animal studies, the GSH-monoethyl ester successfully modulated anticancer drugs such as BCNU, cyclophosphamide, and mitomycin C.134 Primarily, the ester protected liver, lungs, and spleen. At least in the murine system, it afforded no protection to marrow progenitor cells.
The obverse approach to repletion is depletion of GSH, in an attempt to gain a therapeutic advantage through specific effects in tumor cells. A number of agents, including diethylmaleate, phorone, and dimethylfumarate, have been used and, although successful in achieving tumor cell GSH depletion, have proved to be too toxic to use clinically. The toxicities and complications associated with the use of nonspecific depletors of GSH were circumvented by the design and synthesis of agents that acted as inhibitors of certain enzymes involved in the synthesis of GSH. Direct interference with GSH synthetase results in the buildup of 5-oxoproline, and this has the consequence of marked acidosis in patients.135 By far the most effective approach to reducing the GSH biosynthetic capacity of a cell has been achieved by administering amino acid sulfoximines,136, 137 which inhibit g-glutamylcysteine synthetase. The lead compound to emerge from these studies was L-buthionine (SR)-sulfoximine (BSO), the R-stereoisomer of which is the active inhibitor of g-glutamylcysteine synthetase.138 A large number of reports now describe low levels of GSH in unperturbed tumor cells adding to the rationale for BSO use to improve the therapeutic index of alkylating agents. Although BSO caused differential sensitization of tumors in animal models,139 trials in humans failed to clearly demonstrate therapeutic index improvement140, 141 and enthusiasm for clinical use of BSO has waned.
An alternative approach to decreasing GSH effects is to inhibit the enzymes that use GSH as a cofactor. Because GST over-expression was determined to be at least one contributing mechanism to the alkylating agent–resistant phenotype, a rationale was established for the use of GST inhibitors as modulating agents. Because GST P1 often dominates in tumors whereas other subtypes often dominate in normal tissues, inhibitors specific to GST P1 might offer a therapeutic index advantage. Initial studies employed a relatively nonspecific inhibitor of GSTs, ethacrynic acid, a Food and Drug Administration (FDA)-approved diuretic. Preclinical studies were promising,139, 142 but dose escalation was inhibited by diuretic complications. Poor specificity for tumor-associated GST has deterred further development. Specific inhibitors of GST P1 have now been developed and are being studied clinically.143 In a novel attempt to exploit the association between GST P1 and tumors, a unique drug that is activated by GST P1 to produce an alkylating agent in situ is also undergoing study144 and a Phase 3 trial in ovarian cancer is underway.
A very different approach to modulation of alkylator toxicity was suggested in studies by the United States Army, which examined over 4,000 synthetic thiol derivatives as radioprotectors.145 One of these compounds, WR2721, 5-2-(3-aminopropylamino)-has been shown to dephosphorylate selectively in normal tissues through catalysis by alkaline phosphatase.146 In most tumors, the relatively more acidic microenvironment is believed to inhibit WR2721 activation.147 WR2721 enhances the dose-modifying factor of cisplatin, melphalan, cyclophosphamide, nitrogen mustard, BCNU, and 5-fluorouracil in preclinical tumor models.184 In clinical trials WR2721, given as a single dose at 740 mg/m2before 1,500 mg/m2 of cyclophosphamide, does afford protection from granulocytopenia, decreasing the duration and increasing the nadir granulocyte count.148 More recent studies suggest that WR2721 decreases myelosuppression, neurotoxicity, and nephrotoxicity of the cisplatin-cyclophosphamide combination without compromising the antitumor effect. Hospers et al.149 have reviewed the recent clinical trials with WR2721 and alkylating agents. Additional studies suggest that coadministration of WR-2721 with high-dose melphalan and HCT may allow delivery of increased doses of melphalan to be delivered with no increase in toxicity.150
A modulatory approach specific for nitrosoureas has resulted from studies of DNA repair. Alkyl guanine alkyl transferase (AGT) binds irreversibly to O6-guanine alkyl adducts and removes them from DNA, inactivating itself in the process. Because an O6-guanine chloroethyl adduct is produced by BCNU and can lead in a subsequent slow step to DNA cross-linking, AGT inhibitors have been explored in combination with BCNU in cultured human leukemic cells. O6-benzylguanine was ultimately selected for further study based on a variety of structure-activity and binding studies. The enhancement ratios reported for O6-benzylguanine were equivalent or superior to those found for other modulating agents. Clinical evidence that response to nitrosoureas and methylating agents is inversely related to the tumor level of AGT supported further development.151 Unfortunately, a Phase 2 trial of O6-benzylguanine failed to demonstrate significant activity in patients with nitrosourea-resistant glioma.152
Ultimately, studies have shown that alkyl guanine alkyl transferase (AGT) inhibition by DNA methylation of its promoter improves the therapeutic activity of BCNU for brain tumors,101 and further studies of this promising combination are warranted. Recent supporting data indicates that methylation (silencing) of DNA coding for AGT is correlated with improved sensitivity of human gliomas for BCNU.153
The primary characteristics of the clinical pharmacokinetics of standard alkylating agents are given in Table 12.1. Although some agents are too reactive chemically to provide more than momentary exposure of tumor cells to parent drug (the best examples are mechlorethamine and BCNU), others are stable in their parent form and require metabolic activation, as in the case of cyclophosphamide and ifosfamide. The clinician must possess a working knowledge of the chemical and metabolic fate of individual alkylating agents to adjust doses for organ dysfunction and to plan rational treatment regimens.
Activation, Decomposition, and Metabolism
Decomposition Versus Metabolism
A principal route of degradation of most of the reactive alkylating agents is spontaneous hydrolysis of the alkylating entity (i.e., alkylation by water).154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 For example, mechlorethamine rapidly undergoes reaction to produce 2-hydroxyethyl-2-chloroethylmethylamine and bis-2-hydroxyethylmethylamine (Fig. 12.9).154 Likewise, both melphalan and chlorambucil undergo similar hydrolysis to form the monohydroxyethyl and bishydroxyethyl products, although less rapidly than the aliphatic nitrogen mustards.155, 156 The hydroxylated products are less active than their chloroalkyl precursors.
Most alkylating agents also undergo some degree of enzymatic metabolism. For example, if mechlorethamine radiolabeled in the methyl group is administered to mice, approximately 15% of the radioactivity can be recovered as exhaled carbon dioxide, which indicates that enzymatic demethylation is occurring.
Cyclophosphamide and Ifosfamide
The widely used drugs cyclophosphamide and ifosfamide are activated to alkylating and cytotoxic metabolites by cytachrome P-450s, particularly the P-450 3A4 subtype.165 The complex metabolic transformations that cyclophosphamide undergoes are illustrated in Figure 12.10.164, 166 The initial metabolic step is the oxidation of the ring carbon adjacent to the ring nitrogen to produce 4-hydroxycyclophosphamide, which spontaneously ring-opens and establishes equilibrium with aldophosphamide.
The 4-hydroxycyclophosphamide and aldophosphamide may be oxidized by soluble enzymes to produce 4-ketocyclophosphamide and carboxyphosphamide, respectively. These compounds have little cytotoxic activity and represent inactivated urinary excretion products. They account, between them, for approximately 80% of a dose of administered cyclophosphamide.167, 168
Figure 12.9 Hydrolysis products of mechlorethamine.
Figure 12.10 Metabolism of cyclophosphamide.
The 4-hydroxycyclophosphamide/aldophosphamide that has escaped enzymatic oxidation by aldehyde dehydrogenase can eliminate acrolein to produce phosphoramide mustard,169 an active alkylating agent that appears to be responsible for the biologic effects of cyclophosphamide.170, 171 The concentration of aldehyde dehydrogenase in a variety of cell types appears inversely proportional to cytotoxicity, supporting the pivotal role of aldehyde dehydrogenase in determining the cytotoxic specificity of cyclophosphamide.125 The high enzyme concentration in hematopoietic progenitor cells is suggested to explain the ability of cyclophosphamide to produce major myelosuppression without myeloablation in patients receiving high doses without transplantation.172 The 4-hydroxycyclophosphamide/aldophosphamide serves as a transport form to deliver the highly polar phosphoramide mustard efficiently into cells.
A related oxazaphosphorine, ifosfamide (Fig. 12.3), also requires P-450 for activation to its active intermediates, which are found in plasma and urine.173 As with cyclophosphamide, it undergoes hepatic activation to an aldehyde form that decomposes in plasma and peripheral tissues to yield acrolein and its alkylating metabolite.174 Hydroxylation proceeds at a slower rate for ifosfamide than for cyclophosphamide, which results in a longer plasma half-life for the parent compound. Dechloroethylation of ifosfamide produces inactive metabolites and competes with the activation step as a major pathway of elimination.175Both cyclophosphamide (above doses of 4 g/m2) and ifosfamide (above doses of 5 g/m2) exhibit dose-dependent nonlinear pharmacokinetics, with significant delays in elimination at higher doses.176 Interestingly, both drugs also induce their own metabolism, resulting in significant shortening of the elimination half-life for the parent compound when the drugs are administered on multiple consecutive days.177
The decomposition of nitrosoureas to generate the alkylating chloroethyldiazonium hydroxide entity48 has been mentioned, and the products generated by this decomposition in aqueous solution are illustrated in Figure 12.11. The nitrosoureas also undergo metabolic transformation. Hill et al.178 demonstrated that BCNU is enzymatically denitrosated by P-450, a finding of possible clinical significance.179 Enhancement of P-450 activity in vivo by phenobarbital abolished the therapeutic effect of BCNU against the 9L intracerebral rat tumor and decreased the therapeutic activity of CCNU and BCNU against this tumor. The phenobarbital-treated rats had increased plasma clearance of BCNU, with lower plasma levels and lower area under the concentration 3 time curve (AUC) plasma values of BCNU. The plasma clearance of parent BCNU decreases and the plasma half-life increases as doses escalate from standard-dose (150 to 200 mg/2) to high-dose regimens (600 mg/m2) (Table 12.1). CCNU and methyl-CCNU undergo hydroxylation of their cyclohexyl ring to produce a series of metabolites that represent the major circulating species after treatment with these drugs.180, 181 These metabolites have increased alkylating activity but diminished carbamoylating effects.182, 183
Figure 12.11 Decomposition of bischloroethylnitrosourea (BCNU) in buffered aqueous solution.
Because of the lack of definitive techniques for measuring certain specific drug and metabolite molecules, the data on the clinical pharmacology of the alkylating agents have been relatively limited. Recently, however, gas chromatography–mass spectrometry and high-pressure liquid chromatography (HPLC) have generated more definitive pharmacokinetic information (Table 12.1).
Several groups have examined the clinical pharmacology of melphalan. Alberts and colleagues184 studied the pharmacokinetics of melphalan in patients who received 0.6 mg/kg of the drug intravenously. The peak levels of melphalan, as measured by HPLC, were 4.5 to 13 mmol/L (1.4 to 4.1 mg/mL), and the mean terminal-phase half-life (t½β) of the drug in the plasma was 1.8 hours. The 24-hour urinary excretion of the parent drug averaged 13% of the administered dose. Inactive monohydroxy and dihydroxy metabolites appear in plasma within minutes of drug administration. Because renal excretion of drug is an unimportant pathway for drug elimination, full doses of drug are routinely given with hematopoietic cell transplantation in patients with complete renal failure.185 As renal insufficiency often occurs in patients with myeloma, this flexibility allows a standard transplant regimen to be given to these patients.
Other studies have demonstrated low and variable systemic availability of the drug after oral dosing.156, 186 Food slows its absorption. After oral administration of melphalan, 0.6 mg/kg, much lower peak levels of drug of approximately 1 mmol/L (0.3 mg/mL) were seen. The time to achieve peak plasma levels varied considerably and occurred as late as 6 hours after dosing. The low bioavailability was caused by incomplete absorption of the drug from the gastrointestinal tract, because 20 to 50% of an oral dose could be recovered in the feces.186 No drug or drug products were found in the feces after intravenous administration. Not only does oral melphalan show unpredictable bioavailability, but its AUC is reduced one-third by concomitant administration of cimetidine.187 Use of orally administered melphalan has declined steeply because of these bioavailability issues and because more effective therapies have been developed for breast and ovarian carcinomas and myeloma.
Regional administration of melphalan is possible by both intracavitary188 and limb perfusion methods.189
After the oral administration of 0.6 mg/kg of chlorambucil,156, 157 peak levels of 2.0 to 6.3 mmol/L (0.6 to 1.9 mg/mL) occurred within 1 hour. Peak plasma levels of phenylacetic acid mustard, an alkylating metabolite of uncertain but potential importance, ranged from 1.8 to 4.3 mmol/L (0.5 to 1.18 mg/mL), and the peak levels of this metabolite were achieved 2 to 4 hours after dosing. The terminal-phase half-lives for chlorambucil and phenylacetic acid mustard were 92 and 145 minutes, respectively. Less than 1% of the administered dose of chlorambucil was excreted in the urine as either chlorambucil (0.54%) or as phenylacetic acid mustard (0.25%). Approximately 50% of the radioactivity from carbon 14–labeled chlorambucil administered orally was excreted in the urine in 24 hours. Of this material, over 90% appeared to be the monohydroxy and dihydroxy hydrolysis products of chlorambucil and phenylacetic acid mustard. Thus, orally administered chlorambucil is absorbed more completely and more rapidly than melphalan and has a similar terminal-phase half-life.
The study of the clinical pharmacology of cyclophosphamide has been complicated by the inactivity of the parent compound and by the complex array of metabolites. These metabolites have proved difficult to isolate and measure, and their properties are not yet completely established. The pharmacokinetics and bioavailability of the parent compound have been well established by a number of studies177, 190, 191, 192, 193, 194, 195, 196 (Table 12.2).
Cyclophosphamide seems to be reasonably well absorbed after oral administration to humans. D'Incalci et al.177 found the systemic availability of the unchanged drug after oral administration of 100-mg doses (1 to 2 mg/kg) to be 97% of that after intravenous injection of the same dose. Juma and colleagues190 found the systemic availability of the drug to be somewhat less and more variable (mean, 74%; range, 34 to 90%) after oral administration of larger doses of 300 mg (3 to 6 mg/kg). A more recent comparison of oral versus intravenous cyclophosphamide in the same patient revealed no difference in the AUC for the primary cytotoxic metabolites, hydroxycyclophosphamide and phosphoramide mustard, after drug administration by the two different routes.196 After intravenous administration, the peak plasma levels of the parent compound are dose-dependent, with peak levels of 4, 50, and 500 nmol/mL reported after the administration of 1 to 2,177 6 to 15,190 and 60 mg/kg,191 respectively. The terminal-phase half-life of cyclophosphamide varies considerably among patients, with a range of 3 to 10 hours reported by a number of authors. In patients .19 years of age, the primary half-life for cyclophosphamide is 1.5 hours.195 Several investigators have reported increased clearance of cyclophosphamide on successive days of high-dose infusion,193 but this effect can be variable,197 suggesting that strategies of dose-adjustment aimed at providing uniform exposure of drug or metabolites may be difficult to achieve. Less than 15% of the parent drug is eliminated in the urine; the major site of clearance is the liver. The pharmacokinetics of the metabolites have been clarified in recent years. Initial measurements of total plasma alkylating activity showed considerable variation across patients, but similar ranges of alkylating activity of the equivalent of 10 to 80 nmol of nitrogen mustard per milliliter after doses of 40 to 60 mg of cyclophosphamide per kilogram have been found by several investigators.190, 198, 199 Peak alkylating levels are achieved 2 to 3 hours after drug administration, and Juma et al.190 found the terminal half-life of plasma alkylating activity to be 7.7 hours. All investigators have noted a plateau-like level of plasma alkylating activity maintained for at least 6 hours.
TABLE 12.2 CLINICAL PHARMACOKINETICS OF CYCLOPHOSPHAMIDE AND METABOLITES
The predominant metabolites found in plasma are nornitrogen mustard and phosphoramide mustard, with lesser concentrations of the putative transport forms aldophosphamide and 4-hydroxycyclophosphamide (Table 12.2). Of some significance is the fact that reports200 have questioned the reliability and applicability of earlier gas chromatography methods191, 201 for measuring levels of phosphoramide mustard and nornitrogen mustard in patient plasma. Because of these concerns, the actual quantitation of these metabolites and their half-lives (Table 12.2) remain approximate.
Fenselau et al.202 identified aldophosphamide as the cyanohydrin derivative in the plasma of patients receiving cyclophosphamide, and Wagner et al.203identified a mercaptan derivative of 4-hydroxycyclophosphamide in the plasma of cyclophosphamide-treated patients. Because the two primary metabolites are in equilibrium, the formation of either derivative should allow the measurement of the total of the two metabolites. Wagner et al.204 used the mercaptan derivatization technique to estimate that peak plasma levels of 1.4 and 2.6 nmol of total 4-hydroxycyclophosphamide/aldophosphamide per mL are achieved in humans after injection of doses of 10 and 20 mg of radiolabeled cyclophosphamide per kilogram, respectively. Subsequent studies have determined that 4-hydroxycyclophosphamide/aldophosphamide has a half-life of approximately 1.5 hours in children195 and 1 to 5 hours in adults receiving conventional196 or high-dose205 cyclophosphamide. The AUC for 4-hydroxycyclophosphamide and aldophosphamide at conventional doses of drug ranged from 3 to 19 nmol/mL 3 hours and seems to be independent of either peak plasma levels or the plasma half-life of the parent drug or hydroxycyclophosphamide.
Because the initial metabolism of cyclophosphamide is hepatic, modulation of the activity of P-450 in vivo might be expected to alter the pharmacokinetics of the drug. Pretreatment with phenobarbital, a known P-450 inducer, reduces the plasma half-life of the parent compound in both humans and experimental animals.206, 207 Also, with repeated doses of cyclophosphamide, the plasma half-life can be shown to become progressively shorter,177, 192 which indicates that cyclophosphamide can induce the P-450 enzymes responsible for its metabolism. The wide variation in the plasma half-life of cyclophosphamide seen in patients can be partly caused by differing previous drug exposure and the consequent differences in hepatic microsomal activity. For example, Egorin et al.208found consistently short plasma half-lives of cyclophosphamide (,2 hours) in a group of patients with brain tumors who had had long-term phenobarbital exposure. The net effect of P-450 induction on alkylating metabolite AUC's appears to be variable, however,209, 210 so the use of P-450 inducer pretreatment as a method to improve the therapeutic index of cyclophosphamide would require careful study.
Two authors have reported increased and prolonged plasma levels of cyclophosphamide metabolites in patients with renal failure, and on this basis, a reduction in dosage has been recommended for such patients.199, 211 Because the use of cyclophosphamide in patients with severe renal failure has been incompletely studied, and both parent drug and active metabolites are known to be renally excreted, caution should be used when cyclophosphamide is administered to such patients.212
The clinical pharmacology of ifosfamide has been studied by Creaven et al.213 and their colleagues199, 214, 215 and has been summarized by Brade et al.216 and Bagley et al.199 After single doses of 3.8 to 5.0 g/m2, the terminal half-life of ifosfamide was 15 hours, considerably longer than the previously cited values of 3 to 10 hours for cyclophosphamide. At ifosfamide doses of 1.6 to 2.4 g/ m2, however, the half-life of the drug was similar to that of cyclophosphamide. Creaven et al.213 found similar values for alkylating activity in plasma after the administration of 3.8 g/m2 ifosfamide and 1.1 g/m2 cyclophosphamide. Also, the alkylating activity excreted in the urine was similar for these doses of the two analogs and ranged from 6 to 15% for ifosfamide, although urinary excretion may approach 50% at high single doses.216 These findings are consistent with the previous results of Allen and Creaven,173 which indicate that P-450 activation of ifosfamide to alkylating metabolites proceeds more slowly than the activation of cyclophosphamide and that high doses of ifosfamide seem to saturate the activation mechanism. As with cyclophosphamide, ifosfamide clearance increases during continuous infusion or with multiple daily doses, reaching a steady state 2 to 3 days after drug administration is begun.217 Norpoth218 reported that cleavage of the chloroethyl group from the side chain and ring nitrogen is a quantitatively more significant pathway for ifosfamide metabolism than for cyclophosphamide metabolism in humans. Whereas less than 10% of an administered dose of cyclophosphamide is dechlorethylated,168 as much as 50% of a dose of ifosfamide may be excreted in the urine as dechlorethylated products. These findings suggest that the less rapid oxidative activation at C-4 of ifosfamide allows the chloroethyl group cleavage to become a significantly competing pathway in the in vivo metabolism of the drug. Although oxidation at C-4 of both cyclophosphamide and ifosfamide leads to ring opening and creation of compounds with alkylating activity, the products of side-chain cleavage have little alkylating activity. Thus, at doses below 3.8 g/m2, the rates of metabolism of cyclophosphamide and ifosfamide are similar, but a lower proportion of the ifosfamide is converted into alkylating and biologically active metabolites.
Because the slower activation rate of ifosfamide results in more prolonged exposure to the bladder-toxic metabolite acrolein, the glutathione analog MESNA is routinely administered in association with ifosfamide.
Studies using gas chromatographic analysis specific for thiotepa have revealed that it is rapidly desulfurated to TEPA and other alkylating species.219, 220, 221,222, 223, 224 The conversion of thiotepa to TEPA is mediated by P-450 as confirmed in vitro by incubation of thiotepa with hepatic microsomes. P-450-inducing agents increase thiotepa clearance, but the pharmacodynamic effects of this are unclear. Both thiotepa and TEPA have cytotoxic activity. Aside from individual variability, the plasma terminal half-life of intact thiotepa is a relatively consistent 1.2 to 2 hours. TEPA appears in plasma within 5 minutes of thiotepa administration. In 120 minutes its plasma concentration reaches that of thiotepa, but it persists longer, with a half-life of 3 to 21 hours, so that after 24 hours, TEPA concentration × time exceeds that of the parent drug. In 24 hours, only 1.5% of the administered thiotepa is excreted in the urine unchanged, together with 4.2% as TEPA and 23.5% as other alkylating species.219 The pharmacokinetics in children resembles that in adults.220, 223
Levin et al.225 studied the pharmacokinetics of BCNU in humans and found that after short-term infusion (15 to 75 minutes) of 60 to 170 mg/m2, initial peak levels of up to 5 µmol/L of BCNU were achieved. The plasma concentration decay curves were biexponential, with a distribution-phase half-life of 6 minutes and a second-phase half-life of 68 minutes. With high-dose BCNU, longer elimination half-lives of 22 to 45 minutes have been reported.226, 227
The pharmacokinetics of busulfan have been studied using a variety of sensitive methods.228, 229, 230, 231, 232 The parent compound can be measured by gas chromatography after derivatization230 or by HPLC using mass spectroscopic detection.233 Busulfan is routinely administered over 3 to 4 consecutive days, often with dosing every 6 hours. The drug bioavailability is often variable between patients.234 Because of this difficulty, when busulfan is used in high doses with hematopoietic cell transplantation either therapeutic drug monitoring, a recently approved parenteral busulfan formulation, or both are frequently used to assure uniform drug exposure.235 The drug exhibits circadian rhythmicity in its pharmacokinetics, particularly in children, with higher drug levels and slower elimination in the evening. The primary elimination half-life is approximately 2.5 hours in both children and adults, although interpatient variability is considerable at both low and high doses.
The relationship between intravenous dose and AUC within the same patient appears to be predictable over multiple days of administration. Because of variable bioavailability, there is less consistency when the oral formulation is used.236 Clearance declines with age, which leads to underdosing of children in high-dose regimens.237 Busulfan clearance for patients older than 18 years averages 2.64 to 2.9 mL/minute per kilogrAM, whereas for children aged 2 to 14 clearance averages 4.4 to 4.5 Ml/minute per kilogram, and for children age 3 or younger it is 6.8 to 8.4 mL/minute per kilogram.232 Thus, larger doses must be used in the younger age groups to achieve the desired cytotoxic exposure. Although data are incomplete, young children with hepatic disease (e.g., lysosomal storage disease) have a more prolonged half-life (4.9 hours) than their counterparts with normal liver function (2.3 hours).232 Because of its high lipid solubility and low level of protein binding, busulfan penetrates readily into the brain and cerebrospinal fluid. The ratio of drug concentration in cerebrospinal fluid to plasma approximates 1.238 Positron-labeled busulfan has been used to track uptake into the brain, revealing that approximately 20% of a standard dose rapidly enters the CNS.239 This access to the brain may enhance the activity of this drug against leukemia and lymphoma cells in the CNS, but it also may explain its propensity to cause seizures. Prophylaxis with anticonvulsants is required in patients receiving high-dose busulfan. Busulfan enhances the clearance of phenytoin (dilantin) and, in some patients, lowers the drug's plasma concentration below the therapeutic range, which increases the risk of seizures.240 Phenytoin levels should be monitored in the setting of busulfan therapy or an alternative, non–P-450 metabolized anticonvulsant should be used.
Pharmacokinetic studies have provided insight into both toxic and therapeutic effects of busulfan when used in high doses with hematopoietic cell transplantation. In this setting, busulfan produces hepatic veno-occlusive disease (VOD), which can be fatal. Grochow and colleagues231 have shown that excessive busulfan exposure increases VOD risk, and Andersson et al.241 have suggested that excessive exposure also increases the risk of graft-versus-host disease during allotransplantation. Alternatively, inadequate exposures may increase the risk of both leukemia relapse242 or allotransplant rejection.243 These data, taken together, highlight the importance of appropriate busulfan exposure during transplantation and the utility of therapeutic drug monitoring in this setting. The impact of PK-directed dosing of other alkylating agents may merit further study.
The usual dose-limiting toxicity of the alkylating agents is suppression of hematopoiesis. Characteristically, this suppression involves all formed elements of the blood: leukocytes, platelets, and red cells. However, the degree, time course, and cellular pattern of the hematopoietic suppression produced by the various alkylating agents differ. Clinically significant depression of the platelets may be seen when the dose of cyclophosphamide exceeds 30 mg/kg, but a relative platelet sparing is very characteristic of the drug.
Even at the very high doses (200 mg/kg or greater) of cyclophosphamide used in preparation for bone marrow transplantation, recovery of endogenous hematopoietic elements occurs within 21 to 28 days and has allowed these high doses to be used without transplantation in patients with aplastic anemia.172This stem cell-sparing property of cyclophosphamide is further reflected by the fact that cumulative damage to the bone marrow is uncommonly seen when cyclophosphamide is given as a single agent, and repeated high doses of the drug can be given without progressive lowering of leukocyte and platelet counts. In contrast to cyclophosphamide, busulfan seems to be especially damaging to bone marrow stem cells,37, 244 and prolonged or permanent hypoplasia of the bone marrow may be seen after busulfan administration. Melphalan seems to be more damaging to hematopoietic stem cells than cyclophosphamide, in that a longer recovery period for hematopoietic cells is seen both in animals245 and in humans, and a cumulative bone marrow depression may occur with repeated doses of melphalan.
The hematopoietic depression produced by the nitrosoureas is characteristically delayed. The onset of leukocyte and platelet depression occurs 3 to 4 weeks after drug administration and may last an additional 2 to 3 weeks.43 Thrombocytopenia appears earlier and usually is more severe than leukopenia. Even if the nitrosourea is given at 6-week intervals, hematopoietic recovery may not occur between courses, and the drug dose often must be decreased when repeated courses are used.
The biochemical basis for the stem cell-sparing effect of cyclophosphamide is now known to be the presence of a high level of the enzyme aldehyde dehydrogenase in the early bone marrow progenitor cells.246 This enzyme metabolizes the reactive intermediate aldophosphamide. The mechanistic and biochemical bases for the profound effect of busulfan and the nitrosoureas on marrow stem cells remain unknown.
Nausea and Vomiting
Although nausea and vomiting are not usually life-threatening toxic reactions, they are a frequent side effect of alkylating agent therapy and usually require potent antiemetics to control. The effectiveness of the newer centrally acting antiemetics suggests that the emetic effect of these drugs is at most only partially mediated by direct gastrointestinal effects.
Interstitial Pneumonitis, Lung Injury, and Fibrosis
Both BCNU247 and busulfan248, 249 have been observed to produce pulmonary fibrosis when administered in lower doses over prolonged periods. Busulfan is less frequently used in this dosing pattern in recent times, but BCNU is still used in this manner to treat brain tumors. Both drugs are being increasingly used in high dose over 1 to 4 days with hematopoietic cell transplantation. With this dosing pattern, busulfan produces much less lung toxicity. A dose-dependent pattern of acute lung injury from BCNU with HCT is well recognized, varying from a frequency of 5 to 60%, depending on the regimen and dose of BCNU administered.250 This acute injury is manifested by cough, dypnea, occasional fever, and frequently minimal radiographic findings.251 Although these problems can become life-threatening or progress to fibrosis, the acute injury is entirely reversible and treatable with prednisone if diagnosed early. Virtually all other alkylating agents have been associated with similar patterns of acute lung injury or fibrosis, though much less frequently than with BCNU or busulfan.
Renal and Bladder Toxicity
A toxicity that is relatively unique to the oxazaphosphorines (cyclophosphamide and ifosfamide) is hemorrhagic cystitis, which may range from a mild cystitis to severe bladder damage with massive hemorrhage.252, 253, 254, This toxicity is caused by the excretion of acrolein, a metabolite of both cyclophosphamide and ifosfamide in the urine, with subsequent direct irritation of the bladder mucosa.252, 255 This toxicity is usually seen within weeks of the administration of high-dose cyclophosphamide, but can be seen at any time after repeated doses of ifosfamide or lower-dose cyclophosphamide. In HCT-treated patients, bladder hemorrhage occurring later than 4 to 6 weeks after cyclophosphamide treatment is more likely the result of viral cystitis256 or coagulopathy. The most effective agent for preventing oxazaphosphorine-induced cystitis is 2-mercaptoethane sulfonate (MESNA), which dimerizes to an inactive metabolite in plasma but hydrolyzes in urine to yield the active parent that conjugates with alkylating species and prevents cystitis. MESNA should be administered routinely to all patients receiving ifosfamide and to any patient who has a history of drug-induced cystitis.257 MESNA is usually given in divided doses every 4 hours in dosages of 60% of those of the alkylating agent. Experiments in animals and clinical evaluation indicate that the systemic administration of sulfhydryl compounds does not impair the antitumor or immunosuppressive effect of cyclophosphamide.258, 259
Hemorrhagic cystitis is often a devastating, life-threatening, or extremely debilitating treatment complication, so great emphasis should always be placed on prevention. Chronic cystitis caused by cyclophosphamide has been associated with the later development of malignant transitional cell tumors of the bladder.260
At high doses of ifosfamide, severe renal tubular damage with elevation of serum urea and creatinine has been seen, and a Fanconi-like syndrome has been described after ifosfamide therapy.261, 262 High-dose cyclophosphamide can also produce the syndrome of inappropriate antidiuretic hormone excretion, resulting in transient water retention.263 Chronic administration of nitrosoureas can also infrequently produce renal damage.264, 265, 266 High-dose melphalan has also been associated with renal tubular injury and proteinuria.267
Alopecia and Allergic Reactions
Alopecia and allergic reactions have been associated with all alkylating agents. Alopecia is almost universal when alkylating agents are used in high doses with hematopoietic cell transplantation.
Alkylating agents have profound toxic effects on reproductive tissue; these are discussed in greater detail in Chapter 4. A depletion of testicular germ cells but preservation of Sertoli's cells was described by Spitz268 in the first extensive review of the histologic effects of mechlorethamine in patients. This toxic effect and its functional counterpart of aspermia have subsequently been well documented in both animals269 and humans.270, 271 The probability of aspermia increases with increasing dose and cumulative dose of alkylating agents. Because of the widespread availability of sperm banking, patients who will undergo treatment with alkylating agents and who wish to father children should be counseled to be evaluated for sperm banking prior to treatment. Oligospermia or aspermia are also associated with advanced malignancy. There are isolated reports of return of sperm and sperm function after complete aspermia induced by either conventional or high-dose alkylating agents.272, 273 Amenorrhea as a complication of busulfan therapy was reported by Galton et al.274 Several reports subsequently documented the high incidence of amenorrhea and ovarian atrophy associated with cyclophosphamide therapy.275, 276, 277, 278 A high incidence of amenorrhea after melphalan therapy also has been established. Pathologic examination of the ovaries after alkylating agent-induced amenorrhea reveals the absence of mature or primordial follicles. Endocrinologic studies demonstrate the decreased estrogen and progesterone levels and elevated serum follicle-stimulating hormone and luteinizing hormone levels typical of menopause. The risk of amenorrhea and infertility after alkylating agents increases with increasing age, as well as dose and cumulative dose of alkylating agents used. Preliminary data suggest that hormonal suppression of menses during alkylating agent treatment can increase the probability of later return of menstrual function, and further study is needed.
All alkylating agents are teratogenic.279, 280, 281 Studies have been carried out in a number of systems, both in vivo and in embryo culture in vitro.282, 283, 284,285 The teratogenic action seems to be the result of direct cytotoxicity to the developing embryo by the same mechanisms operative in tumor cells.286, 287
Because of the demonstrated teratogenicity of the alkylating agents in animals, appropriate concern has existed about the potential effects of their administration to patients during pregnancy. In 1968, Nicholson288 reviewed literature reports of women treated with cytotoxic agents during pregnancy. In the 25 instances in which the alkylating agents were given during the first trimester of pregnancy and the status of the fetus was recorded, 4 cases of fetal malformation occurred. No instances of malformed fetuses were reported when alkylating agents or other cytotoxic drugs were administered during the second or third trimester. Thus, administration of alkylating agents during the first trimester presents a definite risk of a malformed viable infant, but the administration of such drugs during the second and third trimesters may not increase the risk of fetal malformation above normal. Other reports confirm the risk of malformation in children born to mothers who had received chlorambucil,289 cyclophosphamide,290 or nitrogen mustard and procarbazine291 during the first trimester and the birth of normal infants to mothers receiving alkylating agents during the second or third trimester.292, 293
Carcinogenesis as a complication of cancer chemotherapy is covered in detail in Chapter 5. Case reports began appearing during the early 1970s of development of an aggressive acute myeloid leukemia in patients treated with alkylating agents. These leukemias are often characterized by a preceding phase of myelodysplasia, alteration of chromosome 5, 7 or 11, and a poor response to treatment. They usually occur between 1 and 5 years following alkylating agent treatment. Cases described have been in patients treated with melphalan,294, 295 cyclophosphamide,296, 297, 298 chlorambucil,299, 300 and the nitrosoureas.301 The frequency of so-called “secondary leukemia” varies with alkylating agent regimen, use of other carcinogenic treatments such as radiation, and the dose and schedule of the treatments. Less common use of chronic, low-dose alkylating agent therapy for myeloma and ovarian cancer may alter the incidence of secondary leukemia in these diseases.302 The routine use of sequential chemotherapy and radiation for treatment of Hodgkin's disease is being reduced because of concern for the increased level of secondary leukemia that these regimens produce.303 Other malignancies, including solid tumors, also have been reported to develop in patients treated with alkylating agents.303, 304 High-dose alkylating agent therapy with HCT produces an increased risk for a variety of solid tumors including lung, skin, and breast cancer, which frequently occur between 10 and 20 years following treatment. This observation reflects both the carcinogenic and curative potential of alkylating agents used in these regimens.
Organ Toxicity in High-Dose Chemotherapy
Alkylating agents have become a logical tool, either alone or in combination, for high-dose chemotherapy regimens.305, 306, 307, 308 Their use is often associated with nonoverlapping tumor resistance and log-linear increases in tumor killing with dose. In this high-dose setting, toxicities that affect the gut, lung, liver, and CNS become dose-limiting and life threatening. A list of the dose-limiting extramedullary toxicities of the alkylating agents is given in Table 12.3. Melphalan produces severe gastrointestinal toxicity.309 A number of alkylating agents, including the nitrosoureas, busulfan, thiotepa, and carboplatin produce venoocclusive disease of the liver.235, 310
The highly lipid-soluble alkylators, especially, busulfan, the nitrosoureas, and thiotepa, cause CNS dysfunction, including seizures, altered mental status, cerebellar dysfunction, cranial nerve palsies, and coma.311, 312, 313 High-dose ifosfamide produces neurotoxicity at least partly because of a metabolite chloracetaldehyde314 (Fig. 12.12)
TABLE 12.3 ALKYLATING AGENTS IN HIGH-DOSE CHEMOTHERAPY
Patients with hypoalbuminemia, renal insufficiency, and those treated with higher doses of ifosfamide are at increased risk for neurotoxicity.315 Concomitant use of aggressive intravenous fluids appears to increase the renal excretion of CNS-toxic metabolites and reduce the risk of CNS injury.
The dose-limiting toxicity of cyclophosphamide is cardiac toxicity.316, 317, 318 This toxicity has been reported at doses of over 100 mg/kg administered during a 48-hour period, and has been noted most often in patients receiving total doses of more than 200 mg/kg with HCT.318 High-dose cyclophosphamide produces an acute myopericardial injury within days of administration,319 and life-threatening effects are often produced by pericardial tamponade. Unlike the cumulative and irreversible injury produced by anthracyclines, cyclophosphamide cardiac toxicity is largely reversible if the patient survives for days following the acute toxic episode. No evidence exists for cumulative damage to the heart after repeated moderate or low doses of cyclophosphamide. BCNU and melphalan are also capable of producing reversible cardiac injury in this setting.
Numerous reports have detailed that alkylating agents suppress both humoral and cellular immunity in a variety of experimental systems. Cyclophosphamide is a particularly potent immunosuppressive alkylating agent and has been extensively studied.320, 321, 322, 323, 324 Selective effects of cyclophosphamide on different components of the lymphoid system have been described. In vivo, it has been reported to cause selective suppression of B-lymphocyte function and to deplete B lymphocytes.325, 326 Cyclophosphamide, however, can suppress lymphocyte functions that are mediated by T cells, such as the graft-versus-host response and delayed hypersensitivity.323, 327 Appropriate doses of cyclophosphamide in vivo328 or of activated cyclophosphamide in vitro329 also have been established to enhance immunologic responses by selective inhibition of the function of suppressor T cells. Several lines of evidence suggest that at least some of the immunosuppressive effects of cyclophosphamide may involve mechanisms other than lethal damage to lymphocytes. Shand and Howard330 reported that induction of tolerance in B cells by cyclophosphamide in vivo or by activated cyclophosphamide in vitro occurs, is reversible, and is associated with failure of the cyclophosphamide-treated B cell to regenerate a surface immunoglobulin receptor after capping with anti-immunoglobulin serum. Also, 4-hydroperoxycyclophosphamide, an activated analog of cyclophosphamide, blocks the differentiation of suppressor T-cell precursors at drug levels that are not cytotoxic and do not produce demonstrable DNA cross-linking in drug- sensitive cell lines.331
Figure 12.12 Metabolic activation of ifosfamide to its active form, 4-hydroxyifosfamide, and further metabolic transformation to chloracetaldehyde and other end products. NADPH, reduced form of nicotinamide-adenine dinucleotide phosphate.
The clinical significance of the immunosuppression produced by alkylating agents (and other drugs) in the setting of cancer therapy is variable. The major concerns are the danger of increased susceptibility to infection in the immunosuppressed host and the potential interference with a host immune response to the tumor. There is further uncertainty in this area because the immunologic measurements most appropriate to estimate the risks are not certain. Mullins et al.332 studied the immune responses of patients with solid tumors treated with high-dose cyclophosphamide, 120 mg/kg over a 2-day period. Six of the 12 patients studied became transiently anergic (for 1 to 2 weeks) to skin test antigens to which they had previously been responsive, but the response to these antigens recovered in all patients by 4 weeks after the cyclophosphamide therapy. Nine of the 12 patients showed an adequate antibody response to an antigenic challenge given 24 hours after the cyclophosphamide therapy, despite severe hematopoietic depression. Because most antitumor regimens would not be expected to be as immunosuppressive as the dose of cyclophosphamide used in this study, the results suggest that most intermittent antitumor regimens do not uniformly produce profound immunosuppression and that recovery of the immune response is usually prompt. Continuous drug therapy with cytotoxic agents or high-dose therapy followed by delayed marrow reconstitution are more likely to lead to severe lymphocyte depletion and profound immunosuppression and to be associated with an increased frequency of opportunistic infections.333
The immunosuppressive activity of alkylating agents, and of cyclophosphamide in particular, has been used for two types of clinical application. The first use has been for the suppression of the recipient immune response before allogeneic transplantation. Since the demonstration by Santos and colleagues334 that matched sibling bone marrow can be successfully transplanted into recipients who have been pretreated with large doses of cyclophosphamide, this drug has been one of several agents used for immunosuppressive effect during bone marrow transplantation; the other drugs are methotrexate and calcineurin inhibitors (cyclosporin A, tacrolimus). Cyclophosphamide also has been shown to be effective in controlling kidney graft rejection335 but has been less widely used for this application than the antimetabolite immunosuppressive agents.
The other use of alkylating agents in patients with nonmalignant disease has been in the treatment of immunologic disorders. Osborne et al.,336 in 1947, reported the successful treatment of a patient with systemic lupus erythematosus using nitrogen mustard. Subsequently, the alkylating agents have been tried in a wide variety of diseases thought to be autoimmune in nature, with variable results. Cyclophosphamide has been shown to be an effective agent in the treatment of Wegener's granulomatosis,337 rheumatoid arthritis,338, 339 idiopathic thrombocytopenic purpura,340 and membranous glomerulonephritis.341, 342 Because of the severe side effects, however, including carcinogenesis, the role of alkylating agents in the treatment of nonmalignant disease must be considered carefully. High-dose cyclophosphamide without HCT has been used to treat aplastic anemia, a disease with a possible autoimmune basis.172
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