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

Nonclassic Agents

Henry S. Friedman

Steven D. Averbuch

Joanne Kurtzberg

Classic alkylating agents, such as the prototype nitrogen-mustard compounds, typically contain a chloroethyl group, and their biologic activity results from polyfunctional alkylation of biologic macromolecules (see Chapter 12, “Clinical and High-Dose Alkylating Agents”). Compounds with diverse chemical structures are also capable of covalent binding to biologic macromolecules, and they also have important clinical activity. These compounds, referred to as the nonclassic alkylating agents, include procarbazine (PCB), dacarbazine (DTIC), and temozolomide (TMZ).

Although these agents lack bifunctionality, as Newell and coworkers1 point out, they share a common structural feature, an N-methyl group, which is important for activity. These agents are essentially prodrugs and must undergo complex metabolic transformation to active intermediates; their precise cellular mechanisms of action and clinical pharmacology are not completely understood, but they are clinically useful, and indeed, PCB and DTIC are part of curative regimens for lymphomas. Additionally, TMZ was recently approved for treatment of patients with recurrent anaplastic astrocytoma in 1999, the first new agent approved for malignant gliomas in more than 30 years.

PROCARBAZINE

PCB was synthesized as part of an effort to develop new monoamine-oxidase inhibitors at the Hoffman-LaRoche Laboratories,2 and it was found to have antitumor activity in rodent preclinical testing.3, 4 Early clinical trials demonstrated significant efficacy for PCB in the treatment of Hodgkin's disease and lymphomas, with little activity against solid tumors.5, 6, 7, 8, 9 PCB has been used widely in combination with other agents in the treatment of Hodgkin's and non-Hodgkin's lymphomas,10, 11, 12, 13 and, to a lesser extent, in small cell lung carcinoma14, 15, 16 and melanoma.17, 18 Building on earlier experience with the treatment of brain tumors,19, 20, 21 trials have demonstrated considerable activity against high-grade glioma.22, 23, 24 Nevertheless, there is still little known about PCB's cellular mechanism of action, and information regarding its clinical pharmacology is incomplete.

Mechanism of Action and Cellular Pharmacology

PCB, a prodrug, must undergo metabolism to active species. It enters cells by passive diffusion and thereafter is rapidly converted to cytotoxic metabolites by several possible routes.25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 Although selected tumor cells may contain cytosolic enzymes capable of activating PCB,29 the parent drug is weakly cytotoxic for most tumor cell lines in culture, and its activity is markedly enhanced by allowing chemical decomposition of the drug25 or cocultivation of tumor cells with rat hepatocytes.31 However, it is not clear that the cytotoxic species generated by in vitro incubation with hepatic microsomes or intact hepatocytes is the same as that produced in humans.

As indicated in Figure 13.1, potential pathways of activation include chemical decomposition (I) as well as microsomal oxidation (II–V). The active end products have not been identified with certainty; they may be diazonium ions (R—N+ N), methyl or N-isopropylbenzamide free radicals, or other species capable of covalently binding to DNA.40, 41, 42, 43 Although most evidence favors a cytotoxic pathway involving the production of methyl or benzylazoxy intermediates by liver cytochrome P-450, with release of these metabolites into plasma and their subsequent uptake and further decomposition to diazonium ions in situ, free-radical species (either a methyl radical or an isopropylbenzylamide radical) can be generated by pathways II and V in the presence of rat liver cytochrome P-450.44 Thus, it is not clear which of the several putative and positively identified metabolites are responsible for cytotoxicity. The metabolic pathways will be considered again with respect to PCB pharmacokinetics in humans in a later section, “Clinical Pharmacology.”

Figure 13.1 Chemical and metabolic reactions of procarbazine, leading to the generation of reactive intermediates. I, Chemical breakdown of procarbazine in aqueous solution; II, III, IV, and V, proposed metabolic activation pathways in vivo. Intermediates not identified in vivo or in vitro are indicated by brackets. See text for detailed description. CYT, cytochrome; MAO, monoamine oxidase; NADP, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate.

Several additional cellular effects of PCB and its metabolites have been demonstrated, although it is unclear whether these contribute directly to cytotoxicity. Hydrogen peroxide and formaldehyde are two potentially toxic products generated from PCB and are thought to cause cytotoxicity by interaction with DNA.45However, the data demonstrating azo-PCB activity in the absence of hydrogen peroxide generation strongly argue against a role for this toxic byproduct in the cytotoxic activity of PCB.25, 46 Earlier reports and more recent studies using alkaline elution techniques have demonstrated that PCB and its metabolites are capable of causing chromatid and single-strand DNA breaks in murine tumor cells in vitro.29, 47, 48, 49 The number of breaks depends on the dosage and time elapsed after treatment, and it has been suggested that the breaks occur during, or soon after, DNA synthesis.50 Because the percentage of cells undergoing mitosis is also diminished as a function of dosage and time after PCB,48 it is likely that the most susceptible phase in the cell cycle may be the premitotic G2phase. However, this has not been confirmed using current technology, such as cell sorting by flow cytometry. In addition, a G2 block may simply indicate the physiologic response of cells to DNA injury. Chromatid translocations (sister chromatid exchange) are also observed in murine tumor cells in vivo after PCB treatment, although this effect is not observed in vitro after PCB.47, 48 Again, this result suggests that differences exist in the toxic metabolites generated in vitro versus in vivo. A recent report suggests that procarbazine causes DNA damage through nonenzymatic formation of a Cu(I)-hydroperoxy complex and methyl radicals.51

In addition to these effects on nuclear DNA, PCB can inhibit DNA, RNA, and protein synthesis in vitro and in vivo.26, 47, 52 Single doses of PCB administered to mice bearing transplanted tumors inhibited DNA incorporation of thymidine by 35 to 70%.26, 47 Maximal inhibition occurred within several hours, and complete recovery was achieved by 8 to 24 hours. De novo purine synthesis and pyrimidine-nucleotide synthesis are inhibited, but there is no effect on nucleoside or nucleotide kinases. PCB produces a similar time course and degree of inhibition for the RNA (uracil) incorporation of orotic acid and for nuclear RNA synthesis. Protein synthesis inhibition cuased by PCB is relatively delayed, reaching a maximum at 12 to 16 hours, and this effect is believed to be a result of the inhibition of nucleic acid synthesis.26, 47, 52, 53 PCB seems to inhibit normal transfer RNA (tRNA) methylation, and the resulting altered tRNA synthesis and function may well account for some of the effects on nucleic acid and protein synthesis.43, 54

The most compelling evidence to date suggests that the cytotoxicity of PCB is mediated by its role as a methylating agent. Adult Fisher rats treated with radiolabeled PCB developed large amounts of O6-[14C]methylguanine compared with 7-[14C]methylguanine.55 O6-methylguanine is a known mutagenic and carcinogenic adduct56, 57, 58 also thought to contribute to cytotoxicity.59 Accordingly, the observation that administration of PCB to athymic nude mice bearing xenografts derived from human malignant gliomas and medulloblastoma resulted in greater growth delays in those tumors lacking O6-alkylguanine-DNA alkyl transferase (AGT),60 the enzyme mediating repair of O6-methylguanine,61 is particularly convincing. Four of five tumor lines with AGT levels had growth delays of less than 20 days after PCB, whereas all five lines with undetectable AGT levels had growth delays of more than 30 days. Furthermore, O6-methylguanine was found in significantly higher levels in two sensitive lines with low-AGT levels as compared with O6-methylguanine levels in a resistant line with a high-AGT level.

Mechanisms of Resistance

Recent studies have shed light on the cellular mechanism(s) of resistance to PCB. Resistance develops rapidly in tumor cells3 after exposure to PCB, and one study suggested a direct correlation between the rate of DNA synthesis and the rapidity of resistance development.26 Resistant cells also were found to contain additional chromosomes.62 The previously mentioned inverse correlation between central nervous system (CNS) xenograft response to PCB and AGT activity suggests that resistance to this methylating agent is secondary to AGT-mediated repair of O6-methylguanine, similar to nitrosourea resistance mediated by this enzyme.61 However, an alternative method of resistance has been defined. Friedman et al.63 established a methylator- resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice by serially treating the parent xenograft, D-245 MG, with PCB. D-245 MG xenografts were sensitive to PCB, TMZ, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)- 1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to PCB, TMZ, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. D-245 MG and D-245 MG (PR) xenografts displayed no AGT alkyl transferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch-repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

These results indicate that this resistance to PCB and other methylators was secondary to an in vivo-acquired mismatch repair deficiency. This observation is consistent with other reports demonstrating methylator resistance in human tumor cells resulting from mismatch repair deficiency.64, 65

Drug Interactions

Because PCB undergoes extensive hepatic microsomal metabolism and because it inhibits monoamine oxidase, which is widespread in tissues and plasma, there are many potential drug-drug and drug-food interactions. The activity of other drugs that are inactivated by microsomal metabolism may be enhanced in the presence of PCB, as shown by a prolonged pentobarbital-induced sleep time in animals.66, 67, 68, 69 Therefore, patients taking barbiturates, phenothiazines, narcotics, and other hypnotics or sedatives may experience potentiated effects of these agents. Conversely, these drugs and others, such as cimetidine, that affect hepatic metabolism may increase or decrease PCB metabolism and thereby alter PCB activity and toxicity.45,70, 71 Pretreatment of rats with phenobarbital before PCB administration resulted in increased PCB clearance and a slight decrease in concentrations of the azometabolite. Inasmuch as phenobarbital or phenytoin pretreatment increased the survival of tumor-bearing mice treated with PCB (Table 13.1), it may be presumed that microsomal enzyme induction resulted in increased production of active PCB metabolites.46 It is not known whether this drug interaction may be useful clinically to achieve therapeutic advantage through biochemical modulation of PCB activity.

Monoamine oxidase inhibition72 and pyridoxal phosphate depletion73 by PCB cause CNS depression. This also may potentiate the sedative effects of other CNS depressants. This inhibition of monoamine oxidase also predisposes patients to acute hypertensive reactions after concomitant therapy with tricyclic antidepressants and sympathomimetic drugs, as well as after ingestion of tyramine-rich foods, such as red wine, bananas, ripe cheese, and yogurt. Finally, a disulfiram-like reaction manifested by sweating, facial flushing, and headache may occur in patients who ingest alcohol while taking PCB.

Clinical Pharmacology

PCB hydrochloride is supplied in capsules containing the equivalent of 50 mg of the base for oral administration. As a single agent, the usual dose is 100 to 200 mg/m2 of body surface area, given daily until myelosuppression occurs. As part of the mechlorethamine, vincristine, PCB, and prednisone (MOPP) combination regimen for Hodgkin's disease, the daily dose of PCB is 100 mg/m2 of body surface area daily for 14 days.10

TABLE 13.1 KEY FEATURES OF PROCARBAZINE (PCB) [N-ISOPROPYL-α-(2-METHYLHYDRAZINO)-P-TOLUAMIDE, IBENZMETHYZIN, NATULAN, MATULANE, NSC 77213]

Factor

Result

Mechanism of action

Metabolic activation required: methylation of nucleic acids; inhibition of DNA, RNA, and protein synthesis.

Metabolism

Converted to azo-PCB by erythrocyte and liver microsomes

Subsequent metabolism to N-isopropyl-p-formylbenzamide, N-isopropyl-p-hydroxymethyl benzamide, N-isopropyl-p-toluamide, N-isopropyl-N-isopropylterephthalamic acid (inactive), methane, and carbon dioxide

Possible formation of methyldiazene free radical “active intermediate”

Pharmacokinetics:

Half-life = 7 min

Approximately 100% bioavailability from oral route, peak plasma concentration reached within 60 min

Equilibration between plasma and cerebrospinal fluid in 15–30 min

Elimination

Renal elimination of ≥75% in 24 hr

Drug and food interactions

PCB may inhibit hepatic microsomal drug metabolism and therefore potentiate activity of barbiturates, antihistamines, narcotics, and phenothiazines

Alcohol use may cause “disulfiram-like” reaction

Sympathomimetics, tricyclic antidepressants, or tyramine-rich foods may cause severe hypertension from PCB inhibition of monoamine oxidase

Toxicity

Myelosuppression

Gastrointestinal (nausea and vomiting); rare, hepatic dysfunction

Neurotoxicity (drowsiness, depression, agitation, paresthesias)

Cutaneous or pulmonary hypersensitivity (rare)

Azoospermia; anovulation

Carcinogenesis (associated with secondary malignancy in treated patients)

Teratogenesis

Precautions

Dose modification may be necessary in hepatic and/or renal dysfunction

Avoid alcohol

Avoid tyramine-rich foods, sympathomimetics, tricyclic antidepressants, hypnotics, antihistamines, narcotics, phenothiazines

 

The pharmacokinetics and metabolism of PCB have been studied mostly in laboratory animals, and information regarding pharmacokinetics in humans is incomplete.62, 74, 75, 76, 77 After oral administration, the drug is rapidly and completely absorbed from the gastrointestinal tract. The biodistribution of PCB is not well known; however, earlier studies using drug that was isotopically labeled at different sites on the molecule showed high levels of radioactivity in the liver, kidney, intestine, and skin at 30 and 60 minutes after drug administration.76 There is also rapid equilibration of [14C]PCB (labeled in the benzyl ring) between plasma and cerebrospinal fluid (CSF) in dogs and humans.66 After the intravenous administration of 150 mg [14C]PCB, the plasma half-life (t1/2) of parent drug was approximately 7 minutes in humans, whereas studies in dogs and rats demonstrated t1/2 of 12 and 24 minutes, respectively.74 Because single-bolus intravenous dosages of PCB produce a spectrum of toxicity, primarily neurotoxicity,78 distinct from the myelosuppression seen after oral administration, it is likely that a first-pass effect of orally administered drug through the portal circulation significantly influences drug metabolism and pharmacokinetics. This is supported by the observation of almost complete conversion of PCB to the azometabolite in isolated liver perfusion studies.35, 75After the intraperitoneal injection of 150 mg PCB in rats, the azometabolite appears in plasma within minutes, peaking at 10 to 20 minutes and then decreasing slowly over several hours concomitant with the appearance of the methyl and benzylazoxy isomers28 (Fig. 13.2 A). Preliminary data in humans show that the methylazoxy isomer is the major plasma metabolite after a single 250 mg/kg oral dose of PCB. This compound peaks at approximately 90 minutes and seems to have an initial plasma half-life of approximately 60 minutes. Azo-PCB and the benzylazoxy isomer are present in relatively equal but lesser concentrations compared to the methylazoxy isomer. Interestingly, PCB treatment seems to alter its own metabolism, a change that may, in turn, influence its activity.28, 46, 67 The total and relative plasma concentrations of PCB metabolites are markedly changed after the administration of a fourteenth daily oral dose of PCB28 (Fig. 13.2 B). Of note was the significant increase in azo-PCB concentration, suggesting that prior PCB exposure induces this metabolite's production or delays its clearance. Shiba and Weinkam46 also observed that prior treatment with PCB enhances PCB antitumor activity in rats.

In all species examined, the major urinary metabolite of PCB is the biologically inactive N-isopropylterephthalamic acid.66, 70, 74, 75, 76 Approximately 70% of radioactivity administered in the form of [14C]PCB was recovered, primarily as the acid, in the urine during the first 24 hours. There is minimal fecal excretion (4 to 12% over 96 hours), and approximately 30% of radioactivity labeled in the N-methyl group appears as respiratory 14CO2.76, 79

The complex pharmacokinetic and excretion characteristics of PCB reflect the rapid and extensive enzymatic metabolism of this compound, which is necessary for antitumor activity and presumably is responsible for host organ toxic reactions. The proposed metabolic routes for PCB were discussed in detail in the previous edition of this text80 and were reviewed in detail by Prough and Tweedie29 (Fig. 13.1). The understanding of PCB metabolism is improving as a result of improved experimental techniques and analytical methods, including high-pressure liquid chromatography (HPLC) and mass spectroscopy.27, 28, 33, 34, 35, 36,37, 81, 82 Again, most of the information in this area is derived from studies in animals in vivo and in vitro. There do not seem to be any major discrepancies in these results in animals as compared with the literature describing human metabolism.46, 66, 74, 76, 83 Nonetheless, the information is not sufficient to allow assignment of quantitative importance to the several possible alternate routes of metabolism (Fig. 13.1). However, a recent report detailing an improved assay for procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry may facilitate these needed analyses.84

Figure 13.2 A. Procarbazine (PCB) disappearance and azo and azoxy metabolite kinetics in rat plasma after administration of PCB, 150 mg/kg, intraperitoneally. B. Plasma concentrations of azo and azoxyprocarbasine metabolites in a patient after the administration of PCB, 250 mg/kg per day, orally, on days 1 and 14 of a 14-day treatment schedule. (From Shiba DA, Weinkam RJ. Quantitative analysis of PCB, PCB metabolites and chemical degradation products with application to pharmacokinetic studies. J Chromatogr 1982; 229:397.)

PCB is not stable in aqueous solution, decomposing by rapid metal-catalyzed oxidation to azo-PCB with the production of hydrogen peroxide.29, 33, 45, 66, 81 In the presence of light, isomerization to the biologically inactive hydrazone (N-isopropyl-p-formylbenzamide methylhydrazine) occurs slowly. This is followed by hydrolysis to yield the aldehyde, N-isopropyl-p-formylbenzamide, and methylhydrazine. The former compound is further oxidized to N-isopropylterephthalamic acid. Earlier studies suggested that this route of chemical decomposition was responsible for the biologic activity of PCB,45, 66, 74 but subsequent investigations have shown that the chemical decomposition products are relatively stable under physiologic conditions and that they account for a small proportion of the compounds formed in vitro and in vivo as compared with cytochrome P-450–mediated metabolism.25, 26, 29, 33, 34, 35, 36, 37, 38 Because of PCB's chemical degradation to potentially toxic compounds under common conditions, such as aqueous solvent, trace metal contamination, and air and light exposure, extreme care must be taken in the formulation and storage of PCB solutions intended for parenteral administration. These considerations also apply when evaluating the results of PCB studies in vitro and in vivo.

In biologic systems, the oxidation of PCB to azo-PCB occurs by microsomal cytochrome P-450 oxidoreductase or by mitochondrial monoamine oxidase enzymatic conversion32, 34, 75, 85, 86 (Fig. 13.1). Isolated rat liver perfusion studies, as well as incubation of drug with rat liver microsomes, disclose extensive metabolism of the drug and suggest that the liver is the predominant site of the initial metabolism of PCB.29, 34, 35, 36, 37, 38 The subsequent metabolism of azo-PCB may occur by several different routes. Isozymic cytochrome P-450–mediated N-oxidation results in the formation of methyl and benzylazoxy isomers27, 34, 87, 88 (Fig. 13.1, pathways III and IV). The former is produced in higher quantitative yield during in vitro reactions and is the predominant metabolite of azo-PCB in rat and human plasma.28, 29 It has been proposed that hydroxylation of either carbon atom adjacent to the azoxy function results in unstable compounds that react to produce the reactive alkylating alkyldiazonium ion [R—N+ N]. Further microsomal metabolism of the azoxy compounds results in formation of N-isopropyl-p-formylbenzamide or N-isopropyl- p-hydroxymethylbenzamide. These compounds are then oxidized to the major urinary metabolite, N-isopropylterephthalamic acid.27, 28, 29, 66, 74 Alternatively, Moloney and associates40 recently demonstrated a pathway of metabolic activation of the terminal N-methyl group of azo-PCB that does not involve azoxy formation (Fig. 13.1, pathway V). This pathway involves a P-450–mediated oxidation of the benzyl carbon atom adjacent to the azo function with subsequent formation of N-isopropyl-p-formylbenzamide and a putative unstable methyldiazene intermediate. The proposed intermediate could form either a methyl radical or a carbonium ion, both of which are covalent binding species. If, instead, hydrogen abstraction occurs, as in the presence of reduced glutathione, then methane is formed as a final metabolic product.38, 77 Another pathway that would produce a free-radical intermediate and not involve azoxy formation is the oxidation of the methyl carbon adjacent to the azo function (Fig. 13.1, pathway II). This metabolic route would ultimately lead to formation of CO2 and N-isopropyl-p-toluamide, a metabolite identified in rat plasma and brain after the administration of PCB.33, 75, 76, 77, 79, 80 Because the azoxy metabolites are the predominant products found in plasma, however, it is likely that pathways III and IV predominate in humans.

Toxicity

After oral administration, PCB causes anorexia and mild nausea and vomiting, which is probably of central origin and often abates with continued use.89 In some patients, it is often helpful to escalate the dosage in a stepwise fashion over the first several days of drug administration to minimize these gastrointestinal side effects. Mild-to-moderate myelosuppression in the form of reversible leukopenia and thrombocytopenia is the most common dose-limiting toxicity of PCB given orally. Depression of peripheral leukocyte and platelet counts becomes apparent after 1 week of therapy and may persist for 2 weeks or longer after discontinuation of the drug.90 PCB also may cause hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency.91 PCB generally does not cause mucosal injury to the rapidly proliferating gastrointestinal epithelium.

Patients receiving PCB orally may occasionally experience neurotoxicity manifest by drowsiness, depression, agitation, paresthesias of the extremities,92 and reversible orthostatic hypotension.8 These effects are probably a result of central monoamine oxidase inhibition and may be related to drug-induced depletion of pyridoxal phosphate.72, 73, 93 When PCB is administered intravenously, neurotoxic effects become more pronounced and are dose-limiting. After a single high-dose intravenous bolus (2 g/m2) or a 5-day continuous infusion of PCB, patients experienced severe nausea and vomiting, confusion, and even coma lasting several days.78, 93 Myelosuppression does not occur when PCB is administered in this way. However, there is also a parallel lack of clinical antitumor effect, which emphasizes the importance of first-pass hepatic metabolism for activation of PCB to antiproliferative intermediates. The pattern of toxicity after small, intermittent intravenous doses is more like that seen after oral administration,1, 6 although it is unlikely that this schedule offers any clinical benefit over that of conventional oral dosing.

PCB also may cause hypersensitivity reactions, including maculopapular skin rash, eosinophilia, pulmonary infiltrates, or, rarely, transient hepatic dysfunction.5, 7, 94, 95, 96, 97, 98 The skin rash usually responds to concomitant glucocorticosteroid treatment, and the PCB may be continued without exacerbation of rash or further sequelae. In contrast, PCB-induced interstitial pneumonitis usually necessitates discontinuation of the drug.

PCB has potent immunosuppressive properties that may contribute to the infectious complications.99 These immunosuppressive properties have been used to therapeutic advantage for the treatment of lupus erythematosus and in the suppression of graft-versus-host disease after bone marrow transplantation.100With the use of newer agents developed for these indications and with the increasing concern over serious late toxic reactions to PCB, this drug should probably not be used for non-neoplastic diseases.

The successful use of PCB-containing chemotherapy combinations resulting in curative and long-term disease-free survival has directed increasing attention and concern to the chronic and late toxicities of this agent. PCB has profound azoospermic,101, 102 teratogenic,103 mutagenic,104, 105 and carcinogenic106, 107properties in experimental animals, and most of these effects have been associated with PCB use in humans.

PCB is highly toxic to reproductive organs, causing azoospermia and anovulation.108, 109, 110, 111, 112 More than 90% of men receiving PCB in combination with classic alkylating agents, such as in MOPP combination chemotherapy for Hodgkin's disease, have irreversible azoospermia. Approximately 50% of women thus treated have permanent drug-induced ovarian failure. In pregnant animals, administration of PCB causes congenital skeletal and CNS abnormalities.103, 113Although evidence for direct causation of lethal and nonlethal mutations in human fetuses is lacking, women of childbearing potential should be advised against pregnancy during chemotherapy. In women treated with MOPP chemotherapy and who regain normal ovarian function, there seems to be no impairment of fertility nor any increased birth defects in offspring.111, 112, 114, 115

Mutagenesis and carcinogenesis resulting from PCB have been demonstrated experimentally in vitro and in vivo.104, 105, 106, 107 Nonlymphocytic leukemias and adenocarcinomas developed in rodents and nonhuman primates after PCB administration, and, accordingly, the finite increased incidence of secondary leukemias and solid malignancies in patients after treatment with MOPP combination chemotherapy pointed to PCB as the responsible carcinogen.116, 117, 118Because this regimen also contains an alkylating agent with carcinogenic properties, it is difficult to assign a direct cause of secondary malignancies to PCB alone.118 Indeed, studies in experimental systems suggest that additive or interactive effects of classic alkylating agents with PCB may account for the observed mutagenesis.119

The mechanisms of PCB gonadal toxicity and somatic genotoxicity are mostly thought to be the same as for its antitumor activity. As for the latter, metabolic conversion of PCB is necessary for its toxic and carcinogenic effects on normal tissue, although it is not clear which metabolic pathways are mechanistically important, nor is it known whether there may be separate mechanisms for anticancer activity and for normal organ toxicity. Yost et al.120 and Horstman et al.121 proposed separate mechanisms for PCB spermatotoxicity and anticancer activity based on different activating metabolic pathways. Furthermore, these authors exploited this difference by using antioxidants that protected against PCB spermatotoxicity but did not compromise its antileukemic activity in mice (Table 13.2). These studies, as well as those of Prough and Tweedie29 and Shiba and Weinkam,46 which show improved therapeutic benefit from phenobarbital induction of PCB metabolism, suggest that anticancer and toxic effects of PCB may be separable and, therefore, susceptible to modulation for therapeutic advantage. Further investigations of PCB's metabolism and molecular mechanisms of action are necessary to develop clinically useful approaches to lessen toxicity successfully and improve efficacy.

TABLE 13.2 EXPERIMENTAL BIOCHEMICAL MODULATION OF PROCARBAZINE (PCB) EFFICACY AND TOXICITY IN MICE BEARING L1210 LEUKEMIA, INTRAPERITONEALLY

Treatment

Modulation

Life Span Increase (%)a

Sperm Count (%)b

Reference

PCB 200 mg/kg, IP, daily × 3

None

122

 

43

PCB 200 mg/kg, IP, daily × 3

Phenytoin 60 mg/kg, PO, for 7 d

146c

 

43

PCB 200 mg/kg, IP, daily × 3

Phenobarbital 48 mg/kg, PO, for 7 d

140c

 

43

PCB 400 mg/kg, IP

None

125

45

108

PCB 400 mg/kg, IP

N-Acetylcysteine 189.9 mg/kg, IP

128

85

108

PCB 400 mg/kg, IP

Sodium ascorbate 307.4 mg/kg, IP

125

90

108

a% mean treated/control.
bMean expressed as a percentage of control mice given 0.9% NaCl.
cSignificant at the 95% confidence limit compared with mice treated with PCB alone.
IP, intraperitoneally; PO, per os.

 

DACARBAZINE

History

DTIC was chemically synthesized as a result of a rational attempt to develop agents capable of interfering with the synthesis of purines. As reviewed by Montgomery,122 a series of compounds designed as analogs of aminoimidazole carboxamide (AIC), an intermediate in purine ring synthesis, was synthesized in the late 1950s and had significant antitumor activity in experimental testing. The addition of nitrous acid to form a 5-diazoimidazole derivative seemed to confer this antitumor activity, and the further addition of a third nitrogen group to form the 5-triazene resulted in a light-sensitive compound that spontaneously converted back to the diazo analog. Dimethyl substitution of the triazine resulted in a more stable but still light-sensitive derivative, DTIC, which was highly active and was developed for clinical use.123

DTIC is an active single agent in the treatment of metastatic malignant melanoma, producing remissions in 16 to 31% of patients with this disease,124 and it is also active as a single agent in Hodgkin's disease.125 Thus, in the United States, DTIC is approved for use in these two diseases. It is frequently used alone or in combination with agents such as nitrosoureas, bleomycin, and vinca alkaloids in melanoma,126, 127, 128, 129 and it is most commonly used as part of the doxorubicin, bleomycin, vinblastine, and DTIC (ABVD) and actinomycin D, bleomycin, and vincristine regimens for Hodgkin's disease.130, 131 In addition, DTIC has demonstrated activity in the treatment of sarcomas,132, 133, 134 childhood neuroblastoma,135, 136 and primary brain tumors.137 It may be the most active agent alone or in combination for the treatment of malignant amine precursor uptake and decarboxylation and other neuroendocrine tumors.138, 139, 140 The key features of DTIC are summarized in Table 13.3.

General Mechanism of Action and Cellular Pharmacology

The exact mechanism underlying DTIC's antitumor activity remains an enigma. Although DTIC was developed as a purine antimetabolite, there is abundant evidence that its antitumor activity does not result from interference with purine synthesis. The drug is active against several cell lines resistant to the purine analogs 6-thioguanine and 6-mercaptopurine, and it does not demonstrate cell-cycle schedule dependence observed with other antimetabolites.122 Second, the AIC portion of the molecule is not necessary for antitumor activity.141, 142

There is mounting evidence to suggest that, similar to PCB, the production of O6-methylguanine is the primary cytotoxic event after administration of DTIC. Xenografts, or cell lines with negligible levels of AGT, are more sensitive to DTIC than are xenografts or cell lines with high levels of AGT.143, 144, 145, 146, 147, 148, 149Furthermore, DTIC depletes AGT levels in human colon cancer HT 29 xenografts in athymic mice150 and in human peripheral blood cells in patients treated for metastatic melanoma.151

TABLE 13.3 KEY FEATURES OF DACARBAZINE [5-(3,3-DIMETHYL-1-TRIAZENO) IMIDAZOLE-4-CARBOXAMIDE, DTIC, DIC, NSC-45388]

Factor

Result

Mechanism of action

Metabolic activation probably required; methylation of nucleic acids; direct DNA damage; inhibition of purine synthesis.

Metabolism

Oxidative N-methylation to 5-aminoimidazole-4-carboxamide via formation of 5(3-hydroxymethyl-3-methyltriazen-1-yl) imidazole-4-carboxamide and 5-(3-methyltriazen-2-yl) imidazole-4-carboxamide.

t1/2α = 3 min; t1/2β = 41 min

Vd = 0.6 L/kg; Cl = 15 mL/kg/min

20% protein bound

Variable oral absorption.

Poor CSF penetration (plasma/CSF ratio = 7:1 at equilibrium).

Elimination

Renal excretion: 50% as unchanged dacarbazine and 9–18% as 5-aminoimidazole-4-carboxamide.

Minor hepatobiliary and pulmonary excretion.

Drug and food interactions

Corynebacterium parvum may prolong t1/2.

Toxicity

Myelosuppression.

Gastrointestinal (nausea and vomiting).

Influenza-like syndrome (fever, myalgia, and malaise).

Infrequent alopecia, cutaneous hypersensitivity, or photosensitivity.

Rare hepatic vein thrombosis and hepatic necrosis.

Possible carcinogenesis and teratogenesis.

Precautions

Dose modification may be necessary in hepatic and/or renal dysfunction.

Cl, clearance; CSF, cerebrospinal fluid; t1/2, half-life; Vd, apparent volume of distribution.

The previously mentioned work143, 144, 145, 146, 147, 148, 149 supporting O6-methylguanine as the major cytotoxic lesion produced by DTIC strongly suggests that elevated levels of AGT may be responsible for resistance to this agent. The inverse relationship between AGT levels and response to DTIC in human xenografts also may be operational in clinical tumor resistance to DTIC.143, 144, 145, 146, 147, 148, 149 Furthermore, resistance to all methylators, including DTIC, is seen in the setting of a deficiency of DNA mismatch repair.63, 64, 65 Finally, Lev et al.152 have reported DTIC resistance mediated by up-regulation of interleukin 8 and vascular endothelial growth factor in melanoma cells.

Drug Interactions

At present, there are no known drug or food interactions with DTIC that are of clinical importance. Because DTIC has been used in conjunction with immune adjuvants in the treatment of malignant melanoma, there has been some interest in the influence of these agents on DTIC pharmacology. Farquhar and coworkers153 described an inhibition of DTIC N-demethylase in rats pretreated with bacillus Calmette-Guérin (BCG), suggesting that patients receiving both agents may be less able to activate DTIC. In four patients with melanoma, BCG did not seem to influence DTIC pharmacokinetics, although altered metabolism per se was not examined.154 In contrast, patients receiving Corynebacterium parvum adjuvant immunotherapy did show a prolongation of DTIC serum half-life155 consistent with the ability of C. parvum to depress hepatic microsomal N-demethylation of a variety of drugs.156 Although the initial step for metabolic activation of DTIC is catalyzed by microsomal cytochrome P-450, the interaction of phenobarbital, or other commonly used cytochrome P-450–inducing agents, with DTIC has not been reported.

DTIC activity against L1210 murine leukemia is potentiated by alkylating agents, such as melphalan, and by doxorubicin.122 Activity is also enhanced when DTIC is combined with the nitrosoureas bischloromethyl- nitrosourea (BCNU) and chloroethylcyclo-hexylnitrosourea (CCNU). The mechanism(s) for the potentiation observed using these combinations may be related to the ability of nitrosoureas to deplete AGT and thereby sensitize cells to methylating agents.

Clinical Pharmacology

DTIC is supplied in sterile vials containing 100 or 200 mg DTIC for intravenous administration. As a single agent, a dose of up to 1,500 mg/m2 of body surface area may be given as a single bolus as opposed to the more frequently used schedule of 250 mg/m2 daily for 5 days every 3 to 4 weeks.126, 128, 134, 157 The latter schedule was developed in an attempt to minimize the gastrointestinal toxicity from DTIC, which tends to lessen with repeated administration. Most studies, however, fail to show any significant schedule dependency with respect to antitumor efficacy or toxicity.126, 128 DTIC also has been used by intra-arterial infusion for the regional treatment of malignant melanoma involving liver, pelvis, the maxillofacial region, and extremities with high response rates in uncontrolled series.158, 159, 160, 161 It is not known whether in situ melanoma cells in humans are capable of metabolizing DTIC162; otherwise, these results are difficult to interpret because DTIC requires metabolic activation for its antitumor activity.

Initial studies of DTIC pharmacokinetics and metabolism in rodents, dogs, and humans used radiochemical163, 164 and colorimetric methods.165, 166, 167 More recently, improved experimental methods, such as HPLC154, 155, 168 and mass spectroscopy,169 have been used to study triazine pharmacology. Because of the scarcity of clinical studies using adequately sensitive and specific techniques, knowledge of DTIC pharmacology in humans remains incomplete. After oral administration, the drug is absorbed slowly and variably166, 167; therefore, intravenous administration is the preferred route. Intravenous boluses of 2.65 to 6.85 mg/kg (approximately 120 to 300 mg/m2) produced peak plasma concentrations of nearly 10 to over 30 µg/mL, respectively.154 After intravenous administration of DTIC, Breithaupt and coworkers154 found a biphasic plasma disappearance of the parent drug consistent with a two-compartment model with an initial half-life of 3 minutes and a terminal half-life of 41 minutes (Fig. 13.3). This is in contrast to a terminal half-life of 3.2 hours found in an earlier study using HPLC155 analysis. Approximately 20% of DTIC is loosely bound to plasma protein.166 In humans, the mean volume of distribution for DTIC was 0.6 L/kg, and the total-body clearance was 15.4 mL/kg per minute.154 In one study, approximately 50% of an intravenous dose of DTIC was recovered in the urine as parent drug, and the renal clearance was calculated to be between 5 and 10 mL/kg per minute,154 confirming earlier reports166, 167 that tubular secretion may be involved in the renal excretion of DTIC. Altered schedules of intravenous drug administration did not change the area under the curve (concentration × time), confirming a lack of schedule dependence for DTIC pharmacokinetics.154

In dogs166 and humans,126 DTIC penetrates poorly into the CSF. At equilibrium, the ratio between plasma and spinal fluid was 7:1. This finding may explain the lack of DTIC activity against intracranial L1210 leukemia.122 It fails to explain, however, the observations that DTIC has activity against transplantable murine ependymoblastoma170 and against primary and metastatic brain tumors in humans.124, 127, 137

The major metabolite of DTIC found in plasma and urine is AIC (Fig. 13.3),154, 163, 164, 166 with cumulative excretion in the urine accounting for 9 to 20% of parent compound in several patients studied.154, 164 AIC is also formed from DTIC in the presence of liver microsomes171and by some tumor cells.172 After the intraperitoneal administration of [14CO-methyl]DTIC to rats or mice, 4% of the dose is recovered as respiratory 14CO2 in 6 hours, and 9% of the dose is recovered as 14CO2 in 24 hours.173, 174 Presumably, the expired radiolabeled 14CO2 is derived from the formaldehyde produced after N-demethylation of DTIC. These findings, as well as the identification of 5-(3- hydroxymethyl-3-methyltriazen-1-yl) imidazole-4-carboxamide (HMTIC) as a urinary metabolite of DTIC in rats,175, 176, 177 are consistent with a metabolic pathway for DTIC, as shown in Figure 13.4, in which MTIC is the primary active metabolite, responsible for transferring its methyl group to DNA.

Figure 13.3 Plasma concentrations of dacarbazine (DTIC) and 5-aminoimidazole-4-carboxamide (AIC) in a patient after administration of dacarbazine, 6.34 mg/kg, intravenously. (From Breithaupt H, Dammann A, Aigner K. Pharmacokinetics of dacarbazine [DTIC] and its metabolite 5-aminoimidazole-4-carboxamide [AIC] following different dose schedules. Cancer Chemother Pharmacol 1982;9:103.)

Toxicity

The most frequent toxic reaction to DTIC treatment is moderately severe nausea and vomiting, which occurs in 90% of patients.134, 179 These symptoms appear soon after infusion and may persist for up to 12 hours. The severity of gastrointestinal toxicity decreases with successive doses when the drug is given on a 5-day schedule and if the initial dose is decreased.179 Above 1,200 mg/m2 as a rapid intravenous bolus, DTIC frequently causes severe, but short-lived, watery diarrhea.126, 157 After rapid infusion of a high dose (>1,380 mg/m2) of DTIC, hypotension may occur.157

Myelosuppression is a common dose-related toxicity of DTIC, although the degree of leukopenia and thrombocytopenia is variably mild to moderate. Significant myelosuppression occurs when more than 1,380 mg/m2 is given as a single intravenous bolus,157 whereas studies using a 5-day administration schedule reported increasing frequency of myelosuppression above a total of 1,000 mg/m2.126, 134 In the latter, nadir leukopenia and thrombocytopenia occurred on day 25, with complete recovery by day 40. This delayed bone marrow recovery is not common, however, and usually there is sufficient recovery so that DTIC may be administered every 21 to 28 days.

Figure 13.4 Light-activated and metabolic reactions of dacarbazine leading to the generation of reactive intermediates. CYT, cytochrome; uv, ultraviolet.

Less frequent toxic reactions include a flulike syndrome of fever up to 39°C, myalgias, and malaise lasting several days after DTIC treatment. Headache, facial flushing, facial paresthesias, pain along the injection vein, alopecia, and abnormal hepatic and renal function tests rarely occur. Photosensitivity to DTIC has been reported in several patients, especially after high-dose therapy.157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180 Therefore, patients should be advised to avoid sunlight exposure for several days after DTIC therapy. Cases of hepatic vein occlusion associated with fever, eosinophilia, and hepatic necrosis and resulting in death have been attributed to DTIC as a distinct clinical pathologic syndrome.181, 182, 183, 184, 185 The mechanism for this toxicity is unknown, but an allergic etiology has been suggested.184, 185

DTIC causes a number of immunologic effects in vitro and in vivo. The drug markedly depresses antibody responses and allograft rejection in mice for up to 60 days after a single injection.186 This is probably a specific effect of DTIC because structure activity studies showed different patterns of immunodepression depending on which phenyltriazene analog was tested.187 DTIC apparently does not directly suppress natural killer cell activity in mice.188 After DTIC treatment, L1210 or L5178 lymphomas were found to be highly immunogenic, such that large inocula of the DTIC-resistant tumors were rejected by immunocompetent animals. DTIC-treated cells were actually less susceptible to natural killer cell cytolysis in vitro.189

DTIC has mutagenic, carcinogenic, and teratogenic properties in experimental systems.190, 191 In rodents, DTIC causes lymphoma and tumors of the thymus, lung, uterus, or mammary glands when given orally or by single or multiple injections.142, 192, 193, 194 MTIC treatment also caused similar tumors but in a lower frequency compared with DTIC.193 It is not firmly established whether DTIC is carcinogenic for humans. In a retrospective analysis of patients receiving either MOPP or ABVD (plus or minus radiation therapy) for Hodgkin's disease, Valagussa et al.195 reported no treatment-associated secondary malignancies in patients receiving ABVD. Subsequently, isolated cases of acute leukemia occurring after DTIC therapy have been reported,196, 197 but these remain rare. Finally, DTIC causes dose-dependent fetal malformations and fetal resorptions when administered to pregnant rats and rabbits.198, 199 Teratogenic effects were observed in the urogenital system, skeleton, eye, and cardiovascular system.

TEMOZOLOMIDE

History

Several series of 1,2,4-triazines and 1,2,4-triazinones were synthesized in England in the 1960s and 1970s, and selected compounds proved to have activity against murine tumors.200, 201, 202, 203, 204 The most promising was mitozolomide, which was active against a broad spectrum of murine tumors,203 but it produced severe and unpredictable thrombocytopenia in clinical trials and was abandoned as a clinical candidate.204

Selection of the next generation of imidazotetrazinones focused on TMZ, the 3-methyl derivative of mitozolomide (Fig. 13.5). This compound, with a different spectrum of activity against murine tumors,205 was less active and considerably less toxic than mitozolomide and displayed superb delivery to all body tissues, including the brain.206, 207 TMZ was rationally advanced to clinical trial, partly based on the realization that under physiologic conditions the ring opens with resulting generation of the monomethyl triazine MTIC, the same metabolite formed by metabolic dealkylation of DTIC.208 The inefficient demethylation of DTIC in humans (despite rapid demethylation in mice) coupled with the conversion of TMZ to MTIC without need for this metabolic step suggested a potential benefit for the use of TMZ. Table 13.4 lists the key features of TMZ.

Figure 13.5 Structure of temozolomide.

TABLE 13.4 KEY FEATURES OF TEMOZOLOMIDE {8-CARBAMOYL-3-METHYLIMIDAZO [5,1-D]-1,2,3,5-TETRAZIN-4(3H)-one}

Factor

Result

Mechanism of action

Methylation of nucleic acids

Metabolism

Chemical conversion of 5 (3-methyltriazeno) imidazole-4-carboxamide

Pharmacokinetics (IV or PO)

Volume of distribution: 28.3 L

Elimination half-life: 1.8 hr

Distribution half-life: 0.26 hr

Clearance: 11.76 L/hr253

Drug and food inter-actions

Unknown

Toxicity

Myelosuppression

Nausea and vomiting

Elevated hepatic transaminases

IV, intravenously; PO, per os.

Phase 1 trials of intravenous and subsequently oral TMZ began in 1987 with a single-dose schedule and demonstrated the dose-limiting toxicity to be myelosuppression with trivial clinical benefits observed.209 However, based on preclinical data supporting a multiple-dose regimen, another phase 1 trial using a 5-day schedule was conducted, with myelosuppression again the dose-limiting toxicity. Greater clinical activity was noted with four responses (two partial and two complete) in 23 patients with metastatic melanoma and two partial responses in four patients with high-grade glioma.209

Further evaluation in 28 patients with primary brain tumors revealed five radiographic responses in 10 patients with recurrent astrocytoma (the majority of which were high-grade type). Similarly, four radiographic responses were seen in seven patients with newly diagnosed high-grade glioma.210 It should be noted that radiographic criteria for response were not the conventionally accepted partial or complete response criteria. Nevertheless, these results are provocative and justified further studies in patients with CNS tumors, particularly gliomas.

This study of O'Reilly et al.210 was extended to 75 patients (48 with recurrent disease and 27 with new diagnoses).211 Improvements on computed tomography (CT) were seen in 12 (25%) of the patients with recurrent disease and in eight (30%) of the patients with new diagnoses. Twenty-two percent of patients with recurrences and 43% of those with newly diagnosed tumors survived to 1 year.

The Cancer Research Campaign (CRC) conducted a multicenter phase 2 study in which TMZ demonstrated activity in patients with recurrent and progressive high-grade glioma.212 Objective responses, measured by improvement in neurologic status, were seen in 11 of 103 patients (11%) who received TMZ; five of these patients had improvement on CT or magnetic resonance imaging (MRI) scans.212 Objective responses were observed in patients in whom anaplastic astrocytoma, glioblastoma multiforme (grade IV), and unclassified high-grade astrocytoma (grades III–IV) were diagnosed.

The Schering-Plough Research Institute conducted a randomized, multicenter, open-label phase 2 study of TMZ and PCB in 225 patients with glioblastoma multiforme (GBM) at first relapse.213 The primary objectives were to compare the progression-free survival at 6 months and safety of TMZ and PCB in adult patients with GBM who had failed conventional treatment. The 6-month progression-free survival rate was significantly higher for patients who received TMZ (21%) than for those who received PCB (8%) (P = .008). Median progression-free survival for TMZ patients (12.4 weeks) was significantly longer than for PCB patients (8.32 weeks; P = .0063). The 6-month overall survival rate for TMZ patients was 60% versus 44% for PCB patients (P = .019).

The Schering-Plough Research Institute also conducted an open-label, multicenter phase 2 trial comprising 162 patients with malignant astrocytoma at first relapse.214 The primary protocol end point, progression-free survival at 6 months, was 46% [95% confidence limit (CL), 38 to 54%]. The median progression-free survival was 5.4 months, and 24% of patients remained progression-free at 12 months based on Kaplan-Meier estimates.

Duke University participated in a Schering-Plough Research Institute multicenter phase II trial evaluating the activity of TMZ before radiation therapy in the treatment of newly diagnosed high-grade glioma.215 Eligibility criteria included residual enhancing disease on postoperative MRI and a Karnofsky performance score (KPS) ≥70%. Thirty-three patients with GBM evaluated for tumor response revealed 3 with complete response, 14 with partial response, 4 with stable disease, and 12 with progressive disease. Five patients with anaplastic astrocytoma evaluated for response revealed one with partial response, two with stable disease, and two with progressive disease. These results with patients with glioblastoma multiforme have been extended to phase 3 trials, confirming an increase in survival when TMZ is used in an adjuvant setting.216, 217 Furthermore, TMZ has been shown to be active in the treatment of other primary brain tumors, including low-grade glioma,218 oligodendroglioma,219 and meningioma.220

The efficacy of TMZ has also been evaluated in a study of patients with advanced metastatic melanoma, including patients with brain metastases.221 Among 56 patients (49 with evaluable lesions), complete responses occurred in 3, all with lung metastases only, and partial responses occurred in 9, yielding a response rate of 21%. Stable disease was observed in an additional eight patients.

General Mechanism of Action and Cellular Pharmacology

The spontaneous conversion of TMZ is initiated by the effect of water at the highly electropositive C 4 position of TMZ. This activity opens the ring, releases CO2, and generates the reactive methylating agent MTIC. The initial proposal was that this effect of water was catalyzed in the close environment of the major groove of DNA,222, 223 but confirming this mechanism has been difficult, and it is known that TMZ converts readily to MTIC in free solution in the absence of DNA.224 MTIC degrades to the methyldiazonium cation, which transfers the methyl group to DNA and to the final degradation product AIC, which is excreted via the kidneys.225, 226 The methylation of DNA appears to be the principal mechanism responsible for the cytotoxicity of TMZ to malignant cells (see following discussion). The methyldiazonium cation can also react with RNA and with soluble and cellular protein.227However, the methylation of RNA and the methylation or carbamoylation of protein do not appear to have any known significant role in the antitumor activity of TMZ.227 Further studies are required to clarify the role of these targets in the biochemical mechanism of action of TMZ.

The spontaneous conversion of TMZ and MTIC depends on pH. Under acidic conditions, TMZ is stable; however, its chemical stability decreases at a pH of >7.0 and is converted rapidly to MTIC in that environment.226 In contrast, MTIC is more stable under basic conditions and rapidly degrades to the methyldiazonium cation and AIC at a pH of <7.0.226 A comparison of the half-life of TMZ in phosphate buffer (pH, 7.4; t1/2 = 1.83 hours)209, 226 indicates that the conversion of TMZ to MTIC is a chemically controlled reaction with little or no enzymatic component. The spontaneous conversion of TMZ may contribute to its highly reproducible pharmacokinetics in comparison with other alkylating agents such as DTIC and PCB, which must undergo metabolic conversion in the liver and are thus subject to interpatient variation in metabolic rates of conversion.223, 226

Among the lesions produced in DNA after treatment of cells with TMZ, the most common is methylation at the N7 position of guanine, followed by methylation at the O3 position of adenine and the O6 position of guanine.226 Although the N7-methylguanine and O3-methyladenine adducts probably contribute to the antitumor activity of TMZ in some, if not all, sensitive cells, their role is controversial.228 The critical role of the O6-methylguanine adduct, which accounts for 5% of the total adducts formed by TMZ,226 in the agent's antitumor activity is supported by the correlation between the sensitivity of tumor cell lines to TMZ and the activity of the DNA repair protein AGT, which specifically removes alkyl groups at the O6 position of guanine. Cell lines that have low levels of AGT are sensitive to the cytotoxicity of TMZ, whereas cell lines that have high levels of this repair protein are much more resistant to it.229, 230, 231, 232 This correlation also has been observed in human glioblastoma xenograft models.233, 234, 235 The preferential alkylation of guanine and adenine and the correlation of sensitivity to the drug with the ability to repair the O6- alkylguanine lesion also have been seen with triazine, DTIC, and the nitrosourea alkylating agents BCNU and CCNU.232, 236, 237

The cytotoxic mechanism of TMZ appears to be related to the failure of the DNA mismatch repair system to find a complementary base for methylated guanine. This system involves the formation of a complex of proteins that recognize, bind to, and remove methylated guanine.238, 239, 240 The proposed hypothesis is that when this repair process is targeted to the DNA strand opposite the O6-methylguanine, it cannot find a correct partner, thus resulting in long-lived nicks in the DNA.241 These nicks accumulate and persist into the subsequent cell cycle, where they ultimately inhibit initiation of replication in the daughter cells, blocking the cell cycle at the G2M boundary.241, 242, 243, 244, 245 In murine242 and human246 leukemia cells, sensitivity to TMZ correlates with increased fragmentation of DNA and apoptotic cell death. More recent work has shown that TMZ induces G2-M arrest through activation of Chk1 kinase with subsequent phosphorylation of Ccd 25 phosphatase and cdc2.247, 248 This has been shown to be p53-independent, although p53 status impacts on G2-M arrest duration and outcome. Specifically, p53 wild-type cells undergo prolonged G2-M arrest and senescence, whereas p53-deficient cells bypass cell cycle arrest and die by mitotic catastrophe. Since p53-proficient cells were less sensitive than p53-deficient cells to TMZ, it is possible that targeting the G2 checkpoint might enhance TMZ-induced antitumor activity.247, 248 Additionally, O6-methylguanine induced apoptosis is executed by the mitochondrial damage pathway, requires DNA replication, and is mediated by p53 and Fas/CD95/Apo-1.249 Nevertheless, studies confirming that base excision repair mediates TMZ resistance have not yet been conclusively demonstrated.

DNA adducts formed by TMZ and the subsequent DNA damage or alteration of specific genes may cause cell death or reduce the metastatic potential of tumor cells. For example, mutations caused by adduct formation may result in altered surface antigens on tumor cells that contribute enhanced immunogenicity in the host.250, 251 The effects of enhanced immunologic response range from complete tumor rejection to reduced growth rates and reduced metastatic potential.252 Additional evidence suggests that TMZ can reduce the metastatic potential of Lewis lung carcinoma cells253 and induce differentiation in the K562 erythroleukemia cell line.254 It has been postulated that TMZ- induced DNA damage and subsequent cell-cycle arrest may reduce the metastatic properties of some tumor cells.254

Mechanism of Resistance

AGT DNA Repair Protein

Several studies have shown that AGT is the primary mechanism of resistance to TMZ and other alkylating agents.147, 257 AGT functions as the first line of defense against TMZ by removing the alkyl groups from the O6 position of guanine, in effect reversing the cytotoxic lesion of TMZ.258 AGT levels can be correlated with the sensitivity of tumor cell lines to TMZ and the alkylating agents BCNU and DTIC.237, 246, 259, 260, 261, 262 The role of AGT in resistance to TMZ is also evidenced by the ability of the virally transfected human AGT gene to confer a high level of resistance to TMZ and other methylating and chloroethylating agents on cells that are devoid of endogenous AGT activity.263

AGT levels in human tumor tissues and normal tissue specimens derived from brain, lung, and ovary vary widely over a 100-fold range, with some human tumors having no detectable activity.264, 265, 266, 267 Some specimens from all tumor types examined in these studies have demonstrated a complete absence of AGT activity: as many as 22% of primary brain tumor specimens have no detectable AGT activity.264 Similar findings with respect to AGT levels in brain tumor cells have been observed in in vitro models.268 AGT activity has been localized to both the cytoplasm and the nucleus of the cell, although the function of cytoplasmic AGT and its mechanism of transport to the nucleus are unknown.265 AGT transfers the methyl group to an internal cysteine residue, acting as methyltransferase and methyl acceptor protein. In the process, AGT becomes irreversibly inactivated, and new AGT must be synthesized to restore AGT activity.61 Therefore, the number of O6-methylguanine adducts that can be repaired is limited by the number of AGT molecules of the protein available.61Recent work has confirmed that elevated AGT levels in newly diagnosed glioblastoma multiforme are directly correlated with lack of response to TMZ215 or survival following adjuvant therapy with this methylator.269

Deficiency in Mismatch Repair Pathway

Although AGT is clearly important in the resistance of cells to TMZ, some cell lines that express low levels of AGT are nevertheless resistant, indicating that other resistance mechanisms may be involved.270,271A deficiency in the mismatch repair pathway as a result of mutations in any one of the four proteins that recognize and repair DNA (i.e., GTBP, hMSH2, hPMS2, and hMLH1) can render cells tolerant to methylation and to the cytotoxic effects of TMZ. This deficiency in the mismatch repair pathway results in a failure to recognize and repair the O6-methylguanine adducts produced by TMZ and other methylating agents.63,230, 272 The DNA damage that results from failure to repair the O6-methylguanine adducts produces a particular type of genomic instability, microsatellite instability, that is associated with some familial and sporadic cancers, such as hereditary nonpolyposis colorectal cancer.273, 274 The high level of resistance in tumor cells that are deficient in mismatch repair is unrelated to the level of AGT and is, therefore, unaffected by AGT inhibitors.

Base Excision Repair

A series of studies have shown that two Temodar-initiated adducts, N7-methylguanine and N3-methyladenine, are not susceptible to AGT and produce cytotoxicity (particularly N3-methyladenine), independently of DNA mismatch repair activity.228, 275, 276, 277 These lesions are promptly repaired by a series of enzymatic steps including N-methylpurine-DNA glycosylase, AP endonuclease, poly (ADP-ribose) polymerase (PARP) DNA polymerase β, x-ray repair cross complementing 1, and ligase III. Sensitization of tumor cells resistant to Temodar because of DNA mismatch repair deficiency have been rendered susceptible to this methylator by inhibition of base excision repair. Strategies have included inhibition of PARP275, 276, 277, 278, 279, 280, 281, 282, 283 and use of methoxyamine.228 Intriguingly, moderate enhancement of Temodar activity following base excision repair disruption was also seen in DNA mismatch repair proficient cells.228, 275, 276, 277 Nevertheless, conclusive evidence confirming that base excision repair mediates TMZ resistance has not yet been published.

Drug Interactions

There are no known adverse reactions with other drugs. It is expected that compounds that deplete AGT will increase TMZ toxicity.

Clinical Pharmacology and Toxicity

TMZ is supplied in capsules containing 5, 25, 100, or 250 mg for oral use. In the initial phase 1 trial in the United Kingdom, TMZ was administered as a single intravenous dose at doses of 50 to 200 mg/m2 and subsequently was given orally to fasted patients as a single dose, up to a total dose of 200 to 1,200 mg/m2. Additionally, oral doses of 750 to 1,200 mg/m2 were divided into five equal doses and administered daily for 5 days at 4-week intervals.

The pharmacokinetics of TMZ were evaluated in the United Kingdom phase 1 trials.209 After intravenous administration, plasma TMZ concentrations declined biexponentially consistent with a two-compartment open model and a terminal elimination half-life of 1.8 hours. After oral administration, plasma TMZ concentrations were consistent with a one-compartment oral model, with rapid absorption and maximum plasma concentrations occurring 0.7 hour after treatment. The clearance of TMZ was 11.8 L/hr, and the pharmacokinetics were independent of the dosage (with a linear relationship between dose and area under the time × concentration curve). Oral bioavailability was considered to be complete.

In 1993, Schering-Plough began the worldwide development of TMZ using machine-filled capsules that were prepared according to good manufacturing practices, which differed from the hand-filled capsules used in the initial study. Several phase 1 studies have evaluated the safety and tolerability of that new TMZ formulation (Temodar). Data from these studies have confirmed the safety, tolerability, and pharmacokinetics of TMZ reported in the CRC phase 1 study (Table 13.3).284, 285, 286, 287, 288, 289, 290, 291, 292

Phase 1 studies of TMZ also were expanded to include pediatric cancer patients. A phase 1 study was conducted to define the multiple-dose pharmacokinetics of TMZ in this population. In this study, 19 patients between 3 and 17 years old were given TMZ over a dosage range of 100 to 240 mg/m2 per day. TMZ was absorbed rapidly, had an AUC that increased in a dosage-related manner, and showed no evidence of accumulation. The plasma half-life, whole-body clearance, and volume of distribution were independent of dosage (Table 13.4).284 Compared with adult patients treated with 200 mg/m2 per day, children appeared to have a higher AUC (48.7 versus 34.5 mg/hr per milliliter), most likely because children have a larger ratio of body surface area to volume. Despite higher concentrations at dosages equivalent to those used in adult patients, the bone marrow function in pediatric patients appears to allow greater exposure to the drug before dose-limiting bone marrow toxicity develops.284

The effects of food and gastric pH on the pharmacokinetics and bioavailability of orally administered TMZ also have been evaluated. Administration of TMZ after ingestion of food resulted in a small decrease in its oral bioavailability.289 When TMZ was taken after a meal, a slight (9%), but statistically significant, reduction occurred in the rate and extent of its absorption (Table 13.5). Because AUC confidence levels were within the bioequivalence guidelines of 80 to 125%, it is unlikely that the slight reduction observed in the oral bioavailability of TMZ in the presence of a meal has any clinical effect on the antitumor activity of TMZ.

The oral bioavailability, maximum plasma concentration, and half-life of TMZ were not affected by an increase in gastric pH of 1 to 2 units, resulting from the administration of ranitidine every 12 hours on either the first 2 or the last 2 days of the 5-day TMZ dosing schedule.291

Subsequent phase 1 trials sponsored by Schering-Plough in adult286, 287, 288, 289, 290, 291 and pediatric patients284, 293 with advanced cancer also have confirmed that hematologic toxicity, specifically thrombocytopenia and neutropenia, is dose-limiting. Neutropenia or thrombocytopenia appeared 21 to 28 days after the first dose of each cycle and recovered to grade 1 myelosuppression within 7 to 14 days. Grade 4 toxicity occurred at cumulative oral dosages of more than 1,000 mg/m2 over 5 days, but little other toxicity was seen.287 Grade 3 or 4 myelosuppression occurred in less than 10% of patients studied.

The effect of prior treatment with chemotherapy, radiation, or both, on the maximum tolerated dose (MTD) of TMZ has been evaluated.286, 290 In one of these studies,290 24 patients stratified according to prior exposure to chemotherapy and radiation were given a dosage of 100 mg/m2 per day of TMZ for 5 days, which was escalated to 150 and 200 mg/m2 per day in the absence of myelosuppression. The MTD for TMZ was established as 150 mg/m2 per day.290 The other similar phase 1 study, reported by the National Cancer Institute, evaluated the safety of TMZ in patients who were stratified on the basis of prior exposure to nitrosourea.286 The MTD for patients with prior exposure to nitrosourea was 150 mg/m2 per day, and the MTD for patients without such prior exposure was 250 mg/m2 per day. An evaluation of the pharmacokinetics of TMZ showed that its clearance from the plasma was significantly less in patients with prior exposure to nitrosourea than it was in patients without such prior exposure.286 This may have contributed to the lower dose of TMZ that was tolerated by these patients and had a notable effect on the dosing recommendation for these patients.286

The results of these studies indicated that a dosage of 200 mg/m2 of TMZ given on a 5-day schedule and repeated every 28 days is appropriate for patients who are not pretreated with radiation, chemotherapy, or both. Patients who are pretreated with chemotherapy receive a lower starting dose of TMZ (i.e., 150 mg/m2), which can be escalated to 200 mg/m2 in subsequent courses in the absence of grade 3 or 4 myelosuppression.290

TABLE 13.5 KEY FEATURES OF TEMOZOLOMIDE (8-CARBAMOYL-3-METHYLIMIDAZO [5,1-D]-1,2,3,5-TETRAZIN-4(3H)-ONE)

Mechanism of action:

Methylation of nucleic acids

Metabolism:

Chemical conversion of 5(3-methyltriazeno) imidazole-4-carboxamide

Pharmacokinetic Volume of distribution:

28.3 L

s (i.v. or p.o.):

Elimination half-life: 1.8 h

Distribution half-life: 0.26 h

Clearance: 11.76 L/h253

Drug and food interactions:

Unknown

Toxicity:

Myelosuppression

Nausea and vomiting

Elevated hepatic transaminases

REFERENCES

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