Chapter 61: Cytotoxic Agents

Chemistry. The chemotherapeutic alkylating agents have in common the property of forming highly reactive carbonium ion intermediates. These reactive intermediates covalently link to sites of high electron density, such as phosphates, amines, sulfhydryl, and hydroxyl groups. Their chemotherapeutic and cytotoxic effects are directly related to the alkylation of reactive amines, oxygens, or phosphates on DNA. The N7 atom of guanine is particularly susceptible to the formation of a covalent bond with bifunctional alkylating agents and may represent the key target that determines their biological effects. Other atoms in the purine and pyrimidine bases of DNA, including N1 and N3 of the adenine ring, N3 of cytosine, and O6 of guanine, react with these agents, as do the amino and sulfhydryl groups of proteins and the sulfhydryls of glutathione.

The possible actions of alkylating agents on DNA are illustrated in Figure 61–1 with mechlorethamine (nitrogen mustard). First, one 2-chloroethyl side chain undergoes a first-order (S_N1) intramolecular cyclization, with release of Cl⁻ and formation of a highly reactive ethyleneimine intermediate (Figure 61–1). The unstable quaternary amine then reacts with a variety of electron-dense sites. This latter reaction proceeds as a second-order (S_N2) nucleophilic substitution. Alkylation of the N7 of guanine in DNA, a highly favored reaction, exerts several biologically important effects. Guanine residues in DNA exist predominantly as the keto tautomer and readily make Watson-Crick base pairs by hydrogen bonding with cytosine residues. However, when the N7 of guanine is alkylated (to become a quaternary ammonium nitrogen) the guanine residue is more acidic and the enol tautomer is favored. The modified guanine can mispair with thymine residues during DNA synthesis, leading to the substitution of thymine for cytosine. Second, alkylation of the N7 creates lability in the imidazole ring, leading to opening of the ring and excision of the damaged guanine residue. Mispairing and imidazole ring opening can lead to attempts to repair the damaged stretch of DNA, causing strand breakage. Third, with bifunctional alkylating agents such as nitrogen mustard, the second 2-chloroethyl side chain can undergo a similar cyclization reaction and alkylate a second guanine residue or another nucleophilic moiety, resulting in the cross-linking of two nucleic acid chains or the linking of a nucleic acid to a protein, alterations that would cause a major disruption in nucleic acid function. Any of these effects could contribute to both the mutagenic and cytotoxic effects of alkylating agents. The extreme cytotoxicity of bifunctional alkylators correlates very closely with interstrand cross-linkage of DNA (Garcia et al., 1988).

The ultimate cause of cell death related to DNA damage is not known. Specific cellular responses include cell-cycle arrest and attempts to repair DNA. The specific repair enzyme complex utilized will depend on two factors: the chemistry of the adduct formed and the repair capacity of the cell involved. The process of recognizing and repairing DNA generally requires an intact nucleotide excision repair (NER) complex, but, as discussed in the rest of this section, may differ with each drug and with each tumor. Alternatively, recognition of extensively damaged DNA by p53 can trigger apoptosis. Mutations of p53 lead to alkylating agent resistance (Kastan, 1999).

Structure-Activity Relationships. Although these alkylating agents share the capacity to alkylate biologically important molecules, modification of the basic chloroethylamino structure changes reactivity, lipophilicity, active transport across biological membranes, sites of macromolecular attack, and mechanisms of DNA repair, all of which properties determine drug activity in vivo. With several of the most valuable agents (e.g., cyclophosphamide, ifosfamide), the active alkylating moieties are generated in vivo through hepatic metabolism.

The biological activity of alkylators of the nitrogen mustard type is based on the presence of the bis-(2-chloroethyl) group. Although mechlorethamine has been widely used in the past, linkage of the bis-(2-chloroethyl) group to election-rich substitutions such as unsaturated ring systems has yielded more stable drugs with better pharmacodynamic properties and with greater selective killing of tumor cells. Bis-(2-chloroethyl) groups linked to amino acids (phenylalanine) and substituted phenyl groups (aminophenol butyric acid, as in chlorambucil) create a more stable and orally available form. The structures of important nitrogen mustards are shown in Figure 61–2.

A classical example of the role of host metabolism in the activation and degradation of an alkylating agent is seen with cyclophosphamide, now the most widely used agent of this class. The drug undergoes metabolic activation (hydroxylation) by CYP2B (Figure 61–3), with subsequent transport of the activated intermediate to sites of action. The selectivity of cyclophosphamide against certain malignant tissues may result in part from the capacity of normal tissues to degrade the activated intermediates via aldehyde dehydrogenase, glutathione transferase, and other pathways.

Ifosfamide is an oxazaphosphorine, similar to cyclophosphamide. Cyclophosphamide has two chloroethyl groups on the exocyclic nitrogen atom, whereas one of the 2-chloroethyl groups of ifosfamide is on the cyclic phosphoramide nitrogen of the oxazaphosphorine ring. Ifosfamide is activated in the liver by CYP3A4. The activation of ifosfamide proceeds more slowly, with greater production of dechlorinated metabolites and chloroacetaldehyde. These differences in metabolism likely account for the higher doses of ifosfamide required for equitoxic effects, the greater neurotoxicity of ifosfamide, and the possible differences in antitumor spectrum of the two agents.

The newest approved alkylating agent, bendamustine, has the typical chloroethyl reactive groups attached to a benzimidazole backbone.

The unique properties and activity of this drug may derive from this purine-like structure; the agent produces slowly repaired DNA cross-links, lacks cross-resistance with other classical alkylators (Leoni et al., 2008), and has significant activity in chronic lymphocytic leukemia (CLL) and large-cell lymphomas refractory to standard alkylators.

A unique class of alkylating agents transfers methyl rather than ethyl groups to DNA. The triazene derivative 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, usually referred to as dacarbazine or DTIC, is prototypical of methylating agents.

Dacarbazine requires initial activation by hepatic CYPs through an N-demethylation reaction. In the target cell, spontaneous cleavage of the metabolite, methyl-triazeno-imidazole-carboxamide (MTIC), yields an alkylating moiety, a methyl diazonium ion. A related triazene, temozolomide, undergoes spontaneous, nonenzymatic activation to MTIC and has significant activity against gliomas.

The nitrosoureas, which include compounds such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (carmustine; BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (lomustine; CCNU), and its methyl derivative (semustine; methyl-CCNU), as well as the antibiotic streptozocin (streptozotocin), exert their cytotoxicity through the spontaneous breakdown to an alkylating intermediate, the 2-chloroethyl diazonium ion.

The 2-chloroethyl diazonium ion, a strong electrophile, can alkylate guanine, cytidine, and adenine bases (Ludlum, 1990). Displacement of the halogen atom can then lead to interstrand or intrastrand cross-linking of DNA. The formation of cross-links after the initial alkylation reaction proceeds relatively slowly and can be reversed by the DNA repair enzyme O⁶-alkyl, methyl guanine methyltransferase (MGMT), which displaces the chloroethyl adduct from its binding to guanine in a suicide reaction. The same enzyme, when expressed in human gliomas, produces resistance to nitrosoureas and to other methylating agents, including DTIC, temozolomide, and procarbazine. As with the nitrogen mustards, interstrand cross-linking appears to be the primary lesion responsible for the cytotoxicity of nitrosoureas (Ludlum, 1990). The reactions of the nitrosoureas with macromolecules are shown in Figure 61–4.

Because the formation of the ethyleneimine ion constitutes the initial reaction of the nitrogen mustards, it is not surprising that stable ethyleneimine derivatives have antitumor activity. Several compounds of this type, including triethylenemelamine (TEM) and triethylenethiophosphoramide (thiotepa), have been used clinically. In standard doses, thiotepa produces little toxicity other than myelosuppression; it also is used for high-dose chemotherapy regimens, in which it causes both mucosal and central nervous system toxicity. Altretamine (hexamethylmelamine; HMM) is mentioned here because of its chemical similarity to TEM. The methylmelamines are N-demethylated by hepatic microsomes with the release of formaldehyde, and there is a direct relationship between the degree of the demethylation and their activity against murine tumors.

Esters of alkane sulfonic acids alkylate DNA through the release of methyl radicals. Busulfan is of value in high-dose chemotherapy.

In non-dividing cells, DNA damage activates a checkpoint that depends on the presence of a normal p53 gene. Cells thus blocked in the G₁/S interface either repair DNA alkylation or undergo apoptosis. Malignant cells with mutant or absent p53 fail to suspend cell-cycle progression, do not undergo apoptosis, and exhibit resistance to these drugs.

Although DNA is the ultimate target of all alkylating agents, a crucial distinction must be made between the bifunctional agents, in which cytotoxic effects predominate, and the monofunctional methylating agents (procarbazine, temozolomide), which have greater capacity for mutagenesis and carcinogenesis. This suggests that the cross-linking of DNA strands represents a much greater threat to cellular survival than do other effects, such as single-base alkylation and the resulting depurination and single-chain scission. On the other hand, simple methylation may be bypassed by DNA polymerases, leading to mispairing reactions that permanently modify DNA sequence. These new sequences are transmitted to subsequent generations and may result in mutagenesis or carcinogenesis. Some methylating agents, such as procarbazine, are highly carcinogenic.

DNA repair systems play an important role in removing adducts, and thereby determine the selectivity of action against particular cell types, and acquired resistance to alkylating agents. Alkylation of a single strand of DNA (mono-adducts) is repaired by the nucleotide excision repair pathway, while the less frequent cross-links require participation of nonhomologous end joining, an error-prone pathway, or the error-free homologous recombination pathway. After drug infusion in humans, mono-adducts appear rapidly and peak within 2 hours of drug exposure, while cross-links peak at 8 hours. The t_1/2 for repair of adducts varies among normal tissues and tumors; in peripheral blood mononuclear cells, both mono-adducts and cross-links disappear with a t_1/2 of 12-16 hours (Souliotis et al., 1990).

The homologous end-joining pathway has multiple components (Wang et al., 2001):

• sensors of DNA integrity (such as p53)

• activation signals such as the ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia and rad-related (ATR) proteins

• the activated repair complex composed of Fanconi anemia proteins and BRCA2, all of which localize at the site of DNA damage and initiate removal of the cross-linked segment of DNA

• homologous recombination, which allows resynthesis of the damaged DNA sequence followed by re-ligation of the repaired sequences

The process depends on the presence and accurate functioning of multiple proteins. Their absence or mutation, as in Fanconi anemia or ataxia telangiectasia, leads to extreme sensitivity to DNA cross-linking agents such as mitomycin, cisplatin, or classical alkylators.

Other repair enzymes are specific for removing methyl and ethyl adducts from the O-6 of guanine (MGMT) and for repair of alkylation of the N-3 of adenine and N-7 of guanine (3-methyladenine-DNA glycosylase) (Matijasevic et al., 1993). High expression of MGMT protects cells from cytotoxic effects of nitrosoureas and methylating agents and confers drug resistance, while methylation and silencing of the gene in brain tumors are associated with clinical response to BCNU and temozolomide (Hegi et al., 2008).

Bendamustine differs from classical chloroethyl alkylators in activating base excision repair, rather than the more complex double-strand break repair or MGMT. It impairs physiological arrest of adduct-containing cells at mitotic checkpoints and leads to mitotic catastrophe rather than apoptosis, and does not require an intact p53 to cause cytotoxicity.

Finally, recognition of DNA adducts is an essential step in promoting attempts at repair and ultimately leading to apoptosis. The Fanconi pathway, consisting of 12 proteins, recognizes adducts and signals the need for repair of a broad array of DNA-damaging drugs and irradiation (Chen et al., 2007). Absence or inactivation of components of this pathway leads to increased sensitivity to DNA damage. Conversely, for the methylating drugs, nitrosoureas, cisplatin and carboplatin, and thiopurine analogs, the mismatch repair (MMR) pathway is essential for cytotoxicity, causing strand breaks at sites of adduct formation, creating mispairing of thymine residues, and triggering apoptosis.

Increased activity of the complex NER pathway correlates with resistance to most chloroethyl and platinum adducts. MGMT activity determines response to BCNU and to methylating drugs such as the triazenes, procarbazine, temozolomide, and busulfan. Methylation of the MGMT gene found that pretreatment in 20% of brain tumor patients decreases MGMT expression and strongly correlates with response and survival after BCNU or temozolomide for malignant gliomas. MGMT activity increases with each round of alkylator treatment and with disease progression (Wiewrodt et al., 2008).

The MSH6 component of the MMR system seems particularly susceptible to mutation by exposure to alkylating agents; in studies of brain tumor resistance to therapy, loss of MSH6 conferred resistance to temozolomide and related drugs (Cahill et al., 2008).

Cyclophosphamide has lesser effects on peripheral blood platelet counts than do the other agents. Busulfan suppresses all blood elements, particularly stem cells, and may produce a prolonged and cumulative myelosuppression lasting months or even years. For this reason, it is used as a preparative regimen in allogenic bone marrow transplantation. Carmustine and other chloroethylnitrosoureas cause delayed and prolonged suppression of both platelets and granulocytes, reaching a nadir 4-6 weeks after drug administration and reversing slowly thereafter.

The mucosal effects are particularly damaging in high-dose chemotherapy protocols associated with bone marrow reconstitution, as they predispose to bacterial sepsis arising from the GI tract. In these protocols, cyclophosphamide, melphalan, and thiotepa have the advantage of causing less mucosal damage than the other agents. In high-dose protocols, however, other toxicities become limiting, as delineated in Table 61–1.

Other Organ Toxicities. Although mucosal and bone marrow toxicities occur predictably and acutely with conventional doses of these drugs, other organ toxicities may supervene after prolonged or high-dose use; these effects can appear after months or years and may be irreversible and even lethal. All alkylating agents, including temozolomide, have caused pulmonary fibrosis, usually several months after treatment. In high-dose regimens, particularly those employing busulfan or BCNU, vascular endothelial damage may precipitate veno-occlusive disease (VOD) of the liver, an often fatal side effect that is successfully reversed by the investigational drug defibrotide (Richardson et al., 2003). The nitrosoureas and ifosfamide, after multiple cycles of therapy, may lead to renal failure. Cyclophosphamide and ifosfamide release a nephrotoxic and urotoxic metabolite, acrolein, which causes a severe hemorrhagic cystitis in high-dose regimens. This adverse effect can be prevented by co-administration of 2-mercaptoethanesulfonate (mesna; mesnex), which conjugates acrolein in urine. Ifosfamide in high doses for transplant causes a chronic, and often irreversible, renal toxicity. Proximal and, less commonly, distal tubules may be affected, leading to defective Ca²⁺ and Mg²⁺ reabsorption, glycosuria, and renal tubular acidosis. Nephrotoxicity correlates with the total dose of drug received and increases in frequency in children <5 years of age. The syndrome may be due to chloroacetaldehyde and/or acrolein excreted in the urine.

The more unstable alkylating agents (particularly mechlorethamine and the nitrosoureas) have strong vesicant properties, damage veins with repeated use and, if extravasated, produce ulceration.

Finally, all alkylating agents have toxic effects on the male and female reproductive systems, causing an often permanent amenorrhea, particularly in perimenopausal women, and an irreversible azoospermia in men.

Leukemogenesis. As a class of drugs, the alkylating agents are highly leukemogenic. Acute nonlymphocytic leukemia, often associated with partial or total deletions of chromosome 5 or 7, peaks in incidence ~4 years after therapy and may affect up to 5% of patients treated on regimens containing alkylating drugs. Leukemia often is preceded by a period of neutropenia or anemia and by bone marrow morphology consistent with myelodysplasia. Melphalan, the nitrosoureas, and the methylating agent procarbazine have the greatest propensity to cause leukemia, while it is less common after cyclophosphamide.

Mechlorethamine. Mechlorethamine was the first clinically used nitrogen mustard and is the most reactive of the drugs in this class. It rarely is used in current practice.

Absorption and Fate. Severe local reactions of exposed tissues necessitate rapid intravenous injection of mechlorethamine for most clinical uses. In either water or body fluids, at rates affected markedly by pH, mechlorethamine rapidly undergoes chemical degradation as it combines with either water or cellular nucleophiles, and the parent compound disappears from the bloodstream within minutes.

Therapeutic Uses. Mechlorethamine HCl (mustargen) formerly was used in the combination chemotherapy regimen MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) in patients with Hodgkin's disease. It is given by intravenous bolus administration in doses of 6 mg/m² on days 1 and 8 of the 28-day cycles of each course of treatment. It has been largely replaced by cyclophosphamide, melphalan, and other, more stable alkylating agents.

It also is used topically for treatment of cutaneous T-cell lymphoma (CTCL) as a solution that is rapidly mixed and applied to affected areas of the skin.

Clinical Toxicity. The major acute toxic manifestations of mechlorethamine are nausea and vomiting, lacrimation, and myelosuppression. Leukopenia and thrombocytopenia limit the amount of drug that can be given in a single course.

Cyclophosphamide.

Absorption, Fate, and Excretion. Cyclophosphamide is well absorbed orally and is activated to the 4-hydroxy intermediate (Xie et al., 2003) (Figure 61–3). 4-Hydroxycyclophosphamide exists in equilibrium with the acyclic tautomer aldophosphamide. The rate of metabolic activation of cyclophosphamide exhibits significant interpatient variability and increases with successive doses in high-dose regimens but appears to be saturable at infusion rates of >4 g/90 min and concentrations of the parent compound >150 μM (Chen et al., 1995). 4-Hydroxycyclophosphamide may be oxidized further by aldehyde oxidase, either in liver or in tumor tissue, to inactive metabolites. The hydroxyl metabolite of ifosfamide similarly is inactivated by aldehyde dehydrogenase. 4-Hydroxycyclophosphamide and its tautomer, aldophosphamide, travel in the circulation to tumor cells where aldophosphamide cleaves spontaneously, generating stoichiometric amounts of phosphoramide mustard and acrolein. Phosphoramide mustard is responsible for antitumor effects, while acrolein causes hemorrhagic cystitis often seen during therapy with cyclophosphamide. Cystitis can be reduced in intensity or prevented by the parenteral co-administration of mesna. Mesna does not negate the systemic antitumor activity of the drug.

Patients should receive vigorous intravenous hydration during high-dose treatment. Brisk hematuria in a patient receiving daily oral therapy should lead to immediate drug discontinuation. Refractory bladder hemorrhage can become life-threatening, and cystectomy may be necessary for control of bleeding.

Inappropriate secretion of antidiuretic hormone has been observed in patients receiving cyclophosphamide, usually at doses >50 mg/kg (see Chapter 25). It is important to be aware of the possibility of water intoxication, because these patients usually are vigorously hydrated to prevent bladder toxicity.

Pretreatment with CYP inducers such as phenobarbital enhances the rate of activation of the azoxyphosphorenes but does not alter total exposure to active metabolites over time and does not affect toxicity or therapeutic activity in humans. Cyclophosphamide can be used in full doses in patients with renal dysfunction, because it is eliminated by hepatic metabolism. Patients with mild hepatic dysfunction (bilirubin <3 mg/dL) can be treated with full doses of this drug, but those with more significant hepatic dysfunction should receive reduced doses.

Urinary and fecal excretion of unchanged cyclophosphamide is minimal after intravenous administration. Maximal concentrations in plasma are achieved 1 hour after oral administration, and the t_1/2 of parent drug in plasma is ~7 hours.

Therapeutic Uses. Cyclophosphamide (lyophilized cytoxan, others) is administered orally or intravenously. Recommended doses vary widely, and standard protocols for determining the schedule and dose of cyclophosphamide in combination with other chemotherapeutic agents should be consulted.

As a single agent, a daily oral dose of 100 mg/m² for 14 days has been recommended for patients with lymphomas and CLL. Higher doses of 500 mg/m² intravenously every 2-4 weeks are used in combination with other drugs in the treatment of breast cancer and lymphomas. The neutrophil nadir of 500-1000 cells/mm³ generally serves as a lower limit for dosage adjustments in prolonged therapy. In regimens associated with bone marrow or peripheral stem cell rescue, cyclophosphamide may be given in total doses of 5-7 g/m² over a 3- to 5-day period. GI ulceration, cystitis (counteracted by mesna and diuresis), and, less commonly, pulmonary, renal, hepatic, and cardiac toxicities (a hemorrhagic myocardial necrosis) may occur after high-dose therapy with total doses >200 mg/kg.

The clinical spectrum of activity for cyclophosphamide is very broad. It is an essential component of many effective drug combinations for non-Hodgkin's lymphomas, other lymphoid malignancies, breast and ovarian cancers, and solid tumors in children. Complete remissions and presumed cures have been reported when cyclophosphamide was given as a single agent for Burkitt's lymphoma. It frequently is used in combination with doxorubicin and a taxane as adjuvant therapy after surgery for carcinoma of the breast.

Because of its potent immunosuppressive properties, cyclophosphamide has been used to treat auto-immune disorders, including Wegener's granulomatosis, rheumatoid arthritis, and the nephrotic syndrome. Caution is advised when the drug is considered for non-neoplastic conditions, not only because of its acute toxic effects but also because of its potential for inducing sterility, teratogenic effects, and leukemia.

Therapeutic Uses. Ifosfamide is approved for treatment of relapsed germ cell testicular cancer and is frequently used for first-time treatment of pediatric and adult sarcomas. It is a common component of high-dose chemotherapy regimens with bone marrow or stem cell rescue; in these regimens, in total doses of 12-14 g/m², it may cause severe neurological toxicity, including hallucinations, coma, and death, with symptoms appearing 12 hours to 7 days after beginning the ifosfamide infusion. This toxicity is thought to result from a metabolite, chloroacetaldehyde. In addition, ifosfamide causes nausea, vomiting, anorexia, leukopenia, nephrotoxicity, and VOD of the liver.

In nonmyeloablative regimens, ifosfamide is infused intravenously over at least 30 minutes at a dose of ≤1.2 g/m²/day for 5 days. Intravenous mesna is given as bolus injections in a dose equal to 20% of the ifosfamide dose concomitantly and an additional 20% again 4 and 8 hours later, for a total mesna dose of 60% of the ifosfamide dose. For ifosfamide doses ≤2 g/m², oral mesna, at a dose equal to 40% of the ifosfamide dose, can be substituted for the second and third IV mesna doses, given at 2 and 6 hours after each dose of ifosfamide, for a total mesna dose equal to the ifosfamide dose. Alternatively, mesna may be given concomitantly in a single dose equal to the ifosfamide dose. Patients also should receive at least 2 L of oral or intravenous fluid daily. Treatment cycles are repeated every 3-4 weeks after hematological recovery.

Pharmacokinetics. The parent compound, ifosfamide, has an elimination t_1/2 in plasma of ~1.5 hours after doses of 3.8-5 g/m² and a somewhat shorter t_1/2 at lower doses, although its pharmacokinetics are highly variable from patient to patient due to variable rates of hepatic metabolism. Hydroxylation by CYPs generates an active phosphoramide mustard.

Toxicity. Ifosfamide has virtually the same toxicity profile as cyclophosphamide, although it causes greater platelet suppression, neurotoxicity, nephrotoxicity, and in the absence of mesna, urothelial damage.

Pharmacological and Cytotoxic Actions. The general pharmacological and cytotoxic actions of melphalan are similar to those of other bifunctional alkylators. The drug is not a vesicant.

Absorption, Fate, and Excretion. Oral melphalan is absorbed in an inconsistent manner and, for most indications, is given as an intravenous infusion. The drug has a t_1/2 in plasma of ~45-90 minutes, and 10-15% of an administered dose is excreted unchanged in the urine. Patients with decreased renal function may develop unexpectedly severe myelosuppression.

Therapeutic Uses. Melphalan (alkeran) for multiple myeloma is given in doses of 4-10 mg orally for 4-7 days every 28 days, with dexamethasone or thalidomide. Treatment is repeated at 4-week intervals based on response and tolerance. Dosage adjustments should be based on blood cell counts. Melphalan also may be used in myeloablative regimens followed by bone marrow or peripheral blood stem cell reconstitution. For this use, the dose is 180-200 mg/m².

Clinical Toxicity. The clinical toxicity of melphalan is mostly hematological and is similar to that of other alkylating agents. Nausea and vomiting are less frequent. The drug causes less alopecia and, rarely, renal or hepatic dysfunction.

Pharmacological and Cytotoxic Actions. The cytotoxic effects of chlorambucil on the bone marrow, lymphoid organs, and epithelial tissues are similar to those observed with other nitrogen mustards. As an orally administered agent, chlorambucil is well tolerated in small daily doses and provides flexible titration of blood counts. Nausea and vomiting may result from single oral doses of ≥20 mg.

Absorption, Fate, and Excretion. Oral absorption of chlorambucil is adequate and reliable. The drug has a t_1/2 in plasma of ~1.5 hours, and is hydrolyzed to inactive products.

Therapeutic Uses. In treating CLL, the standard initial daily dose of chlorambucil (leukeran) is 0.1-0.2 mg/kg, given once daily and continued for 3-6 weeks. With a fall in the peripheral total leukocyte count or clinical improvement, the dosage is titrated to maintain neutrophils and platelets at acceptable levels. Maintenance therapy (usually 2 mg daily) often is required to maintain clinical response.

Clinical Toxicity. In CLL, chlorambucil treatment may continue for months or years, achieving its effects gradually and often without significant toxicity to a compromised bone marrow.

Although it is possible to induce marked hypoplasia of the bone marrow with excessive doses, its myelosuppressive effects are moderate, gradual, and rapidly reversible. GI discomfort, azoospermia, amenorrhea, pulmonary fibrosis, seizures, dermatitis, and hepatotoxicity rarely may be encountered. A marked increase in the incidence of acute myelocytic leukemia (AML) and other tumors was noted in the treatment of polycythemia vera and in patients with breast cancer receiving chlorambucil as adjuvant chemotherapy (Lerner, 1978).

Bendamustine is given as a 30-minute intravenous infusion in dosages of 100 mg/m²/day on days 1 and 2 of a 28-day cycle. Lower doses may be indicated in heavily pretreated patients. It is rapidly degraded through sulfhydryl interaction and adduct formation with macromolecules, and <5% of the parent drug is excreted in the urine intact. N-demethylation and oxidation produces metabolites that have antitumor activity, but less than that of the parent molecule. The parent drug has a t_1/2 in plasma of ~30 minutes (Teichert et al., 2007).

The clinical toxicity pattern of bendamustine is typical of classical alkylators, with a rapidly reversible myelosuppression and mucositis, both generally tolerable in the 28-day regimen.

The usual dosage of altretamine as a single agent in ovarian cancer is 260 mg/m²/day in four divided doses, for 14 or 21 consecutive days out of a 28-day cycle, for up to 12 cycles.

Absorption, Fate, and Excretion. Following oral administration, altretamine is well absorbed from the GI tract and undergoes rapid demethylation in the liver. The principal metabolites are pentamethylmelamine and tetramethyl melamine, and the elimination t_1/2 is 4-10 hours.

Clinical Toxicities. The main toxicities of altretamine are myelosuppression and neurotoxicity. Altretamine causes both peripheral and central neurotoxicity (ataxia, depression, confusion, drowsiness, hallucinations, dizziness, and vertigo). Neurological symptoms abate upon discontinuation of therapy. Peripheral blood counts and a neurological examination should be performed prior to the initiation of each course of therapy. Therapy should be interrupted for at least 14 days and subsequently restarted at a lower dosage of 200 mg/m²/day if the white cell count falls to <2000 cells/mm³ or the platelet count falls to <75,000 cells/mm³ or if neurotoxic or intolerable GI symptoms occur. If neurological symptoms fail to stabilize on the reduced-dose schedule, altretamine should be discontinued. Nausea and vomiting also are common side effects and may be dose limiting. Renal dysfunction may necessitate discontinuing the drug. Other rare adverse effects include rashes, alopecia, and hepatic toxicity. Severe, life-threatening orthostatic hypotension may develop in patients who receive monoamine oxidase (MAO) inhibitors, amitriptyline, imipramine, or phenelzine concurrently with altretamine.

Absorption, Fate, and Excretion. TEPA becomes the predominant form of the drug present in plasma within hours of thiotepa administration. The parent compound has a plasma t_1/2 of 1.2-2 hours, as compared to a longer t_1/2 of 3-24 hours for TEPA. Thiotepa pharmacokinetics essentially are the same in children as in adults at conventional doses (≤80 mg/m²), and drug and metabolite t_1/2 are unchanged in children receiving high-dose therapy of 300 mg/m²/day for 3 days. Less than 10% of the administered drug appears in urine as the parent drug or the primary metabolite. Multiple secondary metabolites and chemical degradation products account for the remainder of the administered dose.

Clinical Toxicities. The toxicities of thiotepa essentially are the same as those of the other alkylating agents, namely myelosuppression, and to a lesser extent mucositis. Myelosuppression tends to develop somewhat later than with cyclophosphamide, with leukopenic nadirs at 2 weeks and platelet nadirs at 3 weeks. In high-dose regimens, thiotepa may cause neurotoxic symptoms, including coma and seizures.

Carmustine (BCNU) and lomustine (CCNU) are highly lipophilic and thus readily cross the blood-brain barrier, an important property in the treatment of brain tumors. Unfortunately, with the exception of streptozocin, nitrosoureas cause profound and delayed myelosuppression with recovery 4-6 weeks after a single dose. Long-term treatment with the nitrosoureas, especially semustine (methyl-CCNU), has resulted in renal failure. As with other alkylating agents, the nitrosoureas are highly carcinogenic and mutagenic. They generate both alkylating and carbamylating moieties as illustrated in Figure 61–4.

Absorption, Fate, and Excretion. Carmustine is unstable in aqueous solution and in body fluids. After intravenous infusion, it disappears from the plasma with a highly variable t_1/2 of ≥15-90 minutes. Approximately 30-80% of the drug appears in the urine within 24 hours as degradation products. The alkylating metabolites enter rapidly into the cerebrospinal fluid (CSF), and their concentrations in the CSF reach 15-30% of the concurrent plasma values.

Therapeutic Uses. When used alone, carmustine (bicnu) is administered intravenously at doses of 150-200 mg/m², given by infusion over 1-2 hours and repeated every 6 weeks.

Because of its ability to cross the blood-brain barrier, carmustine has been used in the treatment of malignant gliomas but has been increasingly replaced by temozolomide. An implantable carmustine wafer (gliadel) is available for use as an adjunct to surgery and radiation in newly diagnosed high-grade malignant glioma patients and as an adjunct to surgery for recurrent glioblastoma multiforme.

Absorption, Fate, and Excretion. Streptozocin is rapidly degraded following intravenous administration. The t_1/2 of the drug is ~15 minutes. Only 10-20% of a dose is recovered intact in the urine.

Therapeutic Uses. Streptozocin (zanosar) is used exclusively in the treatment of human pancreatic islet cell carcinoma and malignant carcinoid tumors. It is administered intravenously, 500 mg/m² once daily for 5 days; this course is repeated every 6 weeks. Alternatively, 1000 mg/m² can be given weekly for 2 weeks, and the weekly dose then can be increased to a maximum of 1500 mg/m², depending on tolerance and response.

Clinical Toxicity. Nausea is frequent. Mild, reversible renal or hepatic toxicity occurs in approximately two-thirds of cases; in <10% of patients, renal toxicity may be cumulative with each dose and may lead to irreversible renal failure. Proteinuria is an early sign of tubular damage and impending renal failure. Streptozocin should not be given with other nephrotoxic drugs. Hematological toxicity—anemia, leukopenia, or thrombocytopenia—occurs in 20% of patients.

Absorption, Fate, and Excretion. Dacarbazine is administered intravenously. After an initial rapid phase of disappearance (t_1/2 of ~20 minutes), dacarbazine is cleared from plasma with a terminal t_1/2 of ~5 hours. The t_1/2 is prolonged in the presence of hepatic or renal disease. Almost 50% of the compound is excreted intact in the urine by tubular secretion. Elevated urinary concentrations of 5-aminoimidazole-4-carboxamide reflect from the catabolism of dacarbazine, rather than inhibition of de novo purine biosynthesis.

Therapeutic Uses. Dacarbazine (dtic-dome) for malignant melanoma is given intravenously in dosages of 2-4.5 mg/kg/day for a 10-day period, repeated every 28 days. Alternatively, 250 mg/m² can be given daily for 5 days and repeated every 3 weeks. Extravasation of the drug may cause tissue damage and severe pain. In combination with other drugs for Hodgkin's disease, dacarbazine is given in dosages of 150 mg/m²/day for 5 days, repeated every 4 weeks, or a single dose of 375 mg/m² is given and repeated every 15 days.

At present, the primary clinical indication for dacarbazine is in the combination chemotherapy of Hodgkin's disease. It is modestly effective against malignant melanoma and adult sarcomas.

Clinical Toxicity. DTIC induces nausea and vomiting in >90% of patients; vomiting usually develops 1-3 hours after treatment and may last up to 12 hours. Myelosuppression, with both leukopenia and thrombocytopenia, is mild and readily reversible within 1-2 weeks. A flulike syndrome consisting of chills, fever, malaise, and myalgias may occur during treatment with DTIC. Hepatotoxicity, alopecia, facial flushing, neurotoxicity, and dermatological reactions are less common adverse effects.

Absorption, Fate, and Excretion. Temozolomide is administered orally in dosages of ~200 mg/day; it has a bioavailability approaching 100%. Maximum drug concentration reaches 5 μg/mL, or ~10 μM in plasma, ~1 hour after administration of a dose of 200 mg and declines with an elimination t_1/2 of 1-2 hours. The primary active metabolite MTIC reaches a maximum plasma concentration of 150 ng/mL 90 minutes after a dose and declines with a t_1/2 of 2 hours. Little intact drug is recovered in the urine, the primary urinary metabolite being the inactive imidazole carboxamide (Baker et al., 1999).

Clinical Toxicity. Temozolomide is available for oral and intravenous administration. It is administered cyclically, and hematological monitoring is necessary to guide dosing adjustments. The toxicities of temozolomide mirror those of DTIC.

Cytotoxic Action. The antineoplastic activity of procarbazine results from its conversion by CYP-mediated hepatic oxidative metabolism to highly reactive alkylating species, which methylate DNA. The activation pathways are complex and not fully understood; free-radical intermediates may be cytotoxic. Activated procarbazine can produce chromosomal damage, including chromatid breaks and translocations, consistent with its mutagenic and carcinogenic actions. Resistance to procarbazine develops rapidly when it is used as a single agent. One mechanism results from the increased expression of MGMT, which repairs methylation of guanine.

Absorption, Fate, and Excretion. The pharmacokinetic behavior of procarbazine has not yet been thoroughly defined. The drug is extensively metabolized by CYP isoenzymes to azo, methylazoxy, and benzylazoxy intermediates, which are found in the plasma and yield the alkylating metabolites in tumor cells. In brain cancer patients, it is surprising that the concurrent use of anti-seizure drugs that induce hepatic CYPs does not significantly alter the pharmacokinetics of the parent drug (He et al., 2004).

Therapeutic Uses. The recommended dosage of procarbazine (matulane) for adults is 100 mg/m²/day for 10-14 days in combination regimens such as MOPP (nitrogen mustard, oncovin, procarbazine, and prednisone) for Hodgkin's disease. The drug rarely is used in current practice, the MOPP combination having been replaced by alternative drugs.

Clinical Toxicity. The most common toxic effects include leukopenia and thrombocytopenia, which begin during the second week of therapy and reverse within 2 weeks off treatment. GI symptoms such as mild nausea and vomiting occur in most patients; diarrhea and rash are noted in 5-10% of cases. Behavioral disturbances also have been reported. Because procarbazine augments sedative effects, the concomitant use of CNS depressants should be avoided. The drug is a weak MAO inhibitor; it blocks the metabolism of catecholamines, sympathomimetics, and dietary tyramine and may provoke hypertension in patients concurrently exposed to these. Procarbazine has disulfiram-like actions, and therefore the ingestion of alcohol should be avoided. The drug is highly carcinogenic, mutagenic, and teratogenic and is associated with a 5-10% risk of acute leukemia in patients treated with MOPP. The highest risk is for patients who also receive radiation therapy. In addition, procarbazine is a potent immunosuppressive agent. It causes infertility, particularly in males.

Chemistry. Cisplatin and carboplatin are divalent, inorganic, water-soluble, platinum-containing complexes. Oxaliplatin, a tetravalent complex, does not display cross-resistance to the divalent compounds in some experimental tumors. The coordination of di- or tetravalent platinum with various organic adducts reduces its renal toxicity and stabilizes the metal ion, as compared to the inorganic divalent platinum ion.

Aquation of cisplatin is favored at the low concentrations of chloride inside the cell and in the urine. High concentrations of chloride stabilize the drug, explaining the effectiveness of chloride diuresis in preventing nephrotoxicity (see "Clinical Toxicities"). The activated platinum complexes can react with electron-rich molecules, such as sulfhydryls, and with various sites on DNA, forming both intrastrand and interstrand cross-links. The N-7 of guanine is a particularly reactive site, leading to platinum cross-links between adjacent guanines (GG intrastrand cross-links) on the same DNA strand; guanine–adenine cross-links also form and may contribute to cytotoxicity. Interstrand cross-links form less frequently. DNA-platinum adducts inhibit replication and transcription, lead to single- and double-stranded breaks and miscoding, and if recognized by p53 and other checkpoint proteins, cause induction of apoptosis. Although no quantitative relationship between platinum-DNA adduct formation and efficacy has been documented, the ability of patients to form and sustain platinum adducts appears to be an important predictor of clinical response (Reed et al., 1988). The analogs differ in the conformation of their adducts and the effects of adduct on DNA structure and function. Oxaliplatin and carboplatin are slower to form adducts. The oxaliplatin adducts are bulkier and less readily repaired, create a different pattern of distortion of the DNA helix, and differ from cisplatin adducts in the pattern of hydrogen bonding to adjacent segments of DNA (Sharma et al., 2007).

Unlike the other platinum analogs, oxaliplatin exhibits a cytotoxicity that does not depend on an active MMR system, which may explain its greater activity in colorectal cancer. It also seems less dependent on the presence of high mobility group (HMG) proteins that are required by the other platinum derivatives. Testicular cancers have a high concentration of HMG proteins and are quite sensitive to cisplatin. Basal-type breast cancers, such as those with BRCA1 and BRCA2 mutations, lack Her 2 amplification and hormone-receptor expression and appear to be uniquely susceptible to cisplatin through their upregulation of apoptotic pathways governed by p63 and p73 (Deyoung and Ellisen, 2007). The specificity of cisplatin with regard to phase of the cell cycle differs among cell types, although the effects of cross-linking are most pronounced during the S phase.

The platinum analogs are mutagenic, teratogenic, and carcinogenic. Cisplatin- or carboplatin-based chemotherapy for ovarian cancer is associated with a 4-fold increased risk of developing secondary leukemia.

Resistance to Platinum Analogs. Resistance to the platinum analogs likely is multifactorial, and the compounds differ in their degree of cross-resistance. Carboplatin shares cross-resistance with cisplatin in most experimental tumors, while oxaliplatin does not. A number of factors influence sensitivity to platinum analogs in experimental cells, including intracellular drug accumulation, as determined by the uptake and efflux transporters; intracellular levels of glutathione and other sulfhydryls such as metallothionein that bind to and inactivate the drug (Meijer et al., 1990); and rates of repair of DNA adducts. Repair of platinum-DNA adducts requires participation of the NER pathway. Inhibition or loss of NER increases sensitivity to cisplatin in ovarian cancer patients, while overexpression of NER components is associated with poor response to cisplatin or oxaliplatin-based therapy in lung, colon, and gastric cancer (Paré et al., 2008). Higher levels of expression of the NER component, ERCC1, in tumor cells and peripheral white blood cells are associated with a lower response rate in patients with solid tumors (Dolan and Fitch, 2007).

Resistance to cisplatin, but not oxaliplatin, appears to be partly mediated through loss of function in the MMR proteins (hMLH1, hMLH2, or hMSH6), which recognize platinum-DNA adducts and initiate apoptosis.

In the absence of effective repair of DNA-platinum adducts, sensitive cells cannot replicate or transcribe affected portions of the DNA strand. However, it is clear that some DNA polymerases can bypass adducts, especially those created by cisplatin. Oxaliplatin adducts are less easily bypassed. It remains unproven whether these polymerases contribute to resistance. Cisplatin resistance related to loss of active uptake has been demonstrated in yeast; overexpression of copper efflux transporters, ATP7A and ATP7B, correlates with poor survival after cisplatin-based therapy for ovarian cancer (Kruh, 2003).

Absorption, Fate, and Excretion. After intravenous administration, cisplatin has an initial plasma elimination t_1/2 of 25-50 minutes; concentrations of total (bound and unbound) drug fall thereafter, with a t_1/2 of ≥24 hours. More than 90% of the platinum in the blood is covalently bound to plasma proteins. The unbound fraction, composed predominantly of parent drug, diminishes within minutes. High concentrations of cisplatin are found in the tissues of the kidney, liver, intestine, and testes but poorly penetrate into the CNS. Only a small portion of the drug is excreted by the kidney during the first 6 hours; by 24 hours, up to 25% is excreted, and by 5 days, up to 43% of the administered dose is recovered in the urine, mostly covalently bound to protein and peptides. Biliary or intestinal excretion of cisplatin is minimal.

Therapeutic Uses. Cisplatin (platinol, others) is given only by the intravenous route. The usual dosage is 20 mg/m²/day for 5 days, 20-30 mg weekly for 3-4 weeks, or 100 mg/m² given once every 4 weeks. To prevent renal toxicity, it is important to establish a chloride diuresis by the infusion of 1-2 L of normal saline prior to treatment. The appropriate amount of cisplatin then is diluted in a solution containing dextrose, saline, and mannitol and administered intravenously over 4-6 hours. Because aluminum reacts with and inactivates cisplatin, the drug should not come in contact with needles or other infusion equipment that contain aluminum during its preparation or administration.

Cisplatin, in combination with bleomycin, etoposide, ifosfamide, or vinblastine, cures 90% of patients with testicular cancer. Used with paclitaxel, cisplatin or carboplatin induces complete response in the majority of patients with carcinoma of the ovary. Cisplatin produces responses in cancers of the bladder, head and neck, cervix, and endometrium; all forms of carcinoma of the lung; anal and rectal carcinomas; and neoplasms of childhood. Interestingly, the drug also sensitizes cells to radiation therapy and enhances control of locally advanced lung, esophageal, and head and neck tumors when given with irradiation.

Clinical Toxicities. Cisplatin-induced nephrotoxicity has been largely abrogated by adequate pretreatment hydration and chloride diuresis. Amifostine (ethyol, others) is a thiophosphate cytoprotective agent that reduces renal toxicity associated with repeated administration of cisplatin, but is not commonly used. Ototoxicity caused by cisplatin is unaffected by diuresis and is manifested by tinnitus and high-frequency hearing loss. The ototoxicity can be unilateral or bilateral, tends to be more frequent and severe with repeated doses, and may be more pronounced in children. Marked nausea and vomiting occur in almost all patients and usually can be controlled with 5-HT₃ antagonists, NK1-receptor antagonists, and high-dose corticosteroids (see Chapter 46).

At higher doses, or after multiple cycles of treatment, cisplatin causes a progressive peripheral motor and sensory neuropathy, which may worsen after discontinuation of the drug and may be aggravated by subsequent or simultaneous treatment with taxanes or other neurotoxic drugs. Cisplatin causes mild to moderate myelosuppression, with transient leukopenia and thrombocytopenia. Anemia may become prominent after multiple cycles of treatment. Electrolyte disturbances, including hypomagnesemia, hypocalcemia, hypokalemia, and hypophosphatemia, are common. Hypocalcemia and hypomagnesemia secondary to tubular damage and renal electrolyte wasting may produce tetany if untreated. Routine measurement of Mg²⁺ concentrations in plasma is recommended. Hyperuricemia, hemolytic anemia, and cardiac abnormalities are rare side effects. Anaphylactic-like reactions, characterized by facial edema, bronchoconstriction, tachycardia, and hypotension, may occur within minutes after administration and should be treated by intravenous injection of epinephrine and with corticosteroids or antihistamines. Like other DNA adduct–forming drugs, cisplatin has been associated with the development of AML, usually ≥4 years after treatment.

The mechanisms of action and resistance and the spectrum of clinical activity of carboplatin (CBDCA, JM-8) are similar to cisplatin (see "Cisplatin"). However, the two drugs differ significantly in their chemical, pharmacokinetic, and toxicological properties.

Because carboplatin is much less reactive than cisplatin, the majority of drug in plasma remains in its parent form, unbound to proteins. Most drug is eliminated via renal excretion, with a t_1/2 in plasma of ~2 hours. A small fraction of platinum binds irreversibly to plasma proteins and disappears slowly, with a t_1/2 of ≥5 days.

Carboplatin is relatively well tolerated clinically, causing less nausea, neurotoxicity, ototoxicity, and nephrotoxicity than cisplatin. Instead, the dose-limiting toxicity is myelosuppression, primarily thrombocytopenia. It is more likely to cause a hypersensitivity reaction; in patients with a mild reaction, premedication, graded doses of drug, and more prolonged infusion lead to desensitization.

Carboplatin and cisplatin are equally effective in the treatment of suboptimally debulked ovarian cancer, non–small cell lung cancer, and extensive-stage small cell lung cancer; however, carboplatin may be less effective than cisplatin in germ cell, head and neck, and esophageal cancers (Go and Adjei, 1999). Carboplatin is an effective alternative for responsive tumors in patients unable to tolerate cisplatin because of impaired renal function, refractory nausea, significant hearing impairment, or neuropathy, but doses must be adjusted for renal function. In addition, it may be used in high-dose therapy with bone marrow or peripheral stem cell rescue. The dose of carboplatin should be adjusted in proportion to the reduction in creatinine clearance (CrCl) for patients with a CrCl <60 mL/min. The following formula is useful for calculation of dose:

where the target AUC (area under the plasma concentration–time curve) is ~5-7 min/mg/mL for acceptable toxicity in patients receiving single-agent carboplatin (GFR = glomerular filtration rate).

Carboplatin (paraplatin) is administered as an intravenous infusion over at least 15 minutes, using the above-mentioned formula for dose calculation, and is given once every 21-28 days.

History. Antifolate chemotherapy occupies a special place in the history of cancer treatment: This class of drugs produced the first striking, although temporary, remissions in leukemia (Farber et al., 1948) and the first cure of a solid tumor, choriocarcinoma (Berlin et al., 1963). Interest in folate antagonists further increased with the development of curative combination therapy for childhood acute lymphocytic leukemia; in this therapy, methotrexate played a critical role in both systemic treatment and intrathecal therapy. Introduction of high-dose regimens with "rescue" of host toxicity by the reduced folate, leucovorin (folinic acid, citrovorum factor, 5-formyl tetrahydrofolate, N⁵-formyl FH₄), further extended the effectiveness of this drug to both systemic and CNS lymphomas, osteogenic sarcoma, and leukemias. Most recently, pemetrexed, an analog that differs from methotrexate in its transport properties and sites of action, has proven useful in treating mesothelioma and lung cancer.

Recognition that methotrexate, an inhibitor of dihydrofolate reductase (DHFR), also directly inhibits the folate-dependent enzymes of de novo purine and thymidylate synthesis led to development of antifolate analogs that specifically target these other folate-dependent enzymes (Figure 61–5). New inhibitors have resulted from synthetic modifications of the basic folate molecule (replacement of the N5 and/or N8 nitrogens of the pteridine ring and the N10 nitrogen of the bridge between the pteridine and benzoate rings of folate, as well as various side-chain substitutions). These new agents have greater capacity for transport into tumor cells (pralatrexate) and exert their primary inhibitory effect on TS (raltitrexed, tomudex), early steps in purine biosynthesis (lometrexol), or both (the multitargeted antifolate, pemetrexed, alimta) (Vogelzang et al., 2003; O'Connor et al., 2007).

Aside from its antineoplastic activity, methotrexate also has been used with benefit in the therapy of psoriasis (see Chapter 65). Additionally, methotrexate inhibits cell-mediated immune reactions and is employed to suppress graft-versus-host disease in allogenic bone marrow and organ transplantation and for the treatment of dermatomyositis, rheumatoid arthritis, Wegener's granulomatosis, and Crohn's disease (see Chapters 35 and 47).

Because folic acid and many of its analogs are polar, they cross the blood-brain barrier poorly and require specific transport mechanisms to enter mammalian cells. Three inward folate transport systems are found on mammalian cells: 1) a folate receptor, which has high affinity for folic acid but much reduced ability to transport methotrexate and other analogs; 2) the reduced folate transporter, the major transit protein for methotrexate, raltitrexed, pemetrexed, and most analogs (Westerhof et al., 1995); and 3) a transporter that is active at low pH. The importance of transport in determining drug sensitivity is illustrated by the finding that the reduced folate transporter is highly expressed in the hyperdiploid subtype of acute lymphoblastic leukemia, due to the presence of multiple copies of chromosome 21, on which its gene resides; these cells have extreme sensitivity to methotrexate (Pui et al., 2004). Once in the cell, additional glutamyl residues are added to the molecule by the enzyme folylpolyglutamate synthetase. Intracellular methotrexate polyglutamates have been identified with up to six glutamyl residues. Because these higher polyglutamates are strongly charged and cross cellular membranes poorly, if at all, polyglutamation serves as a mechanism of entrapment and may account for the prolonged retention of methotrexate in chorionic epithelium (where it is a potent abortifacient); in tumors derived from this tissue, such as choriocarcinoma cells; and in normal tissues subject to cumulative drug toxicity, such as liver. Polyglutamylated folates and analogs have substantially greater affinity than the monoglutamate form for folate-dependent enzymes that are required for purine and thymidylate synthesis and have at least equal affinity for DHFR.

New folate antagonists that are better substrates for the reduced folate carrier have been identified. In efforts to bypass the obligatory membrane transport system and to facilitate penetration of the blood-brain barrier, lipid-soluble folate antagonists also have been synthesized. Trimetrexate (neutrexin), a lipid-soluble analog that lacks a terminal glutamate, was one of the first to be tested for clinical activity. The analog has modest antitumor activity, primarily in combination with leucovorin rescue. However, it is beneficial in the treatment of P. jiroveci (Pneumocystis carinii) pneumonia (Allegra et al., 1987a), where leucovorin provides differential rescue of the host but not the parasite.

The most important new folate analog, MTA or pemetrexed (alimta) (Figure 61–6), is a pyrrole–pyrimidine structure. It is avidly transported into cells via the reduced folate carrier. It readily is converted to polyglutamates that inhibit TS and glycine amide ribonucleotide transformylase, as well as DHFR. It has shown activity against ovarian cancer, mesothelioma, and non–small cell adenocarcinomas of the lung (Vogelzang et al., 2003).

Inhibitors of DHFR differ in their relative potency for blocking enzymes from different species. Agents have been identified that have little effect on the human enzyme but have strong activity against bacterial and parasitic infections (see discussions of trimethoprim, Chapter 52, and pyrimethamine, Chapter 49). By contrast, methotrexate effectively inhibits DHFR in all species investigated. Crystallographic studies have revealed the structural basis for the high affinity of methotrexate for DHFR (Blakley and Sorrentino, 1998) and the species specificity of the various DHFR inhibitors.

Pemetrexed and its polyglutamates have a somewhat different spectrum of biochemical actions. Like methotrexate, pemetrexed inhibits DHFR, but as a polyglutamate, it even more potently inhibits glycinamide ribonucleotide formyltransferase (GART) and thymidylate synthase (TS). Unlike methotrexate, it produces little change in the pool of reduced folates, indicating that the distal sites of inhibition (TS and GART) predominate. Its pattern of deoxynucleotide depletion, as studied in cell lines, also differs from methotrexate, as it causes a greater fall in thymidine triphosphate (TTP) than in other triphosphates (Chen et al., 1998). Like methotrexate, it induces p53 and cell-cycle arrest, but this effect does not seem to depend on downstream induction of p21. Pralatrexate is more effectively taken up and polyglutamated than methotrexate. Pralatrexate is approved for treatment of cutanous T-cell lymphoma (O'Connor et al, 2007).

DHFR levels in leukemic cells increase within 24 hours after treatment of patients with methotrexate; this likely reflects induction of new enzyme synthesis. The unbound DHFR protein may bind to its own message and inhibit translational efficiency of its own synthesis, while the DHFR-MTX complex is ineffective in blocking the DHFR translation. With longer periods of drug exposure, tumor cell populations that contain markedly increased levels of DHFR emerge. These cells contain multiple gene copies of DHFR either in mitotically unstable double-minute chromosomes (extrachromosomal elements formed by amplification of DHFR genes in response to methotrexate treatment) or in stably integrated, homogeneously staining chromosomal regions or amplicons. First identified as an explanation for resistance to methotrexate (Schimke et al., 1978), gene amplification of a target protein has since been implicated in the resistance to many antitumor agents, including 5-FU and pentostatin (2′-deoxycoformycin) and has been observed in patients with lung cancer (Curt et al., 1985) and leukemia. Impaired transport and polyglutamation have been implicated as mechanisms of resistance in childhood leukemia.

To overcome resistance, high doses of methotrexate may permit entry of the drug into transport-defective cells and may permit the intracellular accumulation of methotrexate in concentrations that inactivate high levels of DHFR.

The understanding of resistance to pemetrexed is incomplete. In various cell lines, resistance to this agent seems to arise from loss of influx transport, TS amplification, changes in purine biosynthetic pathways, or loss of polyglutamation.

Absorption, Fate, and Excretion. Methotrexate is readily absorbed from the GI tract at doses of <25 mg/m², but larger doses are absorbed incompletely and are routinely administered intravenously. Peak concentrations of 1-10 μM in the plasma are obtained after doses of 25-100 mg/m², and concentrations of 0.1-1 mM are achieved after high-dose infusions of 1.5-20 g/m². After intravenous administration, the drug disappears from plasma in a triphasic fashion (Sonneveld et al., 1986). The rapid distribution phase is followed by a second phase, which reflects renal clearance (t_1/2 of ~2-3 hours). A third phase has a t_1/2 of ~8-10 hours. This terminal phase of disappearance, if unduly prolonged by renal failure, may be responsible for major toxic effects of the drug on the marrow, GI epithelium, and skin. Distribution of methotrexate into body spaces, such as the pleural or peritoneal cavity, occurs slowly. However, if such spaces are expanded (e.g., by ascites or pleural effusion), they may act as a site of storage and slow release of the drug, resulting in prolonged elevation of plasma concentrations and more severe bone marrow toxicity.

Approximately 50% of methotrexate binds to plasma proteins and may be displaced from plasma albumin by a number of drugs, including sulfonamides, salicylates, tetracycline, chloramphenicol, and phenytoin; caution should be used if these drugs are given concomitantly. Up to 90% of a given dose is excreted unchanged in the urine within 48 hours, mostly within the first 8-12 hours. Metabolism of methotrexate in humans usually is minimal. After high doses, however, metabolites are readily detectable; these include 7-hydroxy-methotrexate, which is potentially nephrotoxic. Renal excretion of methotrexate occurs through a combination of glomerular filtration and active tubular secretion. Therefore, the concurrent use of drugs that reduce renal blood flow (e.g., nonsteroidal anti-inflammatory agents), that are nephrotoxic (e.g., cisplatin), or that are weak organic acids (e.g., aspirin, piperacillin) can delay drug excretion and lead to severe myelosuppression. Particular caution must be exercised in treating patients with renal insufficiency. In such patients, the dose should be adjusted in proportion to decreases in renal function, and high-dose regimens should be avoided.

Methotrexate is retained in the form of polyglutamates for long periods—for example, for weeks in the kidneys and for several months in the liver.

It is important to emphasize that concentrations of methotrexate in CSF are only 3% of those in the systemic circulation at steady state; hence, neoplastic cells in the CNS probably are not killed by standard dosage regimens. When high doses of methotrexate are given (>1.5 g/m²; see "Therapeutic Uses"), cytotoxic concentrations of methotrexate reach the CNS.

Pharmacogenetics may influence the response to antifolates and their toxicity. The C677T substitution in methylenetetrahydrofolate reductase reduces the activity of the enzyme that generates methylenetetrahydrofolate, the cofactor for TS, and thereby increases methotrexate toxicity (Pullarkat et al., 2001). The presence of this polymorphism in leukemic cells confers increased sensitivity to methotrexate and might also modulate the toxicity and therapeutic effect of pemetrexed, a predominant TS inhibitor. Likewise, polymorphisms in the promoter region of TS govern the translation efficiency of this message and, by governing the intracellular levels of TS, modulate the response and toxicity of both antifolates (Pui et al., 2004) and fluoropyrimidines.

Therapeutic Uses. Methotrexate (Amethopterin; rheumatrex, trexall, others) has been used in the treatment of severe, disabling psoriasis in doses of 2.5 mg orally for 5 days, followed by a rest period of at least 2 days, or 10-25 mg intravenously weekly. It also is used at low dosage to induce remission in refractory rheumatoid arthritis (Hoffmeister, 1983). Awareness of the pharmacology, toxic potential, and drug interactions associated with methotrexate is a prerequisite for its use in these non-neoplastic disorders.

Methotrexate is a critical drug in the management of acute lymphoblastic leukemia (ALL) in children. High-dose methotrexate is of great value in remission induction and consolidation and in the maintenance of remissions in this highly curable disease. A 6- to 24-hour infusion of relatively large doses of methotrexate may be employed every 2-4 weeks (≥1-7.5 g/m²) but only when leucovorin rescue follows within 24 hours of the methotrexate infusion. Such regimens produce cytotoxic concentrations of drug in the CSF and protect against leukemic meningitis. For maintenance therapy, it is administered weekly in doses of 20 mg/m² orally. Outcome of treatment in children correlates inversely with the rate of drug clearance. During methotrexate infusion, high steady-state levels are associated with a lower leukemia relapse rate (Pui et al., 2004). Methotrexate is of limited value in adults with AML, except for treatment and prevention of leukemic meningitis. The intrathecal administration of methotrexate has been employed for treatment or prophylaxis of meningeal leukemia or lymphoma and for treatment of meningeal carcinomatosis. This route of administration achieves high concentrations of methotrexate in the CSF and also is effective in patients whose systemic disease has become resistant to methotrexate. The recommended intrathecal dose in all patients >3 years of age is 12 mg (Bleyer, 1978). The dose is repeated every 4 days until malignant cells no longer are evident in the CSF. Leucovorin may be administered to counteract the potential toxicity of methotrexate that escapes into the systemic circulation, although this generally is not necessary. Because methotrexate administered into the lumbar space distributes poorly over the cerebral convexities, the drug may be given via an intraventricular Ommaya reservoir in the treatment of active intrathecal disease. Methotrexate is of established value in choriocarcinoma and related trophoblastic tumors of women; cure is achieved in ~75% of advanced cases treated sequentially with methotrexate and dactinomycin and in >90% when early diagnosis is made. In the treatment of choriocarcinoma, 1 mg/kg of methotrexate is administered intramuscularly every other day for four doses, alternating with leucovorin (0.1 mg/kg every other day). Courses are repeated at 3-week intervals, toxicity permitting, and urinary β-human chorionic gonadotropin titers are used as a guide for persistence of disease.

Beneficial effects also are observed in the combination therapy of Burkitt's and other non-Hodgkin's lymphomas. Methotrexate is a component of regimens for carcinomas of the breast, head and neck, ovary, and bladder. High-dose methotrexate with leucovorin rescue (HDM-L) is a standard agent for adjuvant therapy of osteosarcoma and produces a high complete response rate in CNS lymphomas. The administration of HDM-L has the potential for renal toxicity, probably related to the precipitation of the drug, a weak acid, in the acidic tubular fluid. Thus, vigorous hydration and alkalinization of urine pH are required prior to drug administration. HDM-L should be performed only by experienced clinicians who are familiar with hydration regimens and who have access to laboratories that monitor concentrations of methotrexate in plasma. If methotrexate values measured 48 hours after drug administration are 1 μM or higher, higher doses (100 mg/m²) of leucovorin must be given until the plasma concentration of methotrexate falls to <50 nM (Stoller et al., 1977). With appropriate hydration and urine alkalinization, and in patients with normal renal function, the incidence of nephrotoxicity following HDM-L is <2%. In patients who become oliguric, intermittent hemodialysis is ineffective in reducing methotrexate levels. Continuous-flow hemodialysis can eliminate methotrexate at ~50% of the clearance rate in patients with intact renal function (Wall et al., 1996). Alternatively, a methotrexate-cleaving enzyme, carboxypeptidase G2, can be obtained from the Cancer Therapy Evaluation Program at the National Cancer Institute. When administered intravenously, it rapidly clears the drug (DeAngelis et al., 1996). Methotrexate concentrations in plasma fall by ≥99% within 5-15 minutes following enzyme administration, with insignificant rebound. Systemically administered carboxypeptidase G2 has little effect on methotrexate levels in the CSF.

Additional toxicities of methotrexate include alopecia, dermatitis, an allergic interstitial pneumonitis, nephrotoxicity (after high-dose therapy), defective oogenesis or spermatogenesis, abortion, and teratogenesis. Low-dose methotrexate may lead to cirrhosis after long-term continuous treatment, as in patients with psoriasis. Intrathecal administration of methotrexate often causes meningismus and an inflammatory response in the CSF. Seizures, coma, and death may occur rarely. Leucovorin does not reverse neurotoxicity.

A toxicity of particular significance in chronic administration to patients with psoriasis or rheumatoid arthritis is hepatic fibrosis and cirrhosis. Increased hepatic portal fibrosis is detected with higher frequency than in control patients after ≥6 months of continuous oral methotrexate treatment of psoriasis. Its presence mandates discontinuation of methotrexate. High-dose administration regularly causes acute elevation of hepatic transaminases, but these changes rapidly reverse and are not associated with permanent liver damage.

Folic acid antagonists are toxic to developing embryos. Methotrexate is highly effective when used with the prostaglandin analog misoprostol in inducing abortion in first-trimester pregnancy (Hausknecht, 1995).

Pemetrexed toxicity mirrors that of methotrexate, with the additional feature of a prominent erythematous and pruritic rash in 40% of patients. Dexamethasone, 4 mg twice daily on days –1, 0, and +1, markedly diminishes this toxicity. Unpredictably severe myelosuppression with pemetrexed, seen especially in patients with pre-existing homocystinemia and possibly reflecting folate deficiency, largely is eliminated by concurrent administration of low dosages of folic acid, 350-1000 mg/day, beginning 1-2 weeks prior to pemetrexed and continuing while the drug is administered. Patients should receive intramuscular vitamin B₁₂ (1 mg) with the first dose of pemetrexed to correct possible B₁₂ deficiency. These small doses of folate and B₁₂ do not compromise the therapeutic effect.

To understand the role of these drugs, it is useful to review the nomenclature of the DNA bases and their metabolic intermediates. Four bases, shown in Figure 61–7, form DNA; these are two pyrimidines, thymine and cytosine, and two purines, guanine and adenine. Some bases (guanine) are found in mammalian cells as free bases, while others (the pyrimidines) are present in their active form only as nucleosides (a base attached to a ribose or deoxyribose). These precursor forms then are converted to a nucleoside triphosphate (base, sugar, and 5′-phosphate, also known as a nucleotide). Mammalian cells lack the ability to utilize cytosine, thymine, and adenine as bases, and so these bases are found in the bloodstream as nucleosides and in cells as nucleosides or nucleotides. The various purine and pyrimidine triphosphates form the intracellular pool of precursors for both RNA (with ribose sugars) and DNA (with deoxyribose sugars). The composition of RNA also differs from DNA in that RNA incorporates uracil instead of thymine as one of its bases.

Strategies for inhibiting DNA synthesis are based on the ability to create analogs of these precursors that readily enter tumor cells and become activated by intracellular enzyme. As an example, the first successful pyrimidine analog, 5-FU, is converted to a deoxynucleotide, fluorodeoxyuridine monophosphate (FdUMP), which in turn blocks the enzyme; TS, required for the physiological conversion of dUMP to dTMP, a component of DNA. Other analogs incorporate into DNA itself and thereby block its function.

Cells can make the purine and pyrimidine bases de novo and convert them to their active triphosphates (dNTPs). The dNTPs then act as substrates for DNA polymerase and become linked in 3′-5′ phosphate ester bonds to form DNA strands; their base sequence provides the code for subsequent RNA and protein sequences.

As an alternative to synthesis of new precursor molecules, cells can salvage either free bases or their deoxynucleosides (Figure 61–7) from the bloodstream, presumably the products of degradation of DNA. Certain bases, such as uracil, guanine, and their analogs, can be taken up by cells and converted intracellularly to (deoxy) nucleotides by the addition of deoxyribose and phosphate groups. Antitumor analogs of these bases (5-FU, 6-thioguanine) can be formulated as simple substituted bases. Other bases, including cytosine, thymine, and adenine, and their analogs can only be utilized as deoxynucleosides, which are readily transported into cells and activated to deoxynucleotides by intracellular kinases. Thus, cytarabine (cytosine arabinoside; Ara-C), gemcitabine, 5-azacytidine, and adenosine analogs (cladribine), are nucleosides readily taken up by cells, converted to nucleotides, and incorporated into DNA (Figure 61–8).

Fludarabine phosphate, a nucleotide, is dephosphorylated rapidly in plasma, releasing the nucleoside that is readily taken up by cells. Analogs may differ from the physiological bases in a variety of ways: by altering in the purine or pyrimidine ring; by altering the sugar attached to the base, as in the arabinoside, Ara-C; or by altering both the base and sugar, as in fludarabine phosphate (Figure 61–7). These alterations produce inhibitory effects on vital enzymatic pathways and prevent DNA synthesis.

Mechanism of Action. 5-FU requires enzymatic conversion to the nucleotide status (ribosylation and phosphorylation) in order to exert its cytotoxic activity (Figure 61–9). Several routes are available for the formation of floxuridine monophosphate (FUMP). 5-FU may be converted to fluorouridine by uridine phosphorylase and then to FUMP by uridine kinase, or it may react directly with 5-phosphoribosyl-1-pyrophosphate (PRPP), as catalyzed by orotate phosphoribosyl transferase, to form FUMP. Further metabolic pathways are available to FUMP. As the triphosphate FUTP, it is incorporated into RNA. In an alternative reaction sequence crucial for antineoplastic activity, it is reduced to FUDP by ribonucleotide reductase (RNR) to the deoxynucleotide level and forming of FdUMP. 5-FU also may be converted by thymidine phosphorylase to the deoxyriboside fluorodeoxyuridine (FUdR) and then by thymidine kinase to FdUMP, a potent inhibitor of thymidylate synthesis. This complex metabolic pathway for the generation of FdUMP may be bypassed through administration of floxuridine (fluorodeoxyuridine; FUdR), which is converted directly to FdUMP by thymidine kinase. FUdR rarely is used in clinical practice.

FdUMP inhibits TS and blocks the synthesis of TTP, a necessary constituent of DNA (Figure 61–10). The folate cofactor, 5,10-methylenetetrahydrofolate, and FdUMP form a covalently bound ternary complex with TS. This inhibited complex resembles the transition state formed during the enzymatic conversion of dUMP to thymidylate. The physiological complex of TS-folate-dUMP progresses to the synthesis of thymidylate by transfer of the methylene group and two hydrogen atoms from folate to dUMP, but this reaction is blocked in the inhibited complex of TS-FdUMP-folate by the stability of the fluorine carbon bond on FdUMP; sustained inhibition of the enzyme results.

5-FU is incorporated into both RNA and DNA. In 5-FU-treated cells, both fluorodeoxyuridine triphosphate (FdUTP) and deoxyuridine triphosphate (dUTP) (the substrate that accumulates behind the blocked TS reaction) incorporate into DNA in place of the depleted physiological TTP. The significance of the incorporation of FdUTP and dUTP into DNA is unclear. Presumably, the incorporation of deoxyuridylate and/or fluorodeoxyuridylate into DNA would call into action the excision-repair process. This process may result in DNA strand breakage because DNA repair requires TTP, but this substrate is lacking as a result of TS inhibition. 5-FU incorporation into RNA also causes toxicity as the result of major effects on both the processing and functions of RNA.

Resistance to the cytotoxic effects of 5-FU or FUdR has been ascribed to loss or decreased activity of the enzymes necessary for activation of 5-FU, amplification of TS (Washtein, 1982), mutation of TS to a form that is not inhibited by FdUMP (Barbour et al., 1990), and high levels of the degradative enzymes dihydrouracil dehydrogenase and thymidine phosphorylase (Van Triest et al., 2000). TS levels are finely controlled by an autoregulatory feedback mechanism wherein the unbound enzyme interacts with and inhibits the translational efficiency of its own mRNA, which provides for the rapid TS modulation needed for cellular division. When TS is bound to FdUMP, inhibition of translation is relieved, and levels of free TS rise, restoring thymidylate synthesis. Thus, TS autoregulation may be an important mechanism by which malignant cells become insensitive to the effects of 5-FU (Chu et al., 1991).

Some malignant cells appear to have insufficient concentrations of 5,10-methylenetetrahydrofolate, and thus cannot form maximal levels of the inhibited ternary complex with TS. Addition of exogenous folate in the form of N⁵-formyl FH₄ (leucovorin) increases formation of the complex and enhances responses to 5-FU in clinical trials (Grogan et al., 1993).

In addition to leucovorin, a number of other agents have been combined with 5-FU in attempts to enhance the cytotoxic activity through biochemical modulation. Methotrexate, by inhibiting purine synthesis and increasing cellular pools of PRPP, enhances the activation of 5-FU and increases antitumor activity of 5-FU when given prior to but not following 5-FU. In clinical trials, the combination of cisplatin and 5-FU has yielded impressive responses in tumors of the upper aerodigestive tract, but the molecular basis of their interaction is not well understood. Oxaliplatin, which downregulates TS expression, is commonly used with 5-FU and leucovorin for treating metastatic colorectal cancer. Perhaps the most important interaction is the enhancement of irradiation by fluoropyrimidines, the basis for which is unclear. 5-FU with simultaneous irradiation cures anal cancer and enhances local tumor control in head and neck, cervical, rectal, gastroesophageal, and pancreatic cancer.

Absorption, Fate, and Excretion. 5-FU is administered parenterally, because absorption after oral ingestion of the drug is unpredictable and incomplete. Metabolic degradation occurs in many tissues, particularly the liver. 5-FU is inactivated by reduction of the pyrimidine ring in a reaction carried out by dihydropyrimidine dehydrogenase (DPD), which is found in liver, intestinal mucosa, tumor cells, and other tissues. Inherited deficiency of this enzyme leads to greatly increased sensitivity to the drug (Milano et al., 1999). The rare individual who totally lacks this enzyme may experience profound drug toxicity following conventional doses of the drug. DPD deficiency can be detected either by enzymatic or molecular assays using peripheral white blood cells or by determining the plasma ratio of 5-FU to its metabolite, 5-fluoro-5,6-dihydrouracil.

Intravenous administration of 5-FU produces peak plasma concentrations of 0.1-0.5 mM; plasma clearance is rapid (with a t_1/2 of 10-20 minutes). Only 5-10% of a single intravenous dose of 5-FU is excreted intact in the urine. Although the liver contains high concentrations of DPD, the dose does not have to be modified in patients with hepatic dysfunction, presumably because of degradation of the drug at extrahepatic sites. Given by continuous intravenous infusion for 24-120 hours, 5-FU achieves steady-state plasma concentrations in the range of 0.5-0.8 μM. 5-FU enters the CSF in minimal amounts.

For average-risk patients in good nutritional status with adequate hematopoietic function, the weekly dosage regimen employs 500-600 mg/m² with leucovorin once each week for 6 of 8 weeks. Other regimens use daily doses of 500 mg/m² for 5 days, repeated in monthly cycles. When used with leucovorin, doses of daily 5-FU for 5 days must be reduced to 375-425 mg/m² because of mucositis and diarrhea. 5-FU increasingly is used as a biweekly infusion, in which a loading dose is followed by a 48-hour continuous infusion, a schedule that has less overall toxicity as well as superior response rates and progression-free survival for patients with metastatic colon cancer (De Gramont et al., 1998).

Intrahepatic arterial infusion for 14-21 days causes minimal systemic toxicity; however, there is a significant risk of biliary sclerosis if this route is used for multiple cycles of therapy. Treatment should be discontinued at the earliest manifestation of toxicity (usually stomatitis or diarrhea) because the maximal effects of bone marrow suppression and gut toxicity will not be evident until days 7-14.

The recommended dosage is 2500 mg/m²/day, given in two divided doses with food, for 2 weeks, followed by a rest period of 1 week. Capecitabine is well absorbed orally. It is rapidly de-esterified and deaminated, yielding high plasma concentrations of an inactive prodrug 5′-deoxyfluorodeoxyuridine (5′-dFdU), which disappears with a t_1/2 of ~1 hour. The conversion of 5′-dFdU to 5-FU by thymidine phosphorylase occurs in liver tissues, peripheral tissues, and tumors. 5-FU levels are <10% of those of 5′-dFdU, reaching a maximum of 0.3 mg/L or 1 μM at 2 hours. Liver dysfunction delays the conversion of the parent compound to 5′-dFdU and 5-FU, but there is no consistent effect on toxicity (Twelves et al., 1999).

The combination of 5-FU and oxaliplatin or irinotecan has become the standard first-line treatment for patients with metastatic colorectal cancer, although equivalent results are reported for capecitabine and oxaliplatin. 5-FU in combination regimens has improved survival in the adjuvant treatment for breast cancer and, with oxaliplatin and leucovorin, for colorectal cancer. 5-FU also is a potent radiation sensitizer. Beneficial effects also have been reported when combined with irradiation as primary treatment for locally advanced cancers of the esophagus, stomach, pancreas, cervix, anus, and head and neck. 5-FU produces very favorable results for the topical treatment of premalignant keratoses of the skin and multiple superficial basal cell carcinomas.

Clinical Toxicities. The clinical manifestations of toxicity caused by 5-FU and floxuridine are similar. The earliest untoward symptoms during a course of therapy are anorexia and nausea, followed by stomatitis and diarrhea, which constitute reliable warning signs that a sufficient dose has been administered. Mucosal ulcerations occur throughout the GI tract and may lead to fulminant diarrhea, shock, and death, particularly in patients who are DPD deficient. The major toxic effects of bolus-dose regimens result from the myelosuppressive action of 5-FU. The nadir of leukopenia usually is between days 9 and 14 after the first injection of drug. Thrombocytopenia and anemia also may occur. Loss of hair, occasionally progressing to total alopecia, nail changes, dermatitis, and increased pigmentation and atrophy of the skin may be encountered. Hand-foot syndrome, a particularly prominent adverse effect of capecitabine, consists of erythema, desquamation, pain, and sensitivity to touch of the palms and soles. Acute chest pain with evidence of ischemia in the electrocardiogram may result from coronary artery vasospasms during or shortly after 5-FU infusion. In general, myelosuppression, mucositis, and diarrhea occur less often with infusional regimens than with bolus regimens, while hand-foot syndrome occurs more often with infusional regimens than with bolus regimens. The significant risk of toxicity with fluoropyrimidines emphasizes the need for very skillful supervision by physicians familiar with the action and possible hazards.

Capecitabine causes much the same spectrum of toxicities as 5-FU (diarrhea, myelosuppression), but the hand-foot syndrome occurs more frequently and may require dose reduction or cessation of therapy.

Ara-C penetrates cells by a carrier-mediated process shared by physiological nucleosides. Several candidate carriers bring nucleosides into cells, but the hENT1 transporter appears to be the primary mediator of Ara-C influx. In infants and adults with ALL and t(4;11) mixed-lineage leukemia (MLL) translocation, high-dose Ara-C is particularly effective; in these patients, the nucleoside transporter, hENT1, is highly expressed (Pui et al., 2004), and its expression correlates with sensitivity to Ara-C. A single-point mutation in hENT1 conferred Ara-C resistance in a leukemic cell line (Cai et al., 2008). At extracellular drug concentrations >10 μM (levels achievable with high-dose Ara-C), the nucleoside transporter no longer limits drug accumulation, and intracellular metabolism to a triphosphate becomes rate limiting.

Ara-C must be "activated" by conversion to the 5′-monophosphate nucleotide (Ara-CMP), a reaction catalyzed by CdK. Polymorphisms of CdK may influence the rate of activation in individual patients (Lamba et al., 2007). Ara-CMP then reacts with appropriate deoxynucleotide kinases to form diphosphate and triphosphates (Ara-CDP and Ara-CTP). Ara-CTP competes with the physiological substrate deoxycytidine 5′-triphosphate (dCTP) for incorporation into DNA by DNA polymerases. The incorporated Ara-CMP residue is a potent inhibitor of DNA polymerase, both in replication and repair synthesis, and blocks the further elongation of the nascent DNA molecule. The block in elongation activates checkpoint kinases (ATR and chk-1), which initiate attempts to remove the offending nucleotide. If DNA breaks are not repaired, apoptosis ensues. Ara-C cytotoxicity correlates with the total Ara-C incorporated into DNA. Thus, incorporation of about five molecules of Ara-C per 10⁴ bases of DNA decreases cellular clonogenicity by ~50% (Kufe et al., 1984).

Ara-C exposure activates a complex system of secondary intracellular signals that determine whether a cell survives or undergoes apoptosis. It activates the transcription factor AP-1 and stimulates the formation of ceramide, a potent inducer of apoptosis. It causes an increase in the cell damage response factor NF-κB in leukemic cells and activates checkpoint kinases. Additionally, the level of expression of the anti-apoptotic BCL-2 and BCL-X_L proteins correlates inversely with relative sensitivity to Ara-C (Ibrado et al., 1996). Thus, the lethality depends on the complex interplay of multiple factors, including transport, intracellular metabolism, and cellular response to DNA damage.

Low concentrations of Ara-C induce terminal differentiation of leukemic cells in tissue culture, an effect that is accompanied by decreased c-myc oncogene expression; molecular analysis of bone marrow specimens from some leukemic patients in remission after Ara-C therapy has revealed persistence of leukemic markers, suggesting that differentiation may have occurred.

Continuous inhibition of DNA synthesis for a duration equivalent to at least one cell cycle or 24 hours is necessary to expose most tumor cells during the S, or DNA-synthetic, phase of the cycle. The optimal interval between bolus doses of Ara-C is ~8-12 hours, a schedule that maintains intracellular concentrations of Ara-CTP at inhibitory levels during a multi-day cycle of treatment. Typical schedules for administration of Ara-C employ bolus doses every 12 hours or continuous drug infusion for 5-7 days.

Particular subtypes of AML derive benefit from high-dose Ara-C treatment; these include t(8;21), inv(16), t(9;16), and del(16), all of which involve core-binding factors that regulate hematopoiesis (Widemann et al., 1997). Approximately 20% of AML patients have leukemic cells with a k-RAS mutation, and these patients seem to derive greater benefit from high dose Ara-C regimens than do patients with wild type k-RAS (Neubauer et al., 2008).

Less than 10% of the injected dose is excreted unchanged in the urine within 12-24 hours, while most appears as the inactive deaminated product, Ara-U. Higher concentrations of Ara-C are found in CSF after continuous infusion than after rapid intravenous injection, but are far lower (≤10%) than concentrations in plasma. After intrathecal administration of the drug at a dose of 50 mg/m², deamination proceeds slowly, with a t_1/2 of 3-4 hours, and peak concentrations of 1-2 μM are achieved. CSF concentrations remain above the threshold for cytotoxicity (0.4 μM) for ≥24 hours. A depot liposomal formulation of Ara-C (depocyt) provides sustained release into the CSF. After a standard 50-mg dose, liposomal Ara-C remains above cytotoxic levels for an average of 12 days, thus avoiding the need for frequent lumbar punctures (Cole et al., 2003).

Ara-C is indicated for induction and maintenance of remission in AML and is useful in the treatment of other leukemias, such as ALL, CML in the blast phase, acute promyelocytic leukemia, and high-grade lymphomas. Because drug concentration in plasma rapidly falls below the level needed to saturate transport and intercellular activation, clinicians have employed high-dose regimens (2-3 g/m² every 12 hours for 6-8 doses) to achieve 20- to 50 times higher serum levels, with improved results in remission induction and consolidation for AML. Injection of the liposomal formulation is indicated for the intrathecal treatment of lymphomatous meningitis.

Onset of dyspnea, fever, and pulmonary infiltrates on chest computed tomography scans may follow 1-2 weeks after high-dose Ara-C and may be fatal in 10-20% of patients, especially in patients being treated for relapsed leukemia (Forghieri et al., 2007). Infectious or other causes of pulmonary infiltrates must be excluded in evaluating such patients. No specific therapy, other than Ara-C discontinuation, is indicated. Intrathecal Ara-C, either the free drug or the liposomal preparation, may cause arachnoiditis, seizures, delirium, myelopathy, or coma, especially if given concomitantly with systemic high-dose methotrexate or systemic Ara-C (Jabbour et al., 2007).

Cerebellar toxicity, manifesting as ataxia and slurred speech, and cerebral toxicity (seizures, dementia, and coma) may follow intrathecal administration or high-dose systemic administration, especially in patients >40 years of age and/or patients with poor renal function.

The aza-nucleosides enter cells by the human equilibrative transporter, the presence of which correlates with sensitivity of cell lines (Stresemann and Lyko, 2008). The drugs incorporate into DNA, where they become covalently bound to the methyltransferase through their N-5 position, depleting intracellular enzyme and leading to global demethylation of DNA and tumor cell differentiation and apoptosis. Decitabine also induces double-strand DNA breaks, perhaps as a consequence of the effort to repair the protein-DNA adduct. The azacytidine ring spontaneously opens in alkaline solution and has greater stability (t_1/2 of 7 hours) at neutral pH. After subcutaneous administration of the standard dose of 75 mg/m², 5-azacytidine achieves peak drug levels of 10 μM and undergoes very rapid deamination by cytidine deaminase (plasma t_1/2 of 20-40 minutes), with the product hydrolyzing to inactive metabolites. Due to the formation of intracellular nucleotides, which become incorporated into DNA, the effects of the aza-nucleosides persist for many hours.

The major toxicities of the aza-nucleosides include myelosuppression and mild GI symptoms. 5-Azacytidine produces rather severe nausea and vomiting when given intravenously in large doses (150-200 mg/m²/day for 5 days). The usual regimen for 5-azacytidine in myelodysplastic syndrome (MDS) is 75 mg/m²/day for 7 days every 28 days, while decitabine is given in a dose of 20 mg intravenously every day for 5 days every 4 weeks (Oki et al., 2007). Best responses may become apparent only after two to five courses of treatment.

Mechanism of Action. Gemcitabine is carried into cells by three distinct nucleoside transporters: hENT, hCNT, and a nucleobase transporter found in malignant mesothelioma cells. The hENT transporter appears to be the major carrier. Intracellularly, deoxycytidine kinase phosphorylates gemcitabine to produce difluorodeoxycytidine monophosphate (dFdCMP), from which point it is converted to difluorodeoxycytidine di- and triphosphate (dFdCDP and dFdCTP). Although gemcitabine's anabolism and effects on DNA in general mimic those of cytarabine, there are distinct differences in kinetics of inhibition, additional enzymatic sites of action, different effects of incorporation into DNA, and a distinct spectrum of clinical activity. Unlike that of cytarabine, the cytotoxicity of gemcitabine is not confined to the S phase of the cell cycle, and the drug is equally effective against confluent cells and cells in logarithmic growth phase. The cytotoxic activity may be a result of several actions on DNA synthesis. dFdCTP competes with dCTP as a weak inhibitor of DNA polymerase. dFdCDP is a stoichiometric inhibitor of RNR (Wang et al., 2007). It forms a complex with the RNR subunits and with ATP, resulting in depletion of deoxyribonucleotide pools necessary for DNA synthesis. Most important, dFdCTP incorporates into DNA and, after the incorporation of one more nucleotide, causes DNA strand termination (Heinemann et al., 1988). This "extra" nucleotide may be important in hiding the dFdCTP from DNA repair enzymes, as the incorporated dFdCMP appears resistant to repair. The ability of cells to incorporate dFdCTP into DNA is critical for gemcitabine-induced apoptosis. Gemcitabine is inactivated by cytidine deaminase, which is found both in tumor cells and throughout the body.

Preliminary evidence suggests that response to gemcitabine in pancreatic carcinoma tumors correlates positively with high expression of hENT (Giovannetti et al., 2006) and low expression of subunits of RNR, but resistance is found in tumors with low levels of deoxycytidine kinase and high levels of cytidine deaminase, the inactivating enzyme (Ohhashi et al., 2008). In mouse tumors, forced expression of the HMGA1 transcription factor also caused gemcitabine resistance (Liau and Whang, 2008), possibly through its effect on Akt-dependent survival pathways.

Absorption, Fate, and Elimination. Gemcitabine is administered as an intravenous infusion. The pharmacokinetics of the parent compound are largely determined by deamination in liver, plasma, and other organs, and the predominant urinary elimination product is difluorodeoxyuridine (dFdU). Although dFdU has modest toxicity at the usual concentrations found in plasma, in patients with significant renal dysfunction, dFdU and its triphosphate accumulate to high and potentially toxic levels (Koolen et al., 2009). Gemcitabine has a short plasma t_1/2 of ~15 minutes, with women and elderly patients clearing the drug more slowly (Abbruzzese et al., 1991). Peak levels reach 20-60 μM with doses of 1000 mg/m² given over 30 minutes intravenously. Clearance can vary widely among individuals but is not affected by renal failure.

Similar to that of cytarabine, conversion of gemcitabine to dFdCMP by deoxycytidine kinase is saturated at infusion rates of ~10 mg/m²/min, which produce plasma drug concentrations of ~10-20 μM. In an attempt to increase dFdCTP formation, the duration of infusion at this maximum concentration has been extended to 100-150 minutes at a fixed rate of 10 mg/min. The 150-minute infusion produces a higher level of dFdCTP within peripheral blood mononuclear cells and increases the degree of myelosuppression but does not improve antitumor activity (Gandhi, 2007). The inhibition of DNA repair by gemcitabine may increase cytotoxicity of other agents, particularly platinum compounds, and with radiation therapy. Preclinical studies of tumor cell lines show that gemcitabine enhances formation of cisplatin-DNA adducts, presumably through suppression of nuclear excision repair.

Therapeutic Uses. The standard dosing schedule for gemcitabine (gemzar) is a 30-minute intravenous infusion of 1-1.25 g/m² on days 1, 8, and 15 of each 21- to 28-day cycle, depending on the indication.

Clinical Toxicities. The principal toxicity of gemcitabine is myelosuppression. Longer-duration infusions lead to greater myelosuppression and hepatic toxicity. Nonhematological toxicities include a flu-like syndrome, asthenia, and rarely a posterior leukoencephalopathy syndrome. Mild elevation in liver transaminases may occur in ≥40% of patients and are reversible. Interstitial pneumonitis, at times progressing to acute respiratory distress syndrome (ARDS), may occur within the first two cycles of treatment and usually responds to corticosteroids. Rarely, patients on gemcitabine treatment for many months may develop a slowly progressive hemolytic uremic syndrome, necessitating drug discontinuation (Humphreys et al., 2004). Gemcitabine is a very potent radiosensitizer, likely the result of its inhibition of RNR, depletion of dATP, and inhibition of DNA repair (Flanagan et al., 2007) and should not be used with radiotherapy except in closely monitored clinical trials.

Mechanism of Action. Both 6-TG and 6-MP are excellent substrates for hypoxanthine guanine phosphoribosyl transferase (HGPRT) and are converted in a single step to the ribonucleotides 6-thioguanosine-5′-monophosphate (6-thioGMP) and 6-thioinosine-5′-monophosphate (T-IMP), respectively. Because T-IMP is a poor substrate for guanylyl kinase, the enzyme that converts guanosine monophosphate (GMP) to guanosine diphosphate (GDP), T-IMP accumulates intracellularly and in a second step is converted to 6-TGMP. T-IMP inhibits the new formation of ribosyl-5-phosphate, as well as conversion of inosine-5′-monophosphate (IMP) to adenine and guanine nucleotides. Of these, the most important point of attack seems to be the reaction of glutamine and PRPP to form ribosyl-5-phosphate, the first committed step in the de novo pathway. 6-Thioguanine nucleotide is incorporated into DNA, where it induces strand breaks and base mispairing. Strand breaks depend on the presence of an intact MMR system, the absence of which leads to resistance.

Mechanisms of Resistance to the Thiopurine Antimetabolites. The most common mechanism of 6-MP resistance observed in vitro is deficiency or complete lack of the activating enzyme, HGPRT, or increased alkaline phosphate activity. Other mechanisms for resistance include 1) decreased drug uptake, or increased efflux due to one of several active transporters; 2) alteration in allosteric inhibition of ribosylamine 5-phosphate synthase; and 3) impaired recognition of DNA breaks and mismatches due to loss of a component (MSH6) of MMR (Karran and Attard, 2008; Cahill et al., 2007).

Mercaptopurine Pharmacokinetics and Toxicity. Absorption of mercaptopurine is incomplete (10-50%) after oral ingestion; the drug is subject to first-pass metabolism by xanthine oxidase in the liver. Food or oral antibiotics decrease absorption. Oral bioavailability is increased when mercaptopurine is combined with high-dose methotrexate.

After an intravenous dose, the t_1/2 of the drug in plasma is relatively short (~50 minutes in adults), due to rapid metabolic degradation by xanthine oxidase and by thiopurine methyltransferase (TPMT). Restricted brain distribution of mercaptopurine results from an efficient efflux transport system in the blood-brain barrier. In addition to the HGPRT-catalyzed anabolism of mercaptopurine, there are two other pathways for its metabolism. The first involves methylation of the sulfhydryl group and subsequent oxidation of the methylated derivatives. Activity of the enzyme TPMT reflects the inheritance of polymorphic alleles; up to 15% of the Caucasian population has decreased enzyme activity. Low levels of erythrocyte TPMT activity are associated with increased drug toxicity in individual patients and a lower risk of relapse (Pui et al., 2004). In patients with auto-immune disease treated with mercaptopurine, those with polymorphic alleles may experience bone marrow aplasia and life-threatening toxicity. Testing for these polymorphisms prior to treatment is recommended in this patient population.

High concentrations of 6-methylmercaptopurine nucleotides are formed in white blood cells and bone marrow following 6-MP administration. They are less potent than 6-MP nucleotides as metabolic inhibitors, and their significance in contributing to the activity of 6-MP is not known.

A relatively large percentage of the administered sulfur appears in the urine as inorganic sulfate, the result of enzymatic desulfuration. The second major pathway for 6-MP metabolism involves its oxidation by xanthine oxidase to 6-thiouric acid, an inactive metabolite. Oral doses of 6-MP should be reduced by 75% in patients receiving the xanthine oxidase inhibitor, allopurinol. No dose adjustment is required for intravenous dosing.

Therapeutic Uses. In the maintenance therapy of ALL, the initial daily oral dose of mercaptopurine (6-MP; purinethol, others) is 50-100 mg/m² and is thereafter adjusted according to white blood cell and platelet count. The combination of methotrexate and 6-MP appears to be synergistic. By inhibiting the earliest steps in purine synthesis, methotrexate elevates the intracellular concentration of PRPP, a cofactor required for 6-MP activation.

Clinical Toxicities. The principal toxicity of 6-MP is bone marrow depression, although in general, this side effect develops more gradually than with folic acid antagonists; accordingly, thrombocytopenia, granulocytopenia, or anemia may not become apparent for several weeks. When depression of normal bone marrow elements occurs, dose reduction usually results in prompt recovery, although myelosuppression may be severe and prolonged in patients with a polymorphism affecting TPMT. Anorexia, nausea, or vomiting is seen in ~25% of adults, but stomatitis and diarrhea are rare; manifestations of GI effects are less frequent in children than in adults. Jaundice and hepatic enzyme elevations occur in up to one-third of adult patients treated with 6-MP and usually resolve upon discontinuation of therapy. Their appearance has been associated with bile stasis and hepatic necrosis on biopsy. 6-MP and its derivative, azathioprine, predispose to opportunistic infection such as reactivation of hepatitis B, fungal infection, and Pneumocystis pneumonia and an increased incidence of squamous cell malignancies of the skin. 6-MP has teratogenic effects during the first trimester of pregnancy, and AML has been reported after prolonged 6-MP therapy for Crohn's disease (Heizer and Peterson, 1998).

After rapid extracellular dephosphorylation to the nucleoside fludarabine, it is rephosphorylated intracellularly by deoxycytidine kinase to the active triphosphate derivative. This antimetabolite inhibits DNA polymerase, DNA primase, DNA ligase, and RNR and becomes incorporated into DNA and RNA. The nucleotide is an effective chain terminator when incorporated into DNA (Kamiya et al., 1996), and the incorporation of fludarabine into RNA inhibits RNA function, RNA processing, and mRNA translation (Plunkett and Gandhi, 1993).

In experimental tumors, resistance to fludarabine is associated with decreased activity of deoxycytidine kinase (the enzyme that phosphorylates the drug) (Mansson et al., 2003), increased drug efflux, and increased RNR activity. Its mechanism of immunosuppression and paradoxical stimulation of auto-immunity stems from the particular susceptibility of lymphoid cells to purine analogs and the specific effects on the CD4⁺ subset of T cells, as well as its inhibition of regulatory T-cell responses.

Absorption, Fate, and Excretion. Fludarabine phosphate is administered both intravenously and orally and rapidly converts to fludarabine in the plasma. The median time to reach maximal concentrations of drug in plasma after oral administration is 1.5 hours, and oral bioavailability averages 55-60%. The terminal t_1/2 of fludarabine in plasma is ~10 hours. The compound is primarily (40-50%) eliminated by renal excretion.

Therapeutic Uses. Fludarabine phosphate (fludara, oforta) is approved for intravenous and oral use and is equally active by both routes (Forconi et al., 2008). The recommended dose of fludarabine phosphate is 25 mg/m² daily for 5 days by intravenous infusion, or 40 mg/m² daily for 5 days by mouth. The drug is administered intravenously over 30 minutes to 2 hours. Dosage should be reduced in patients with renal impairment in proportion to the reduction in CrCl. Treatment may be repeated every 4 weeks, and gradual improvement in CLL usually occurs within two to three cycles.

Fludarabine phosphate is highly active alone or with rituximab and cyclophosphamide for the treatment of patients with CLL. Activity in CLL as a single agent is equal by either the oral or intravenous routes, as overall response rates in previously untreated patients approximate 80% and the duration of response averages 22 months. The synergy of fludarabine with alkylators may stem from the observation that it blocks the repair of double-strand DNA breaks and interstrand cross-links induced by alkylating agents. It also is effective in follicular B-cell lymphomas refractory to standard therapy. It is increasingly used as a potent immunosuppressive agent in nonmyeloablative allogeneic bone marrow transplantation, where it suppresses the host response and may encourage alloreactive donor T cells.

Clinical Toxicities. Oral and intravenous therapy cause myelosuppression (World Health Organization grade 3 or 4) in about half of patients, nausea and vomiting in a minor fraction, and uncommonly chills and fever, malaise, anorexia, peripheral neuropathy, and weakness. Lymphopenia and thrombocytopenia and cumulative side effects are expected. Depletion of CD4⁺ T cells with therapy predisposes to opportunistic infections. Tumor lysis syndrome, a relatively infrequent complication, occurs primarily in previously untreated patients with CLL. Altered mental status, seizures, optic neuritis, and coma have been observed at higher doses and in older patients.

Auto-immune events may occur after fludarabine treatment. CLL patients may develop an acute hemolytic anemia or pure red cell aplasia during or following fludarabine treatment. Prolonged cytopenias, probably mediated by auto-immunity, also complicate fludarabine treatment. Myelodysplasia and acute leukemias may arise as late complications (Tam et al., 2008). Pneumonitis is an occasional side effect and responds to corticosteroids. Because a significant fraction of drug is eliminated in the urine, patients with compromised renal function should be treated with caution, and initial doses should be reduced in proportion to the reduction in CrCl.

For these patients, it produces complete remissions in 20-30 % of patients. It has activity as well in pediatric and adult AML and in myelodysplasia. Its uptake and metabolic activation in tumor cells follow the same path as cladribine and the other purine nucleosides, although it is more readily phosphorylated by dCK. It incorporates into DNA, where it terminates DNA synthesis and leads to apoptosis.

Absorption, Fate, and Distribution. Clofarabine triphosphate has a long intracellular t_1/2 of 24 hours. Its antitumor activity is ascribed to its incorporation into DNA, resulting in chain termination. Like fludarabine, clofarabine inhibits RNR (Bonate et al., 2006). In children, following administration of usual doses of 52 mg/m² given as a 2-hour infusion daily for 5 days, clofarabine has a primary elimination t_1/2 in plasma of 6.5 hours, and the majority of drug is excreted unchanged in the urine. Doses should be adjusted according to reductions in CrCl, although precise guidelines are not available.

Clinical Toxicities. The primary toxicities are myelosuppression; a clinical syndrome of hypotension, tachyphemia, pulmonary edema, organ dysfunction, and fever, all suggestive of capillary leak syndrome and cytokine release; elevated hepatic enzymes and increased bilirubin; nausea, vomiting, and diarrhea; and hypokalemia and hypophosphatemia. Evidence of capillary leak should lead to immediate discontinuation of the drug.

Absorption, Fate, and Distribution. Following infusion, the parent methoxy compound is rapidly activated in blood and tissues by adenosine deaminase–mediated cleavage of the methyl group, yielding the phosphorylase resistant Ara-G, which has a longer plasma t_1/2 of 3 hours. The active metabolite is transported into tumor cells, where it is activated by CdK to the monophosphate and thence to a triphosphate. Ara-G triphosphate (Ara-GTP) is incorporated into DNA and terminates DNA synthesis (Sanford and Lyseng-Williamson, 2008). The drug and its metabolite, Ara-G, are primarily eliminated by metabolism to guanine, and a smaller fraction is eliminated by renal excretion of Ara-G. The dose should not be adjusted for mild to moderate (CrCl >50 mg/mL) renal dysfunction (Sanford and Lyseng-Williamson, 2008), but the drug should be used with close clinical monitoring in patients with more severe renal impairment. Adults are given a dose of 1500 mg/m² intravenously as a 2-hour infusion on days 1, 3, and 5 of a 21-day cycle, and children are given a lower dose of 650 mg/m²/day intravenously for 5 days and repeated every 21 days.

Clinical Toxicities. Side effects include myelosuppression and liver function test abnormalities, as well as frequent, serious neurological sequelae, such as seizures, delirium, somnolence, peripheral neuropathy, or Guillain-Barré syndrome. Neurological side effects may not be reversible.

Pentostatin (2′-deoxycoformycin; see Figure 61–11), a transition-state analog of the intermediate in the adenosine deaminase (ADA) reaction, potently inhibits ADA. Its effects mimic the phenotype of genetic ADA deficiency (severe immunodeficiency affecting both T- and B-cell functions). It was isolated from fermentation cultures of Streptomyces antibioticus. Inhibition of ADA by pentostatin leads to accumulation of intracellular adenosine and deoxyadenosine nucleotides, which can block DNA synthesis by inhibiting RNR. Deoxyadenosine also inactivates S-adenosyl homocysteine hydrolase. The resulting accumulation of S-adenosyl homocysteine is particularly toxic to lymphocytes. Pentostatin also can inhibit RNA synthesis, and its triphosphate derivative is incorporated into DNA, resulting in strand breakage. Although the precise mechanism of cytotoxicity is not known, it is probable that the imbalance in purine nucleotide pools accounts for its antineoplastic effect in hairy cell leukemia and T-cell lymphomas.

Absorption, Fate, and Excretion. Pentostatin is administered intravenously, and a single dose of 4 mg/m² has a mean terminal t_1/2 of 5.7 hours. The drug is eliminated almost entirely by renal excretion. Proportional reduction of dosage is recommended in patients with renal impairment as measured by reduced CrCl.

Therapeutic Uses. The recommended dose is 4 mg/m² administered every other week intravenously. After hydration with 500-1000 mL of 5% dextrose in half-normal (0.45%) saline, the drug is administered by rapid intravenous injection or by infusion over a period of ≤30 minutes, followed by an additional 500 mL of fluids.

Pentostatin is extremely effective in producing complete remissions (58%) and partial responses (28%) in hairy cell leukemia (Grever et al., 2003). It largely has been superseded by cladribine.

Clinical Toxicities. Toxic manifestations include myelosuppression, GI symptoms, skin rashes, and abnormal liver function studies. Depletion of normal T cells occurs, and neutropenic fever and opportunistic infections may result. Immunosuppression may persist for several years after discontinuation of pentostatin therapy. At high doses (10 mg/m²), major renal and neurological complications are encountered. Pentostatin in combination with fludarabine phosphate may result in severe or even fatal pulmonary toxicity.

History. The beneficial properties of the Madagascar periwinkle plant, Catharanthus roseus (formerly called Vinca rosea), a species of myrtle, have been described in medicinal folklore. Periwinkle extracts attracted interest because of their hypoglycemic effects in diabetes. Purified alkaloids, including vinblastine and vincristine, caused regression of an acute lymphocytic leukemia in mice and were among the earliest clinical agents for treatment of leukemias, lymphomas, and testicular cancer. A closely related derivative, vinorelbine, has important activity against lung and breast cancer.

Chemistry. The vinca alkaloids are asymmetrical dimeric compounds formed by condensation of the vindoline and catharanthine subunits.

When cells are incubated with vinblastine, the microtubules dissolve and highly regular crystals form, containing 1 mole of bound vinblastine per mole of tubulin. Cell division arrests in metaphase. In the absence of an intact mitotic spindle, duplicated chromosomes cannot align along the division plate. They disperse throughout the cytoplasm (exploded mitosis) or may clump in unusual groupings, such as balls or stars. Cells blocked in mitosis undergo changes characteristic of apoptosis.

In addition to their key role in the formation of mitotic spindles, microtubules are found in high concentration in the brain and contribute to other cellular functions such as movement, phagocytosis, and axonal transport. Side effects of the vinca alkaloids, such as their neurotoxicity, may relate to disruption of these functions.

Drug Resistance. Despite their structural similarity, the vinca alkaloids have unique individual patterns of clinical effectiveness (see the individual vinca alkaloid sections). However, in most experimental systems, they share cross-resistance. Their antitumor effects are blocked by multidrug resistance mediated by the mdr gene and its glycoprotein. Tumor cells become cross-resistant to a wide range of chemically dissimilar agents (the vinca alkaloids, epipodophyllotoxins, anthracyclines, and taxanes). Chromosomal abnormalities consistent with gene amplification have been observed in resistant cells in culture, and the cells contain markedly increased levels of the P-glycoprotein, a membrane efflux transporter (Endicott and Ling, 1989). Ca²⁺ channel blockers such as verapamil can reverse resistance of this type in vitro; however, clinical trials of resistance-reversing agents have been disappointing. Other membrane transporters, such as the MRP and the closely related breast cancer resistance protein, may mediate multidrug resistance. Still other forms of resistance to vinca alkaloids stem from mutations in β tubulin or in the relative expression of isoforms of β tubulin; both changes prevent the inhibitors from effectively binding to their target.

Cytotoxic Actions. The very limited myelosuppressive action of vincristine makes it a valuable component of several combination therapy regimens for leukemia and lymphoma, while the lack of severe neurotoxicity of vinblastine is a decided advantage in lymphomas and in combination with cisplatin against testicular cancer. Vinorelbine, which causes a mild neurotoxicity as well as myelosuppression, has an intermediate toxicity profile. Vincristine is a standard component of regimens for treating pediatric leukemias, lymphomas, and solid tumors, such as Wilms tumor, neuroblastoma, and rhabdomyosarcoma. In large-cell non-Hodgkin's lymphomas, vincristine remains an important agent, particularly when used in the CHOP regimen with cyclophosphamide, doxorubicin, and prednisone. Vinblastine is employed in treating bladder cancer, testicular carcinomas, and Hodgkin's disease. Vinorelbine has activity against non–small cell lung cancer and breast cancer.

Absorption, Fate, and Excretion. The liver cytochromes extensively metabolize all three agents, and the metabolites are excreted in the bile (Robieux et al., 1996). Only a small fraction of a dose (<15%) is found in the urine unchanged. In patients with hepatic dysfunction (bilirubin >3 mg/dL), a 50-75% reduction in dose of any of the vinca alkaloids is advisable, although firm guidelines for dose adjustment have not been established. The pharmacokinetics of each of the three drugs are similar, with an elimination t_1/2 of 20 hours for vincristine, 23 hours for vinblastine, and 24 hours for vinorelbine.

Therapeutic Uses. Vinblastine sulfate (velban, others) is given intravenously; special precautions must be taken against subcutaneous extravasation, because this may cause painful irritation and ulceration. The drug should not be injected into an extremity with impaired circulation. After a single dose of 0.3 mg/kg of body weight, myelosuppression reaches its maximum in 7-10 days. If a moderate level of leukopenia (~3000 cells/mm³) is not attained, the weekly dose may be increased gradually by increments of 0.05 mg/kg of body weight. In regimens designed to cure testicular cancer, vinblastine is used in doses of 0.3 mg/kg every 3 weeks. Doses should be reduced by 50% for patients with plasma bilirubin >1.5 mg/dL.

One important clinical use of vinblastine is with bleomycin and cisplatin (see "Therapeutic Uses" under "Bleomycin") in the curative therapy of metastatic testicular tumors, although it has been supplanted by etoposide or ifosfamide in this disease. It is a component of the standard curative regimen for Hodgkin's disease [doxorubicin (adriamycin), bleomycin, vinblastine, and dacarbazine (ABVD)]. It also is active in Kaposi sarcoma, neuroblastoma, Langerhans cell histiocytosis, carcinoma of the breast, and choriocarcinoma.

Clinical Toxicities. The nadir of the leukopenia that follows the administration of vinblastine usually occurs within 7-10 days, after which recovery ensues within 7 days. Other toxic effects of vinblastine include mild neurological manifestations. GI disturbances including nausea, vomiting, anorexia, and diarrhea may be encountered. The syndrome of inappropriate secretion of antidiuretic hormone has been reported. Loss of hair, stomatitis, and dermatitis occur infrequently. Extravasation during injection may lead to cellulitis and phlebitis.

Chemistry. Paclitaxel is a diterpenoid compound that contains a complex eight-member taxane ring as its nucleus (Figure 61–12). The side chain linked to the taxane ring at C13 is essential for its antitumor activity. Modification of the side chain has led to identification of the more potent analog, docetaxel (Figure 61–12), which shares the same spectrum of clinical activity as paclitaxel, but differs in its toxicity. Originally purified as the parent molecule from yew bark, paclitaxel now can be obtained for commercial purposes by semisynthesis from 10-desacetylbaccatin, a precursor found in yew needles. It also has been successfully synthesized (Nicolaou et al., 1994) in a complex series of reactions. Paclitaxel has very limited water solubility and is administered in a vehicle of 50% ethanol and 50% polyethoxylated castor oil (cremophor el); this vehicle likely is responsible for a high rate of hypersensitivity reactions. Patients receiving this formulation are protected by pretreatment with a histamine H₁-receptor antagonist such as diphenhydramine, an H₂-receptor antagonist such as cimetidine (see Chapter 32), and a glucocorticoid such as dexamethasone (see Chapter 42).

Nab-paclitaxel is soluble in aqueous solutions and can be administered safely without prophylactic antihistamines or steroids. This form of paclitaxel has increased cellular uptake via an albumin-specific mechanism.

Docetaxel, somewhat more soluble than paclitaxel, is administered in polysorbate 80 and is associated with a lower incidence of hypersensitivity reactions than paclitaxel dissolved in cremophor. However, pretreatment with dexamethasone for 3 days starting 1 day prior to therapy is required to prevent progressive fluid retention and minimize the severity of hypersensitivity reactions.

Mechanism of Action. Interest in paclitaxel was stimulated by the drug's unique ability to promote microtubule formation at cold temperatures and in the absence of GTP. It binds specifically to the β-tubulin subunit of microtubules and antagonizes the disassembly of this key cytoskeletal protein, with the result that bundles of microtubules and aberrant structures derived from microtubules appear in the mitotic phase of the cell cycle. Arrest in mitosis follows. Cell killing is dependent on both drug concentration and duration of cell exposure. Drugs that block cell-cycle progression prior to mitosis antagonize the toxic effects of taxanes.

Drug interactions have been noted; the sequence of cisplatin preceding paclitaxel decreases paclitaxel clearance and produces greater toxicity than the opposite schedule (Rowinsky and Donehower, 1995). Paclitaxel decreases doxorubicin clearance and enhances cardiotoxicity, while docetaxel has no apparent effect on anthracycline pharmacokinetics.

In cultured tumor cells, resistance to taxanes is associated in some lines with increased expression of the mdr-1 gene and its product, P-glycoprotein; other resistant cells have β-tubulin mutations, and these latter cells may display heightened sensitivity to vinca alkaloids (Cabral, 1983). Other resistant cell lines display an increase in survivin, an anti-apoptotic factor, α aurora kinase, an enzyme that promotes completion of mitosis. The taxanes preferentially bind to the βII-tubulin subunit of microtubules; therefore, cells may become resistant by upregulating the βIII-isoform of tubulin (Ranganathan et al., 1998). The basis of clinical drug resistance is not known. Cell death occurs by apoptosis, but the effectiveness of paclitaxel against experimental tumors does not depend on an intact p53 gene product.

Preclinical studies have suggested that nab-paclitaxel has an increased antitumor effect in breast cancer and a higher intratumoral drug concentration compared to cremophor-delivered paclitaxel. The reasons are not clear but may relate to maintenance of the drug in the nanoparticle micellar system or to increased expression of SPARC [Secreted Protein, Acidic and Rich in Cysteine; aka osteonectin, a matri-cellular linkage protein expressed in pro-fibrotic states and linked to myriad pathologies [Kos and Wilding, 2010; Chlenski and Cohn, 2010]) on tumor cells, leading to an increased drug uptake.

Absorption, Fate, and Excretion. Paclitaxel is administered as a 3-hour infusion of 135-175 mg/m² every 3 weeks or as a weekly 1-hour infusion of 80-100 mg/m². Prolonged infusions (96 hours) also have been evaluated in different tumor histologies and are active. The drug undergoes extensive metabolism by hepatic CYPs (primarily CYP2C8 with a contribution of CYP3A4); <10% of a dose is excreted in the urine intact. The primary metabolite identified thus far is 6-OH paclitaxel, which is inactive, but multiple additional hydroxylation products are found in plasma (Cresteil et al., 1994).

Paclitaxel clearance is nonlinear and decreases with increasing dose or dose rate. In studies of 96-hour infusions of 140 mg/m² (35 mg/m²/day), the presence of hepatic metastases >2 cm in diameter decreased clearance and led to high drug concentrations in plasma and greater myelosuppression. Paclitaxel disappears from the plasma compartment with a t_1/2 of 10-14 hours and a clearance of 15-18 L/hr/m². The critical plasma concentration for inhibiting bone marrow elements depends on duration of exposure but likely lies at ~50-100 nM (Huizing et al., 1993).

Nab-paclitaxel achieves a higher serum concentration of paclitaxel compared to cremophor-solubilized paclitaxel, but the increased clearance of nab-paclitaxel results in a similar drug exposure (Gardner et al., 2008). Nab-paclitaxel is most often administered intravenously over 30 minutes at 260 mg/m² every 3 weeks; however, alternate dosing regimens are being evaluated. Like the other taxanes, nab-paclitaxel should not be given to patients with an absolute neutrophil count <1500 cells/mm³. Docetaxel pharmacokinetics are similar to those of paclitaxel, with an elimination t_1/2 of ~12 hours. Clearance is primarily through CYP3A4- and CYP3A5-mediated hydroxylation, leading to inactive metabolites (Clarke and Rivory, 1999). In contrast to paclitaxel, the pharmacokinetics of docetaxel are linear for doses ≤115 mg/m².

Dose reductions in patients with abnormal hepatic function have been suggested, and 50-75% doses of taxanes should be used in the presence of hepatic metastases >2 cm in size or in patients with abnormal serum bilirubin. Drugs that induce CYP2C8 or CYP3A4, such as phenytoin and phenobarbital, or those that inhibit the same cytochromes, such as antifungal imidazoles, significantly alter drug clearance and toxicity.

Paclitaxel clearance is markedly delayed by cyclosporine A and a number of other drugs employed experimentally as inhibitors of the P-glycoprotein. This inhibition may be due to a block of CYP-mediated metabolism or effects on biliary excretion of the parent drug or metabolites.

Therapeutic Uses. The taxanes have become central components of regimens for treating metastatic ovarian, breast, lung, GI, genitourinary, and head and neck cancers. In current regimens, these drugs are administered once weekly or once every 3 weeks. The appropriate use of the steroid-sparing nab-paclitaxel still is being evaluated in clinical trials; in a randomized phase III study comparing 175 mg/m² of paclitaxel to 260 mg/m² of nab-paclitaxel in women with metastatic breast cancer, the nab-paclitaxel arm had a higher response rate and longer time to progression compared to the paclitaxel arm (Gradishar et al., 2005).

Clinical Toxicities. Paclitaxel exerts its primary toxic effects on the bone marrow. Neutropenia usually occurs 8-11 days after a dose and reverses rapidly by days 15-21. Used with filgrastim [granulocyte-colony stimulating factor (G-CSF)], doses as high as 250 mg/m² over 24 hours are well tolerated, and peripheral neuropathy becomes dose limiting. Many patients experience myalgias for several days after receiving paclitaxel. In high-dose schedules, or with prolonged use, a stocking-glove sensory neuropathy can be disabling, particularly in patients with underlying diabetic neuropathy or concurrent cisplatin therapy. Mucositis is prominent in 72- or 96-hour infusions and in the weekly schedule.

Hypersensitivity reactions occurred in patients receiving paclitaxel infusions of short duration (1-6 hours) but have largely been averted by pretreatment with dexamethasone, diphenhydramine, and histamine H₂-receptor antagonists, as noted above. Premedication is not necessary with 96-hour infusions. Many patients experience asymptomatic bradycardia, and occasional episodes of silent ventricular tachycardia also occur and resolve spontaneously during 3- or 24-hour infusions.

Nab-paclitaxel produces increased rates of peripheral neuropathy compared to cremophor-delivered paclitaxel but rarely causes hypersensitivity reactions.

Docetaxel causes greater degrees of neutropenia than paclitaxel but less peripheral neuropathy and asthenia and less frequent hypersensitivity. Fluid retention is a progressive problem with multiple cycles of docetaxel therapy, leading to peripheral edema, pleural and peritoneal fluid, and pulmonary edema in extreme cases. Oral dexamethasone, 8 mg/day, begun 1 day prior to drug infusion and continuing for 3 days, greatly ameliorates fluid retention. In rare cases, docetaxel may cause a progressive interstitial pneumonitis, with respiratory failure supervening if the drug is not discontinued (Read et al., 2002).

Absorption, Fate, and Excretion. Following oral administration, at least 75% of a dose of estramustine is absorbed from the GI tract and rapidly dephosphorylated. Estramustine undergoes extensive first-pass metabolism by CYP1A2 and CYP3A4 to an active oxidized 17-keto derivative, estramustine, and to multiple inactive products; both active forms accumulate in the prostate. Some hydrolysis of the carbamate linkage occurs in the liver, releasing estradiol, estrone, and the normustine group. Estramustine and estromustine have a plasma t_1/2 of 10 and 14 hours, respectively, and are excreted as inactive metabolites, mainly in the feces (Bergenheim and Henriksson, 1998). Estramustine inhibits the clearance of taxanes.

Clinical Toxicities. In addition to myelosuppression, estramustine also possesses estrogenic side effects (gynecomastia, impotence, elevated risk of thrombosis, and fluid retention), hypercalcemia, acute attacks of porphyria, impaired glucose tolerance, and hypersensitivity reactions, including angioedema.

Chemistry. The epothilones are 16-membered polyketides discovered as cytotoxic metabolites from a strain of Sorangium cellulosum, a myxobacterium originally isolated from soil on the bank of the Zambezi River in southern Africa (Gerth et al., 1996).

Six natural epothilones (A-F), and synthetic and semisynthetic analogs are in various stages of development (Lee and Swain, 2008). Most trials of epothilones to date have evaluated compounds of the subtypes A, B, and D, which differ in their functional groups at carbon 12.

Initial studies of the natural epothilone compounds A, B, and D showed good in vitro cytotoxic activity at nanomolar concentrations, epothilone B having roughly twice the potency as epothilones A and D (Lee and Swain, 2008). The early in vivo activity in animals, however, was disappointing due to instability of their lactone ring. Modification of epothilone B by substituting a nitrogen for the lactone oxygen yielded ixabepilone, which is not susceptible to esterases.

Mechanism of Action. The epothilones resemble taxanes in that they bind to β tubulin and trigger microtubule nucleation at multiple sites away from the centriole. This chaotic microtubule stabilization triggers cell-cycle arrest at the G2-M interface and apoptosis. Epothilones bind to a site distinct from that of taxanes. In colon cancer cell lines, p53 and Bax trigger apoptosis in ixabepilone-treated cells.

In vitro studies suggest that ixabepilone is less susceptible to P-glycoprotein-mediated multidrug resistance when compared to taxanes. Other mechanisms implicated in epothilone resistance include mutation of the β-tubulin binding site and upregulation of isoforms of β tubulin.

Absorption, Distribution, and Excretion. Ixabepilone is administered intravenously. Because of its minimal aqueous solubility, it is delivered in the solubilizing agent, polyoxyethylated castor oil/ethanol (cremophor el). cremophor has been implicated as the cause of infusion reactions associated with paclitaxel and with other drug formulations, but such reactions are infrequent when administration is preceded by premedication with H₁ and H₂ antagonists. The drug is cleared by hepatic CYPs and has a plasma t_1/2 of 52 hours.

Therapeutic Uses. In a phase III study for registration, patients with metastatic breast cancer resistant to or pretreated with anthracyclines and resistant to taxanes had an improved progression-free survival of 1.6 months with ixabepilone plus capecitabine compared to capecitabine alone (p = .0003) (Thomas et al., 2007).

Ixabepilone also is indicated as monotherapy for metastatic breast cancer in patients who have previously progressed through treatment with anthracyclines, taxanes, and capecitabine.

The recommended dose of ixabepilone as monotherapy or in combination with capecitabine is 40 mg/m² administered over 3 hours every 3 weeks. Note that because of additive myelosuppression, the phase III trial used an attenuated dose of capecitabine (2000 mg/m²) administered with ixabepilone compared to 2500 mg/m² when capecitabine was administered alone. Patients should be premedicated with both an H₁ and H₂ antagonist before receiving ixabepilone to minimize hypersensitivity reactions.

The combination of ixabepilone and capecitabine is contraindicated in patients with a baseline neutrophil count <1500 cells/mm³, a platelet count <100,000 cells/mm³, serum transaminases >2.5 × ULN or bilirubin above normal. In patients receiving ixabepilone monotherapy with mild to moderate hepatic dysfunction (bilirubin <1.5 × ULN or 1.5-3 × ULN, respectively), starting doses of 32 and 20 mg/m² are recommended due to delayed drug clearance.

Toxicities. Epothilones have toxicities similar to those of the taxanes, namely neutropenia peripheral sensory neuropathy, fatigue, diarrhea, and asthenia. Grade 3/4 peripheral sensory neuropathy was seen in 21% of patients receiving combined therapy with ixabepilone and capecitabine and in 14% of patients receiving monotherapy. Ixabepilone in combination with capecitabine causes a 68% rate of grade 3/4 neutropenia; it causes a 54% rate of grade 3/4 neutropenia when given as monotherapy.

Chemistry. All camptothecins have a fused five-ring backbone that includes a labile lactone ring (Figure 61–13). The hydroxyl group and S-conformation of the chiral center at C20 in the lactone ring are required for biological activity. Appropriate substitutions on the A and B rings of the quinoline subunit enhance water solubility and increase potency for inhibiting topoisomerase I. Topotecan [(S)-9-dimethylaminoethyl-10-hydroxycamptothecin hydrochloride] is a semisynthetic molecule with a basic dimethylamino group that increases its water solubility. Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin, or CPT-11) differs from topotecan in that it is a prodrug. The carbamate bond between the camptothecin moiety and the dibasic bispiperidine side chain at position C10 (which makes the molecule water soluble) is cleaved by a carboxylesterase to form the active metabolite, SN-38 (see Chapter 6).

Mechanism of Action. The DNA topoisomerases are nuclear enzymes that reduce torsional stress in supercoiled DNA, allowing selected regions of DNA to become sufficiently untangled and relaxed to permit replication, repair, and transcription. Two classes of topoisomerase (I and II) mediate DNA strand breakage and resealing, and both have become the target of cancer chemotherapies. Camptothecin analogs inhibit the function of topoisomerase I, while a number of different chemical entities (e.g., anthracyclines, epipodophyllotoxins, acridines) inhibit topoisomerase II. Topoisomerase I binds covalently to double-stranded DNA through a reversible transesterification reaction. This reaction yields an intermediate complex in which the tyrosine of the enzyme is bound to the 3′-phosphate end of the DNA strand, creating a single-strand DNA break. This "cleavable complex" allows for relaxation of the DNA torsional strain, either by passage of the intact single-strand through the nick or by free rotation of the DNA about the noncleaved strand. Once the DNA torsional strain has been relieved, the topoisomerase I reseals the cleavage and dissociates from the newly relaxed double helix.

The camptothecins bind to and stabilize the normally transient DNA-topoisomerase I cleavable complex. Although the initial cleavage action of topoisomerase I is not affected, the re-ligation step is inhibited, leading to the accumulation of single-stranded breaks in DNA. These lesions are reversible and not by themselves toxic to the cell. However, the collision of a DNA replication fork with this cleaved strand of DNA causes an irreversible double-strand DNA break, ultimately leading to cell death (Tsao et al., 1993). Camptothecins are therefore S phase–specific drugs, because ongoing DNA synthesis is necessary for cytotoxicity. This has important clinical implications. S phase–specific cytotoxic agents generally require prolonged exposures of tumor cells to drug concentrations above a minimum threshold to optimize therapeutic efficacy. In fact, preclinical studies of low-dose, protracted administration of camptothecin analogs have less toxicity, and equal or greater antitumor activity, than shorter, more intense courses.

The precise sequence of events that leads from drug-induced DNA damage to cell death has not been fully elucidated. In vitro, camptothecin-induced DNA damage abolishes the activation of the p34^cdc2/cyclin B complex, leading to cell-cycle arrest at the G2 phase (Tsao et al., 1993). Treatment with camptothecins can induce the transcription of c-fos and c-jun early-response genes, and this occurs in association with internucleosomal DNA fragmentation, a characteristic of programmed cell death.

Mechanisms of Resistance. A variety of mechanisms of resistance to topoisomerase I–targeted agents have been characterized in vitro, although little is known about their significance in the clinical setting. Decreased intracellular drug accumulation may underlie resistance in cell lines. Topotecan, but not SN-38 or irinotecan, is a substrate for P-glycoprotein. However, the clinical relevance of P-glycoprotein-mediated efflux as a mechanism of resistance against topotecan remains unclear, as the magnitude of the effect in preclinical studies was found to be substantially lower than that observed with other MDR substrates, such as etoposide or doxorubicin. Other reports have associated topotecan and irinotecan resistance with the MRP class of transporters (Miyake et al., 1999). Cell lines that lack carboxylesterase activity demonstrate resistance to irinotecan (Van Ark-Otte et al., 1998), but in patients, the liver and red blood cells may have sufficient carboxylesterase activity to convert irinotecan to SN-38. Camptothecin resistance also may result from decreased expression or mutation of topoisomerase I. Although a good correlation has been found in certain tumor cell lines between sensitivity to camptothecin analogs and topoisomerase I levels (Sugimoto et al., 1990), clinical studies have not confirmed this association. Chromosomal deletions or hypermethylation of the topoisomerase I gene are possible mechanisms of decreased topoisomerase I expression in resistant cells. A transient downregulation of topoisomerase I has been demonstrated following prolonged exposure to camptothecins in vitro and in vivo. Mutations leading to reduced topoisomerase I enzyme catalytic activity or DNA-binding affinity have been associated with experimental camptothecin resistance (Tamura et al., 1991). In addition, enzyme phosphorylation or polyADP ribosylation may reduce the activity of topoisomerase I and its susceptibility to inhibition. Finally, exposure of cells to topoisomerase I–targeted agents upregulates topoisomerase II, an alternative enzyme for DNA strand passage.

Very little is known about how the cell deals with the stabilized DNA-topoisomerase complexes. Cellular repair processes may not readily recognize the drug-enzyme-DNA complex. However, an enzyme with specific tyrosyl-DNA phosphodiesterase activity may be involved in the disassembly of topoisomerase I–DNA complexes (Yang et al., 1996).

Absorption, Fate, and Excretion.

Topotecan. Topotecan is approved for intravenous administration. However, there has been interest in developing an oral dosage form for the drug, which has a bioavailability of 30-40% in cancer patients. Topotecan exhibits linear pharmacokinetics, and it is rapidly eliminated from systemic circulation. The biological t_1/2 of total topotecan, which ranges from 3.5-4.1 hours, is relatively short compared to that of other camptothecins. Only 20-35% of the total drug in plasma is found to be in the active lactone form. Within 24 hours, 30-40% of the administered dose appears in the urine. Doses should be reduced in proportion to reductions in CrCl. Although several oxidative metabolites have been identified, hepatic metabolism appears to be a relatively minor route of drug elimination. Unlike most other camptothecins considered for clinical development, plasma protein binding of topotecan is low, at only 7-35%, which may explain its relatively greater CNS penetration.

Irinotecan. The conversion of irinotecan to SN-38 is mediated predominantly by carboxylesterases in the liver. Although SN-38 can be measured in plasma shortly after beginning an intravenous infusion of irinotecan, the AUC of SN-38 is only ~4% of the AUC of irinotecan, suggesting that only a relatively small fraction of the dose is ultimately converted to the active form of the drug. Irinotecan exhibits linear pharmacokinetics at doses evaluated in cancer patients. In comparison to topotecan, a relatively large fraction of both irinotecan and SN-38 are present in plasma as the biologically active intact lactone form. Another potential advantage of this analog is that the t_1/2 of SN-38 is 11.5 hours, which is much longer than the t_1/2 of topotecan. CSF penetration of SN-38 in humans has not been characterized yet, although in rhesus monkeys, it is only 14%, significantly lower than that observed for topotecan.

In contrast to topotecan, hepatic metabolism represents an important route of elimination for both irinotecan and SN-38. Oxidative metabolites have been identified in plasma, all of which result from CYP3A-mediated reactions directed at the bispiperidine side chain. These metabolites are not significantly converted to SN-38. The total body clearance of irinotecan was found to be two times greater in brain cancer patients taking antiseizure drugs that induce hepatic CYPs, further attesting to the importance of oxidative hepatic metabolism as a route of elimination for this drug (Gilbert et al., 2003).

Glucuronidation of the hydroxyl group at position C10 (resulting from cleavage of the bispiperidine promoiety) produces the only known metabolite of SN-38. Biliary excretion appears to be the primary elimination route of irinotecan, SN-38, and their metabolites, although urinary excretion also contributes significantly (14-37%). Uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1), converts SN-38 to its inactive derivative (Iyer et al., 1998). The extent of SN-38 glucuronidation inversely correlates with the risk of severe diarrhea after irinotecan therapy. UGT1A1 also glucuronidates bilirubin. Polymorphisms of this enzyme are associated with familial hyperbilirubinemia syndromes such as Crigler-Najjar syndrome and Gilbert syndrome. Crigler-Najjar syndrome is rare (one in a million births), but Gilbert syndrome occurs in up to 15% of the general population and results in a mild hyperbilirubinemia that may be clinically silent. The presence of UGT enzyme polymorphisms may have a major impact on the clinical use of irinotecan. A positive correlation has been found between baseline serum unconjugated bilirubin concentration and both severity of neutropenia and the AUC of irinotecan and SN-38 in patients treated with irinotecan. Moreover, severe irinotecan toxicity has been observed in cancer patients with Gilbert syndrome, presumably due to decreased glucuronidation of SN-38. The presence of bacterial glucuronidase in the intestinal lumen potentially can contribute to irinotecan's GI toxicity by releasing unconjugated SN-38 from the inactive glucuronide metabolite excreted in the bile.

Therapeutic Uses.

Topotecan. Topotecan (hycamtin) is indicated for previously treated patients with ovarian and small cell lung cancer. Its significant hematological toxicity has limited its use in combination with other active agents in these diseases (e.g., cisplatin).

The recommended dosing regimen of topotecan for ovarian cancer and small cell lung cancer is a 30-minute infusion of 1.5 mg/m²/day for 5 consecutive days every 3 weeks. For cervical cancer in conjunction with cisplatin, the dose of topotecan is 0.75 mg/m² on days 1, 2, and 3, repeated every 21 days. Because a significant fraction of the topotecan administered is excreted in the urine, patients with decreased CrCl may experience increased toxicity (O'Reilly et al., 1996). Therefore, the dose of topotecan should be reduced to 0.75 mg/m²/day in patients with moderate renal dysfunction (CrCl of 20-40 mL/min), and topotecan should not be administered to patients with severe renal impairment (CrCl <20 mL/min). Hepatic dysfunction does not alter topotecan clearance and toxicity. A baseline neutrophil count >1500 cells/mm3 and a platelet count >100,000 is necessary prior to topotecan administration. For small cell lung cancer, oral therapy can be used at a dosage of 2.3 mg/m2/day for 5 consecutive days repeated every 21 days. The oral dose is reduced to 1.8 mg/m2 for patients with a CrCl of 30-49 mL/min.

Irinotecan. Approved single-agent dosage schedules of irinotecan (camptosar, others) in the U.S. include 125 mg/m² as a 90-minute infusion administered weekly (on days 1, 8, 15, and 22) for 4 out of 6 weeks, and 350 mg/m² given every 3 weeks. In patients with advanced colorectal cancer, irinotecan is used as first-line therapy in combination with fluoropyrimidines or as a single agent or in combination with cetuximab following failure of a 5-FU/oxaliplatin regimen.

Clinical Toxicities.

Topotecan. The dose-limiting toxicity with all dosing schedules is neutropenia, with or without thrombocytopenia. The incidence of severe neutropenia at the recommended phase II dose of 1.5 mg/m² daily for 5 days every 3 weeks may be as high as 81%, with a 26% incidence of febrile neutropenia. In patients with hematological malignancies, GI side effects such as mucositis and diarrhea become dose limiting. Other less common and generally mild topotecan-related toxicities include nausea and vomiting, elevated liver transaminases, fever, fatigue, and rash.

Irinotecan. The dose-limiting toxicity with all dosing schedules is delayed diarrhea, with or without neutropenia. In the initial studies, up to 35% of patients experienced severe diarrhea. Adoption of an intensive regimen of loperamide (4 mg of loperamide starting at the onset of any loose stool beginning more than a few hours after receiving therapy, followed by 2 mg every 2 hours) (see Chapter 47) has effectively reduced this incidence by more than half. However, once severe diarrhea occurs, standard doses of antidiarrheal agents tend to be ineffective. Diarrhea generally resolves within a week and, unless associated with fever and neutropenia, rarely is fatal.

The second most common irinotecan-associated toxicity is myelosuppression. Severe neutropenia occurs in 14-47% of the patients treated with the every-3-weeks schedule and is less frequently encountered among patients treated with the weekly schedule. Febrile neutropenia is observed in 3% of patients and may be fatal, particularly when associated with concomitant diarrhea. A cholinergic syndrome resulting from the inhibition of acetylcholinesterase activity by irinotecan may occur within the first 24 hours after irinotecan administration. Symptoms include acute diarrhea, diaphoresis, hypersalivation, abdominal cramps, visual accommodation disturbances, lacrimation, rhinorrhea, and less often, asymptomatic bradycardia. These effects are short lasting and respond within minutes to atropine. Atropine may be prophylactically administered to patients who have previously experienced a cholinergic reaction. Other common and generally manageable toxicities include nausea and vomiting, fatigue, vasodilation or skin flushing, mucositis, elevation in liver transaminases, and alopecia. Finally, there have been case reports of dyspnea and interstitial pneumonitis associated with irinotecan therapy in Japanese patients with lung cancer (Fukuoka et al., 1992).

Chemistry and Structure-Activity Relationships. The actinomycins are chromopeptides. Most contain the same chromophore, the planar phenoxazone actinosin, which is responsible for their yellow-red color. The differences among naturally occurring actinomycins are confined to variations in the structure of the amino acids of the peptide side chains.

Mechanism of Action. The capacity of actinomycins to bind with double-helical DNA is responsible for their biological activity and cytotoxicity. X-ray studies of a crystalline complex between dactinomycin and deoxyguanosine permitted formulation of a model that explains the binding of the drug to DNA (Sobell, 1973). The planar phenoxazone ring intercalates between adjacent guanine–cytosine base pairs of DNA, while the polypeptide chains extend along the minor groove of the helix. The summation of these interactions provides great stability to the dactinomycin-DNA complex, and as a result of the binding of dactinomycin, the transcription of DNA by RNA polymerase is blocked. The DNA-dependent RNA polymerases are much more sensitive to the effects of dactinomycin than are the DNA polymerases. In addition, dactinomycin causes single-strand breaks in DNA, possibly through a free-radical intermediate or as a result of the action of topoisomerase II.

Cytotoxic Action. Dactinomycin inhibits rapidly proliferating cells of normal and neoplastic origin and, on a molar basis, is among the most potent antitumor agents known. The drug may produce alopecia and, when extravasated subcutaneously, causes marked local inflammation. Erythema, sometimes progressing to necrosis, has been noted in areas of the skin exposed to X-ray radiation before, during, or after administration of dactinomycin.

Absorption, Fate, and Excretion. Dactinomycin is administered by intravenous injection. Metabolism of the drug is minimal. The drug is excreted in both bile and urine and disappears from plasma with a terminal t_1/2 of 36 hours. Dactinomycin does not cross the blood-brain barrier.

Therapeutic Uses. A wide variety of single-agent and combination chemotherapy regimens with dactinomycin (actinomycin D; cosmegen) are employed. The usual daily dose of dactinomycin is 10-15 μg/kg; this is given intravenously for 5 days; if no manifestations of toxicity are encountered, additional courses may be given at intervals of 2-4 weeks. In other regimens, 3-6 μg/kg/day, for a total of 125 μg/kg, and weekly maintenance doses of 7.5 μg/kg have been used. If infiltrated during administration, the drug is extremely corrosive to soft tissues.

The most important clinical use of dactinomycin is in the treatment of rhabdomyosarcoma and Wilms tumor in children, where it is curative in combination with primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide. Ewing, Kaposi, and soft-tissue sarcomas also respond. Dactinomycin and methotrexate form a curative therapy for choriocarcinoma.

Clinical Toxicities. Toxic manifestations include anorexia, nausea, and vomiting, usually beginning a few hours after administration. Hematopoietic suppression with pancytopenia may occur in the first week after completion of therapy. Proctitis, diarrhea, glossitis, cheilitis, and ulcerations of the oral mucosa are common; dermatological manifestations include alopecia, as well as erythema, desquamation, and increased inflammation and pigmentation in areas previously or concomitantly subjected to X-ray radiation. Severe injury may occur as a result of local drug extravasation.

Chemistry. The anthracycline antibiotics have a tetracyclic ring structure attached to an unusual sugar, daunosamine. Cytotoxic agents of this class all have quinone and hydroquinone moieties on adjacent rings that permit the gain and loss of electrons. Although there are marked differences in the clinical uses of daunorubicin and doxorubicin, their chemical structures differ only by a single hydroxyl group on C-14 (substituent R₄ on the diagram below). Idarubicin is 4-demethoxydaunorubicin (alteration in substituent R₁), a synthetic derivative of daunorubicin; epirubicin is an epimer at the 4′ position of the sugar. Mitoxantrone, an anthracenedione, lacks a glycosidic side group.

Mechanism of Action. A number of important biochemical effects have been described for the anthracyclines and anthracenediones, all of which could contribute to their therapeutic and toxic effects. These compounds can intercalate with DNA, directly affecting transcription and replication. A more important action is the ability to form a tripartite complex with topoisomerase II and DNA. Topoisomerase II is an ATP-dependent enzyme that binds to DNA and produces double-strand breaks at the 3′-phosphate backbone, allowing strand passage and uncoiling of super-coiled DNA. Following strand passage, topoisomerase II re-ligates the DNA strands. This enzymatic function is essential for DNA replication and repair. Formation of the tripartite complex with anthracyclines or with etoposide inhibits the re-ligation of the broken DNA strands, leading to apoptosis. Defects in DNA double-strand break repair sensitize cells to damage by these drugs, while overexpression of transcription-linked DNA repair may contribute to resistance.

Anthracyclines, by virtue of their quinone groups, also generate free radicals in solution and in both normal and malignant tissues (Myers, 1988). Anthracyclines can form semiquinone radical intermediates that can react with O₂ to produce superoxide anion radicals. These can generate both hydrogen peroxide and hydroxyl radicals, which attack DNA (Serrano et al., 1999) and oxidize DNA bases. The production of free radicals is significantly stimulated by the interaction of doxorubicin with iron (Myers, 1988). Enzymatic defenses such as superoxide dismutase and catalase protect cells against the toxicity of the anthracyclines, and these defenses can be augmented by exogenous antioxidants such as alpha tocopherol or by an iron chelator, dexrazoxane (zinecard, others), which protects against cardiac toxicity (Swain et al., 1997).

Exposure of cells to anthracyclines leads to apoptosis; mediators of this process include the p53 DNA-damage sensor and activated caspases (proteases), although ceramide, a lipid breakdown product, and the Fas receptor-ligand system also have been implicated (Friesen et al., 1996).

As discussed in "Drug Resistance" under "Vinca Alkaloids," multidrug resistance is observed in tumor cell populations exposed to anthracyclines. Attempts to reverse or prevent the emergence of resistance through the simultaneous use of inhibitors of the P-glycoprotein (Ca⁺⁺ channel blockers, steroidal compounds, and others) have yielded inconclusive results, primarily due to confounding effects of these inhibitors on anthracycline pharmacokinetics and metabolism. Anthracyclines also are exported from tumor cells by members of the MRP transporter family and by the breast cancer resistance protein, a "half" transporter (Doyle et al., 1998). Other biochemical changes in resistant cells include increased glutathione peroxidase activity, decreased activity or mutation of topoisomerase II, and enhanced ability to repair DNA strand breaks.

Absorption, Fate, and Excretion. Daunorubicin, doxorubicin, epirubicin, and idarubicin usually are administered intravenously and are cleared by a complex pattern of hepatic metabolism and biliary excretion. The plasma disappearance curves for doxorubicin and daunorubicin are multiphasic, with a terminal t_1/2 of 30 hours. All anthracyclines are converted to an active alcohol intermediate that plays a variable role in their therapeutic activity. Idarubicin has a t_1/2 of 15 hours, and its active metabolite, idarubicinol, has a t_1/2 of 40 hours. The drugs rapidly enter the heart, kidneys, lungs, liver, and spleen. They do not cross the blood-brain barrier.

Daunorubicin and doxorubicin are eliminated by metabolic conversion to a variety of aglycones and other inactive products. Idarubicin is primarily metabolized to idarubicinol, which accumulates in plasma and likely contributes significantly to its activity. Clearance of anthracyclines and their active alcohol metabolites is delayed in the presence of hepatic dysfunction, and at least a 50% initial reduction in dose should be considered in patients with abnormal serum bilirubin levels (Twelves et al., 1998).

Therapeutic Use.

Idarubicin. The recommended dosage for idarubicin (idamycin pfs) is 12 mg/m²/day for 3 days by intravenous injection in combination with cytarabine. Slow injection over 10-15 minutes is recommended to avoid extravasation, as with other anthracyclines. It has less cardiotoxicity than the other anthracyclines.

Daunorubicin. Daunorubicin (daunomycin, rubidomycin; cerubidine, others) is available for intravenous use. The recommended dosage is 25-45 mg/m²/day for 3 days. The agent is administered with appropriate care to prevent extravasation, because severe local vesicant action may result. Total doses of >1000 mg/m² are associated with a high risk of cardiotoxicity. Patients should be advised that daunorubicin may impart a red color to the urine.

Daunorubicin and idarubicin also are used in the treatment of AML in combination with Ara-C.

Clinical Toxicities. The toxic manifestations of daunorubicin as well as idarubicin include bone marrow depression, stomatitis, alopecia, GI disturbances, and rash. Cardiac toxicity is a peculiar adverse effect observed with these agents. It is characterized by tachycardia, arrhythmias, dyspnea, hypotension, pericardial effusion, and congestive heart failure poorly responsive to digitalis (see "Clinical Toxicities" under "Doxorubicin").

Doxorubicin.

Therapeutic Uses. Doxorubicin is available for intravenous use. The recommended dose is 60-75 mg/m², administered as a single rapid intravenous infusion that is repeated after 21 days. Care should be taken to avoid extravasation, because severe local vesicant action and tissue necrosis may result. A doxorubicin liposomal product (doxil) is available for treatment of AIDS-related Kaposi sarcoma and is given intravenously in a dose of 20 mg/m² over 60 minutes and repeated every 3 weeks. The liposomal formulation also is approved for ovarian cancer at a dose of 50 mg/m² every 4 weeks and as a treatment for multiple myeloma (in conjunction with bortezomib), where it is given as a 30-mg/m² dose on day 4 of each 21-day cycle. Patients should be advised that the drug may impart a red color to the urine.

Doxorubicin is effective in malignant lymphomas. In combination with cyclophosphamide, vinca alkaloids, and other agents, it is an important ingredient for the successful treatment of lymphomas. It is a valuable component of various regimens of chemotherapy for adjuvant and metastatic carcinoma of the breast. The drug also is particularly beneficial in pediatric and adult sarcomas, including osteogenic, Ewing, and soft-tissue sarcomas.

Clinical Toxicities. The toxic manifestations of doxorubicin are similar to those of daunorubicin. Myelosuppression is a major dose-limiting complication, with leukopenia usually reaching a nadir during the second week of therapy and recovering by the fourth week; thrombocytopenia and anemia follow a similar pattern but usually are less pronounced. Stomatitis, mucositis, diarrhea, and alopecia are common but reversible. Erythematous streaking near the site of infusion ("adriamycin flare") is a benign local allergic reaction and should not be confused with extravasation. Facial flushing, conjunctivitis, and lacrimation may occur rarely. The drug may produce severe local toxicity in irradiated tissues (e.g., the skin, heart, lung, esophagus, and GI mucosa) even when the two therapies are not administered concomitantly.

Cardiomyopathy is the most important long-term toxicity. Two types of cardiomyopathies may occur:

• An acute form is characterized by abnormal electrocardiographic changes, including ST and T-wave alterations and arrhythmias. This is brief and rarely a serious problem. An acute reversible reduction in ejection fraction is observed in some patients in the 24 hours after a single dose, and plasma troponin T, a cardiac enzyme released with myocardial damage, may increase in a minority of patients in the first few days following drug administration (Lipshultz et al., 2004). Acute myocardial damage, the "pericarditis–myocarditis syndrome," may begin in the days following drug infusion and is characterized by severe disturbances in impulse conduction and frank congestive heart failure, often associated with pericardial effusion.

• Chronic, cumulative dose-related toxicity (usually total doses of ≥550 mg/m²) progress to congestive heart failure. The mortality rate in patients with congestive failure approaches 50%. Total doses of doxorubicin as low as 250 mg/m² can cause pathological changes in the myocardium, as demonstrated by subendocardial biopsies. Nonspecific alterations, including a decrease in the number of myocardial fibrils, mitochondrial changes, and cellular degeneration, are visible by electron microscopy. The most promising noninvasive techniques used to detect the early development of drug-induced congestive heart failure are radionuclide cineangiography, which assesses ejection fraction, and echocardiography, which reveals abnormalities in contractility and ventricular dimensions. Sequential echocardiograms have detected structural abnormalities in 25% of children who received up to 300 mg/m² of doxorubicin, although <10% have clinical manifestations of cardiac disease in long-term follow-up. Although no completely practical and reliable predictive tests are available, the frequency of clinically apparent cardiomyopathy is 1-10% at total doses <450 mg/m². The risk increases markedly, with estimates as high as 20% at total doses of 550 mg/m². This total dosage should be exceeded only under exceptional circumstances or with the concomitant use of dexrazoxane, a cardioprotective iron-chelating agent that appears not to compromise the anticancer activity of the drug (Swain et al., 1997). Cardiac irradiation, administration of high doses of cyclophosphamide or another anthracycline, or concomitant trastuzumab (Slamon et al., 2001) increases the risk of cardiotoxicity. Late-onset cardiac toxicity, with congestive heart failure years after treatment, may occur in both pediatric and adult populations. In children treated with anthracyclines, there is a 3- to 10-fold elevated risk of arrhythmias, congestive heart failure, and sudden death in adult life. A total dose limit of 300 mg/m² is advised for pediatric cases. Concomitant administration of dexrazoxane may reduce troponin T elevations and avert later cardiotoxicity (Lipshultz et al., 2004).

Absorption, Fate, and Excretion. Oral administration of etoposide results in variable absorption that averages ~50%. After intravenous injection, peak plasma concentrations of 30 μg/mL are achieved; there is a biphasic pattern of clearance with a terminal t_1/2 of ~6-8 hours in patients with normal renal function. Approximately 40% of an administered dose is excreted intact in the urine. In patients with compromised renal function, dosage should be reduced in proportion to the reduction in CrCl (Arbuck et al., 1986). In patients with advanced liver disease, increased toxicity may result from a low serum albumin (decreased drug binding) and elevated bilirubin (which displaces etoposide from albumin). However, guidelines for dose reduction in this circumstance have not been defined. Drug concentrations in the CSF average 1-10% of those in plasma.

Therapeutic Uses. The intravenous dose of etoposide (vepesid, others) for testicular cancer in combination therapy is 50-100 mg/m² for 5 days, or 100 mg/m² on alternate days for three doses. For small cell carcinoma of the lung, the dosage in combination therapy is 35 mg/m²/day intravenously for 4 days or 50 mg/m²/day intravenously for 5 days. The oral dose for small cell lung cancer is twice the IV dose. Cycles of therapy usually are repeated every 3-4 weeks. When given intravenously, the drug should be administered slowly over a 30- to 60-minute period to avoid hypotension and bronchospasm, which likely result from the additives used to dissolve etoposide, a relatively insoluble compound.

A disturbing complication of etoposide therapy has emerged in long-term follow-up of patients with childhood acute lymphoblastic leukemia, who develop an unusual form of acute nonlymphocytic leukemia with a translocation in chromosome 11q23. At this locus is a gene (the MLL gene) that regulates the proliferation of pluripotent stem cells. The leukemic cells have the cytological appearance of acute monocytic or monomyelocytic leukemia but may express lymphoid surface markers. Another distinguishing feature of etoposide-related leukemia is the short time interval between the end of treatment and the onset of leukemia (1-3 years), compared to the 4- to 5-year interval for secondary leukemias related to alkylating agents, and the absence of a myelodysplastic period preceding leukemia (Pui et al., 1995). Patients receiving weekly or twice-weekly doses of etoposide, with cumulative doses >2000 mg/m², seem to be at higher risk of leukemia.

Etoposide primarily is used for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin and ifosfamide for small cell carcinoma of the lung. It also is active against non-Hodgkin's lymphomas, acute nonlymphocytic leukemia, and Kaposi sarcoma associated with acquired immunodeficiency syndrome (AIDS). Etoposide has a favorable toxicity profile for dose escalation in that its primary acute toxicity is myelosuppression. In combination with ifosfamide and carboplatin, it frequently is used for high-dose chemotherapy in total doses of 1500-2000 mg/m² (Josting et al., 2000).

Clinical Toxicities. The dose-limiting toxicity of etoposide is leukopenia, with a nadir at 10-14 days and recovery by 3 weeks. Thrombocytopenia occurs less often and usually is not severe. Nausea, vomiting, stomatitis, and diarrhea complicate treatment in ~15% of patients. Alopecia is a common but reversible adverse effect. Hepatic toxicity is particularly evident after high-dose treatment. For both etoposide and teniposide, toxicity increases in patients with decreased serum albumin, an effect related to decreased protein binding of the drug.

Teniposide (vumon) is administered intravenously. It has a multiphasic pattern of clearance from plasma; after distribution, a t_1/2 of 4 hours and another t_1/2 of 10-40 hours are observed. Approximately 45% of the drug is excreted in the urine, but in contrast to etoposide, as much as 80% is recovered as metabolites. Anticonvulsants such as phenytoin increase the hepatic metabolism of teniposide and reduce systemic exposure (Baker et al., 1992). Dosage need not be reduced for patients with impaired renal function. Less than 1% of the drug crosses the blood-brain barrier. Teniposide is available for treatment of refractory ALL in children and is synergistic with cytarabine. It is administered by intravenous infusion in dosages that range from 50 mg/m²/day for 5 days to 165 mg/m²/day twice weekly. The drug has limited utility and primarily is given for acute leukemia in children and monocytic leukemia in infants, as well as glioblastoma, neuroblastoma, and brain metastases from small cell carcinomas of the lung. Myelosuppression, nausea, and vomiting are its primary toxic effects.

Chemistry. The bleomycins are water-soluble, basic glycopeptides (Figure 61–14). The core of the bleomycin molecule assumes a metal-binding cage consisting of a pyrimidine chromophore linked to propionamide, a β-aminoalanine amide side chain, and the sugars, l-gulose and 3-O-carbamoyl-d-mannose. Bound in this complex are either Fe²⁺ or Cu²⁺. Attached to the metal ion binding core are a tripeptide chain and a terminal, DNA-binding bithiazole carboxylic acid.

Mechanism of Action. Bleomycin's cytotoxicity results from its ability to cause oxidative damage to the deoxyribose of thymidylate and other nucleotides, leading to single and double-stranded breaks in DNA. Studies in vitro indicate that bleomycin causes accumulation of cells in the G₂ phase of the cell cycle, and many of these cells display chromosomal aberrations, including chromatid breaks, gaps, and fragments, as well as translocations (Twentyman, 1983).

Bleomycin cleaves DNA by generating free radicals. In the presence of O₂ and a reducing agent, such as dithiothreitol, the metal–drug complex becomes activated and functions as a ferrous oxidase, transferring electrons from Fe²⁺ to molecular oxygen to produce oxygen radicals (Burger, 1998). Metallobleomycin complexes can be activated by reaction with the flavin enzyme, NADPH-cytochrome P450 reductase. Bleomycin binds to DNA, and the activated complex generates free radicals that are responsible for abstraction of a proton at the 3′ position of the deoxyribose backbone of the DNA chain, opening the deoxyribose ring and generating a strand break in DNA. The process for repair of this break is poorly understood, but an excess of breaks generates apoptosis.

Bleomycin is degraded by a specific hydrolase found in various normal tissues, including liver. Hydrolase activity is low in skin and lung, perhaps contributing to the serious toxicity at those sites. Some bleomycin-resistant cells contain high levels of hydrolase activity (Sebti et al., 1991). In other cell lines, resistance has been attributed to decreased uptake, repair of strand breaks, or drug inactivation by thiols or thiol-rich proteins. A polymorphism of the hydrolase gene, SNP A1450G, has been identified in 10% of patients with testicular cancer, and the G/G genotype is associated with a 20% decreased survival in patients treated with bleomycin combination therapy, suggesting that this single nucleotide polymorphism is associated with increased hydrolase activity (de Haas et al., 2008).

Absorption, Fate, and Excretion. Bleomycin is administered intravenously, intramuscularly, or subcutaneously or instilled into the bladder for local treatment of bladder cancer. After intravenous infusion, relatively high drug concentrations are detected in the skin and lungs of experimental animals, and these organs become major sites of toxicity. Having a high molecular mass, bleomycin crosses the blood-brain barrier poorly.

After intravenous administration of a bolus dose of 15 mg/m², peak concentrations of 1-5 mg/mL are achieved in plasma. The half-time for elimination is ~3 hours. About two-thirds of the drug are excreted intact in the urine. Concentrations in plasma are greatly elevated if usual doses are given to patients with renal impairment and if such patients are at high risk of developing pulmonary toxicity. Doses of bleomycin should be reduced in the presence of a CrCl <60 mL/min (Dalgleish et al., 1984).

Therapeutic Uses. The recommended dose of bleomycin (blenoxane, others) is 10-20 units/m² given weekly or twice weekly by the intravenous, intramuscular, or subcutaneous route. A test dose of ≤2 units before the first two doses is recommended for lymphoma patients. A variety of regimens are employed clinically, with bleomycin doses expressed in units. In treating testicular cancer, a standard total dose of 30 mg is given weekly for 3 consecutive weeks, and for three to four cycles of treatment. Total courses exceeding 250 mg should be given with caution, and usually only in high-risk testicular cancer treatment, because of a marked increase in the risk of pulmonary toxicity above this total dose. Bleomycin also may be instilled into the pleural cavity in doses of 5-60 mg (depending on the technique) to ablate the pleural space in patients with malignant effusions.

Bleomycin is highly effective against germ cell tumors of the testis and ovary. In testicular cancer, it is curative when used with cisplatin and vinblastine or cisplatin and etoposide. It is a component of the standard curative ABVD regimen (doxorubicin [adriamycin], bleomycin, vinblastine, and dacarbazine) for Hodgkin's lymphoma.

Clinical Toxicities. Because bleomycin causes little myelosuppression, it has significant advantages in combination with other cytotoxic drugs. However, it does cause a constellation of cutaneous toxicities, including hyperpigmentation, hyperkeratosis, erythema, and even ulceration. Rarely, patients with severe skin toxicity may experience Raynaud's phenomenon. Skin changes may begin with tenderness and swelling of the distal digits and progress to erythematous, ulcerating lesions over the elbows, knuckles, and other pressure areas. Healing of these lesions often leaves a residual hyperpigmentation, and lesions may recur when patients are treated with other antineoplastic drugs. Rarely, bleomycin causes a flagellate dermatitis consisting of bands of pruritic erythema on the arms, back, scalp, and hands. This rash responds readily to topical corticosteroids.

The most serious adverse reaction to bleomycin is pulmonary toxicity, which begins with a dry cough, fine rales, and diffuse basilar infiltrates on X-ray and may progress to life-threatening pulmonary fibrosis. Radiological changes of bleomycin-induced lung disease may be indistinguishable from interstitial infection or tumor, and show strong PET-positivity, but may progress from patchy infiltrates to dense fibrosis, cavitation and pneumothorax, atelectasis, or lobar collapse. Approximately 5-10% of patients receiving bleomycin develop clinically apparent pulmonary toxicity, and ~1% die of this complication (O'Sullivan et al., 2003). Most who recover experience a significant improvement in pulmonary function, but fibrosis may be irreversible. Pulmonary function tests are not of predictive value for detecting early onset of this complication. The CO diffusion capacity declines in patients receiving doses >250 mg. The risk of pulmonary toxicity is related to total dose, with a significant increase in risk in total doses >250 mg and in patients >40 years of age, in those with a CrCl of <80 mL/min, and in those with underlying pulmonary disease; single doses of ≥30 mg/m² also are associated with an increased risk of pulmonary toxicity. Administration of high inspired O₂ concentrations during anesthesia or respiratory therapy may aggravate or precipitate pulmonary toxicity in patients previously treated with the drug. There is no known specific therapy for bleomycin lung injury except for symptomatic management and standard pulmonary care. Steroids are of variable benefit, with greatest effectiveness in the earliest inflammatory stages of the lesion.

The etiology of bleomycin pulmonary toxicity has been investigated in rodent models (Moeller et al., 2008). These studies implicate various factors secreted by macrophages, including cytokines (such as transforming growth factor β [TGFβ] and tumor necrosis factor α [TNFα]), and chemokines (such as CCL2 and CXCLI2), as causative factors in leading to fibrosis in response to epithelial damage. Other contributing factors may be disordered coagulation cascades and imbalances in eicosanoids, leading to overproduction of profibrotic leukotrienes and underproduction of antifibrotic prostaglandins. Fibroblasts are recruited to the site of injury by release of lysophosphatide acid from inflammatory cells and contribute to the development of fibrosis (Tager et al., 2008). Various agents (e.g., thalidomide, anti-Her 2 antibodies, PPAR-γ agonists, N-acetylcysteine, anticoagulants, pirfenidone, and bosentan) have attenuated bleomycin toxicity in the animal model, given either before or after the toxic agent. The last four agents are being evaluated in clinical trials for the treatment of idiopathic pulmonary fibrosis (Walter et al., 2006), for which bleomycin lung disease in rodents is the primary disease model.

Other toxic reactions to bleomycin include hyperthermia, headache, nausea and vomiting, and a peculiar acute fulminant reaction observed in patients with lymphomas. This reaction is characterized by profound hyperthermia, hypotension, and sustained cardiorespiratory collapse; it does not appear to be a classical anaphylactic reaction and possibly may be related to release of an endogenous pyrogen. This reaction has occurred in ~1% of patients with lymphomas or testicular cancer.

Mechanism of Action. After intracellular enzymatic or spontaneous chemical reduction of the quinone and loss of the methoxy group, mitomycin becomes a bifunctional or trifunctional alkylating agent. Reduction occurs preferentially in hypoxic cells in some experimental systems. The drug inhibits DNA synthesis and cross-links DNA at the N6 position of adenine and at the O6 and N7 positions of guanine. Attempts to repair DNA lead to strand breaks. Mitomycin is a potent radiosensitizer, teratogen, and carcinogen in rodents. Resistance has been ascribed to deficient activation, intracellular inactivation of the reduced quinone, and P-glycoprotein-mediated drug efflux (Dorr, 1988).

Absorption, Fate, and Excretion. Mitomycin is administered intravenously. It disappears rapidly from the blood after injection, with a t_1/2 of 25-90 minutes. Peak concentrations in plasma are 0.4 μg/mL after doses of 20 mg/m² (Dorr, 1988). The drug distributes widely throughout the body but is not detected in the CNS. Inactivation occurs by hepatic metabolism or chemical conjugation with sulfhydryls. Less than 10% of the active drug is excreted in the urine or the bile.

Therapeutic Uses. Mitomycin (mitomycin-C; mutamycin, others) is administered by intravenous infusion; extravasation may result in severe local injury. The usual dose (6-20 mg/m²) is given as a single bolus every 6-8 weeks. Dosage is modified based on hematological recovery. Mitomycin also may be used by direct instillation into the bladder to treat superficial carcinomas (Boccardo et al., 1994).

Mitomycin is used in combination with 5-FU and cisplatin, for anal cancer. Mitomycin is used off label (in the form of an extemporaneously compounded eye drop) as an adjunct to surgery to inhibit wound healing and reduce scarring; it appears to have benefit in the management of malignant and nonmalignant ophthalmic pathologies (see the review by Abraham, 2006).

Clinical Toxicities. The major toxic effect is myelosuppression, characterized by marked leukopenia and thrombocytopenia; after higher doses, the nadirs may be delayed and cumulative, with recovery only after 6-8 weeks of pancytopenia. Nausea, vomiting, diarrhea, stomatitis, rash, fever, and malaise also are observed. A hemolytic uremic syndrome represents the most dangerous toxic manifestation of mitomycin and is believed to result from drug-induced endothelial damage. Patients who have received >50 mg/m² total dose may acutely develop hemolysis, neurological abnormalities, interstitial pneumonia, and glomerular damage resulting in renal failure. The incidence of renal failure increases to 28% in patients who receive total doses of ≥70 mg/m². There is no effective treatment for the disorder. It must be recognized early, and mitomycin must be discontinued immediately. Mitomycin causes interstitial pulmonary fibrosis, and total doses >30 mg/m² have infrequently led to congestive heart failure. It also may potentiate the cardiotoxicity of doxorubicin when used in combination with this drug.

Absorption, Fate, and Excretion. Approximately 40% of mitotane is absorbed after oral administration. After daily doses of 5-15 g, concentrations of 10-90 μg/mL of unchanged drug and 30-50 μg/mL of a metabolite are present in the blood. After discontinuation of therapy, plasma concentrations of mitotane are still measurable for 6-9 weeks. Although the drug is found in all tissues, fat is the primary site of storage. A water-soluble metabolite of mitotane found in the urine constitutes 25% of an oral or parenteral dose. About 60% of an oral dose is excreted unchanged in the stool.

Therapeutic Uses. Mitotane (lysodren) is administered in initial daily oral doses of 2-6 g, usually in three or four divided portions, and usually increased to 9-10 g/day if tolerated. The maximal tolerated dose may vary from 2-16 g/day. Treatment should continue for at least 3 months; if beneficial effects are observed, therapy should be maintained indefinitely. Spironolactone should not be administered concomitantly, because it interferes with the adrenal suppression produced by mitotane (Wortsman and Soler, 1977).

Treatment with mitotane is indicated for the palliation of inoperable adrenocortical carcinoma, producing symptomatic benefit in 30-50% of such patients.

Clinical Toxicity. Although the administration of mitotane produces anorexia and nausea in most patients, somnolence and lethargy in ~34%, and dermatitis in 15-20%, these effects do not contraindicate the use of the drug at lower doses. Because this drug damages the adrenal cortex, administration of replacement doses of adrenocorticosteroids is necessary (Hogan et al., 1978).

Absorption, Fate, and Excretion. Trabectedin is administered as a 24-hour infusion of 1.3 mg/m² every 3 weeks. Its approval in Europe was based on a trial in soft-tissue sarcoma in which a superior time to progression was found with the longer infusion, as compared to a more convenient 3-hour infusion. It is administered with dexamethasone, 4 mg BID, starting 24 hours before drug infusion to diminish hepatic toxicity. The drug is slowly cleared by CYP3A4, with a plasma t_1/2 of ~24-40 hours.

Therapeutic Uses. Trabectedin is approved outside the U.S. for second-line treatment of soft-tissue sarcomas and for ovarian cancer in combination with a doxorubicin formulation (doxil). It produces a very high (>50%) disease control rate in myxoid liposarcomas, a tumor characterized by a particular genomic translocation, although the reasons for this sensitivity are unclear (Grosso et al., 2009).

Toxicity. Without dexamethasone pretreatment, trabectedin causes significant hepatic enzyme elevations and fatigue in at least one-third of patients. With the steroid, the increases in transaminase are less pronounced and rapidly reverse (Grosso et al., 2009). Other toxicities include mild myelosuppression and, rarely, rhabdomyolysis.

Mechanism of Action. Most normal tissues are able to synthesize l-asparagine in amounts sufficient for protein synthesis, but lymphocytic leukemias lack adequate amounts of asparagine synthetase, and derive the required amino acid from plasma. l-ASP, by catalyzing the hydrolysis of circulating asparagine to aspartic acid and ammonia, deprives these malignant cells of asparagine, leading to cell death. l-ASP is used in combination with other agents, including methotrexate, doxorubicin, vincristine, and prednisone for the treatment of ALL and for high-grade lymphomas.

Resistance arises through induction of asparagine synthetase in tumor cells. For unknown reasons, hyperdiploid ALL cells or those with translocations involving the TEL oncogene are particularly sensitive to l-ASP (Pui et al., 2004), while cells containing the bcr-abl translocation, more common in adult ALL, are more resistant.

Absorption, Fate, Excretion, and Therapeutic Use. l-Asparaginase (elspar), a 144-kDa tetramer, is given intramuscularly or intravenously, but usually by the former route. After intravenous administration, E. coli–derived l-ASP has a clearance rate from plasma of 0.035 mL/min/kg, a volume of distribution that approximates the volume of plasma in humans, and a t_1/2 of 24 hours (Asselin et al., 1993). It is given in doses of 6000-10,000 IU every third day for 3-4 weeks, although doses up to 25,000 IU once per week may be more effective in childhood ALL (Moghrabi et al., 2007). Enzyme levels must be maintained at >0.2 IU/mL in plasma to deplete asparagine in the bloodstream. Pegaspargase (peg-l-asparaginase; oncaspar) a preparation in which the enzyme is conjugated to 5000-Da units of monomethoxy polyethylene glycol, has much slower clearance from plasma (t_1/2 of 6-7 days), and it is administered in doses of 2500 IU/m² intramuscularly no more frequently than every 14 days, producing rapid and complete depletion of plasma and tumor cell asparagine for 21 days in most patients (Appel et al., 2008). Pegaspargase has much reduced immunogenicity (<20% of patients develop antibodies) (Hawkins et al., 2004) and has been approved for first-line ALL therapy.

Intermittent dosage regimens and longer durations of treatment increase the risk of inducing hypersensitivity. In hypersensitive patients, neutralizing antibodies inactivate l-ASP. Not all patients with neutralizing antibodies experience clinical hypersensitivity, although enzyme may be inactivated and therapy may be ineffective. In previously untreated ALL, pegaspargase produces more rapid clearance of lymphoblasts from bone marrow than does the E. coli preparation and circumvents the rapid antibody-mediated clearance seen with E. coli enzyme in relapsed patients (Avramis et al., 2002). Asparaginase preparations only partially deplete CSF asparagine.

Clinical Toxicity. l-ASP toxicities result from its antigenicity as a foreign protein and its inhibition of protein synthesis. Hypersensitivity reactions, including urticaria and full-blown anaphylaxis, occur in 5-20% of patients and may be fatal. These reactions usually are heralded by the earlier appearance of circulating neutralizing antibody and accelerated enzyme clearance from plasma. In these patients, pegaspargase is a safe and effective alternative. So-called "silent" enzyme inactivation by antibodies occurs in a higher percentage of patients than overt hypersensitivity and may be associated with a negative clinical outcome, especially in high-risk ALL patients (Mann et al., 2007).

Other toxicities result from inhibition of protein synthesis in normal tissues (e.g., hyperglycemia due to insulin deficiency, clotting abnormalities due to deficient clotting factors, hypertriglyceridemia due to effects on lipoprotein production, hypoalbuminemia). Pancreatitis may result from extreme triglyceridemia and has been treated by plasma exchange. The clotting problems may take the form of spontaneous thrombosis—more frequent in thrombophilic patients with underlying deficiencies in factor S, factor C, antithrombin III mutation, or factor V Leiden—or, less frequently, hemorrhagic episodes (Caruso et al., 2006). Thrombosis of cortical sinus vessels frequently goes unrecognized. Brain magnetic resonance imaging studies should be considered in patients treated with l-ASP who present with seizures, headache, or altered mental status. Intracranial hemorrhage in the first week of l-ASP treatment is an infrequent but devastating complication. l-ASP suppresses immune function as well.

l-ASP terminates the antitumor activity of methotrexate when given shortly after the antimetabolite. By lowering serum albumin concentrations, it may decrease protein binding and accelerate plasma clearance of other drugs.

Because cells are highly sensitive to irradiation at the G₁–S boundary, HU and irradiation cause synergistic antitumor effects. Through depletion of physiological deoxynucleotides, HU potentiates the antiproliferative effects of DNA-damaging agents such as cisplatin, alkylating agents, or topoisomerase II inhibitors and facilitates the incorporation of antimetabolites such as Ara-C, gemcitabine, and fludarabine into DNA. It also promotes degradation of the p21 cell-cycle checkpoint and thereby enhances the effects of HDAC (histone deacetylase) inhibitors in vitro (Kramer et al., 2008). The role of nitric oxide release in its differentiating activity and in its antitumor effects is uncertain but intriguing (Cokic et al., 2003).

HU has become the primary drug for improving the control of sickle cell (HbS) disease in adults and is also used for inducing fetal hemoglobin (HbF) in thalassemia HbC and HbC/S patients (Brawley et al., 2008). It reduces vaso-occlusive events, painful cries, hospitalizations, and the need for blood transfusions in patients with sickle cell disease. It does so via several potential mechanisms. Increased synthesis of HbF promotes solubility of hemoglobin and prevents sickling. The mechanism of HbF production is uncertain. It may simply result from suppression of erythroid precursor proliferation with compensatory stimulation of a distinct set of fetal Hb-producing cells. Sar1a, a specific promoter that upregulates in response to HU, induces HbF synthesis (Kumkhaek, 2008). Polymorphisms in this promoter may explain differential responses to HU. An alternative mechanism for HbF production has been offered because of the ability of HU to generate nitric oxide both in vitro and in vivo, causing nitrosylation of small-molecular-weight GTPases, a process that stimulate γ-globin production in erythroid precursors. Another property of HU that may be relevant is its ability to reduce L-selectin expression and thereby to inhibit adhesion of red cells and neutrophils to vascular endothelium. Also, by suppressing the production of neutrophils, it decreases their contribution to vascular occlusion.

Tumor cells become resistant to HU through increased synthesis of the hRRM2 subunit of ribonucleoside diphosphate reductase, thus restoring enzyme activity.

Absorption, Fate, and Excretion. The oral bioavailability of HU is excellent (80-100%), and comparable plasma concentrations are seen after oral or intravenous dosing. Peak plasma concentrations are reached 1-1.5 hours after oral doses of 15-80 mg/kg. HU disappears from plasma with a t_1/2 of 3.5-4.5 hours. The drug readily crosses the blood-brain barrier, and it appears in significant quantities in human breast milk. From 40-80% of the drug is recovered in the urine within 12 hours after either intravenous or oral administration. Although precise guidelines are not available, it is advisable to modify initial doses for patients with abnormal renal function until individual tolerance can be assessed. Animal studies suggest that metabolism of HU does occur, but the extent and significance of its metabolism in humans have not been established.

Therapeutic Uses. In cancer treatment, two dosage schedules for HU (hydrea, droxia, others), alone or in combination with other drugs, are most commonly used in a variety of solid tumors: 1) intermittent therapy with 80 mg/kg administered orally as a single dose every third day or 2) continuous therapy with 20-30 mg/kg administered as a single daily dose. In patients with essential thrombocythemia and in sickle cell disease, HU is given in a daily dose of 15 mg/kg, adjusting that dose upward or downward according to blood counts. The neutrophil count responds within 1-2 weeks to discontinuation of the drug. In treating subjects with sickle cell and related diseases, a neutrophil count of at least 2500 cells/mL should be maintained (Platt, 2008). Treatment typically is continued for 6 weeks in malignant diseases to determine its effectiveness; if satisfactory results are obtained, therapy can be continued indefinitely, although leukocyte counts at weekly intervals are advisable.

The principal use of HU has been as a myelosuppressive agent in various myeloproliferative syndromes, particularly CML, polycythemia vera, myeloid metaplasia, and essential thrombocytosis, for controlling high platelet or white cell counts. Many of the myeloproliferative syndromes harbor activating mutations of JAK2, a gene that is downregulated by HU. In essential thrombocythemia, it is the drug of choice for patients with a platelet count >1.5 million cells/mm³ or with a history of arterial or venous thrombosis. In this disease, it dramatically lowers the risk of thrombosis by lowering the platelet, neutrophil, and red cell counts and by reducing expression of L-selectin and increasing nitric oxide production by neutrophils.

In CML, HU has been largely replaced by imatinib. Although it has produced anecdotal, temporary remissions in patients with solid tumors (e.g., head and neck cancers, cervical cancers), HU rarely is used in such patients as a single agent. HU is a potent radiosensitizer as a consequence of its inhibition of RNR (Flanagan et al., 2007) and has been incorporated into several treatment regimens with concurrent irradiation (i.e., cervical carcinoma, primary brain tumors, head and neck cancer, non–small-cell lung cancer).

Clinical Toxicity. Leukopenia, anemia, and occasionally thrombocytopenia—are the major toxic effects; recovery of the bone marrow is prompt if the drug is discontinued for a few days. Other adverse reactions include a desquamative interstitial pneumonitis, GI disturbances, and mild dermatological reactions; more rarely, stomatitis, alopecia, and neurological manifestations have been encountered. Increased skin and fingernail pigmentation may occur, as well as painful leg ulcers, especially in elderly patients or in those with renal dysfunction. HU does not increase the risk of secondary leukemia in patients with myeloproliferative disorders or sickle cell disease. It is a potent teratogen in all animal species tested and should not be used in women with childbearing potential (Platt, 2008).

Clinical Pharmacology. The usual dosing regimen of orally administered ATRA (vesanoid, others) is 45 mg/m²/day until 30 days after remission is achieved (maximum course of therapy is 90 days). ATRA as a single agent reverses the hemorrhagic diathesis associated with APL and induces a high rate of temporary remission. However, clinical trials have clearly established the benefit of giving ATRA in combination with an anthracycline for remission induction, achieving ≥80% relapse-free long-term survival.

ATRA concentrations reach 400 ng/mL in plasma. ATRA is cleared by a CYP3A4-mediated elimination with a t_1/2 of <1 hour. Treatment with inducers of CYP3A4 leads to more rapid drug disappearance and, in some patients, resistance to ATRA (Gallagher, 2002). Inhibitors, such as antifungal imidazoles, block its degradation and may lead to hypercalcemia and renal failure (Cordoba et al., 2008), which responds to diuresis, bisphosphonates, and ATRA discontinuation. Corticosteroids and chemotherapy sharply decrease the occurrence of "retinoic acid syndrome," which is characterized by fever, dyspnea, weight gain, pulmonary infiltrates, and pleural or pericardial effusions. When used as a single agent for remission induction, especially in patients with >5000 leukemic cells/mm³ in the peripheral blood, ATRA induces an outpouring of cytokines and mature-appearing neutrophils of leukemic origin. These cells express high concentrations of integrins and other adhesion molecules on their surface and clog small vessels in the pulmonary circulation, leading to significant morbidity in 15-20% of patients. The syndrome of respiratory distress, pleural and pericardial effusions, and mental status changes may have a fatal outcome. Pretreatment dexamethasone should be given to patients with leukemic cell counts of >5,000/mL to counteract "retinoic acid syndrome".

Toxicity. Retinoids as a class, including ATRA, cause dry skin, cheilitis, reversible hepatic enzyme abnormalities, bone tenderness, pseudotumor cerebri, hypercalcemia, and hyperlipidemia, and as mentioned in the previous paragraph, the retinoic acid syndrome.

The basis for its antitumor activity remains uncertain. APL cells have high levels of reactive oxygen species (ROS) and are quite sensitive to further ROS induction. ATO inhibits thioredoxin reductase and thereby generates ROS. It inactivates glutathione and other sulfhydryls that scavenge ROS and thereby aggravates ROS damage. Cells exposed to ATO also upregulate p53, Jun kinase, and caspases associated with the intrinsic pathway of apoptosis and downregulate anti-apoptotic proteins such as bcl-2. Of particular relevance to APL, it promotes the phosphorylation, sumoylation, and degradation of the APL fusion protein (Lallemand-Breitenbach et al., 2008), as well as the degradation of NF-κB, a transcription factor that stimulates angiogenesis and dampens apoptotic responses in cells with DNA damage. ATO's cytotoxic effects are antagonized by cell survival signals emanating from activation of components of the PI3 kinase cell survival pathway, including Akt kinase, S6 kinase, and mammalian target of rapamycin (mTOR). Inhibition of mTOR by rapamycin enhances its cytotoxic activity in culture systems.

It induces differentiation of leukemic cell lines in vitro, and in both experimental and human leukemias in vivo, but the mechanisms of differentiation and their relationship to the above pharmacological activities are not known.

Clinical Pharmacology. ATO (trisenox) is well absorbed orally, but in cancer treatment is administered as a 2-hour intravenous infusion in dosages of 0.15 mg/kg/day for up to 60 days, until remission is documented. Consolidation therapy begins after a 3-week break. It enters cells via one of several glucose transporters. The primary mechanism of elimination is through enzymatic methylation. Methylated metabolites have uncertain biological effects. Peak steady-state concentrations of arsenic in plasma reached 5-7 μM in one study in adults, while 20-fold lower levels were reported in children using more specific atomic absorption methods (Fox, 2008). Multiple methylated metabolites form rapidly and are excreted in urine. Less than 20% of administered drug is excreted unchanged in the urine. No dose reductions are indicated for hepatic or renal dysfunction.

Toxicity. Pharmacological doses of ATO are well tolerated. Patients may experience reversible side effects, including hyperglycemia, hepatic enzyme elevations, fatigue, dysesthesias, and light-headedness. Ten percent or fewer of patients will experience a leukocyte maturation syndrome similar to that seen with ATRA, including pulmonary distress, effusions, and mental status changes. Oxygen, corticosteroids, and temporary discontinuation of ATO lead to full reversal of this syndrome (Soignet et al., 1998). Another important and potentially dangerous side effect is lengthening of the QT interval on the electrocardiogram in 40% of patients, but rarely do patients develop torsades de pointes, a dangerous form of ventricular tachycardia. Simultaneous treatment with other QT-prolonging drugs, such as macrolide antibiotics, quinidine, or methadone, should be avoided. QT prolongation by ATO results from inhibition of the rapid K⁺ efflux channels in myocardial tissue by As₂O₃. This change leads to slow repolarization of myocardium, and ventricular arrhythmias. Monitoring of serum electrolytes and repletion of serum K⁺ in patients with hypokalemia are precautionary measures in patients receiving ATO therapy. In patients exhibiting a significantly prolonged QT (>470 milliseconds), treatment should be suspended, K⁺ supplemented, and therapy resumed only if the QT returns to normal. Torsades de pointes requires treatment with intravenous magnesium sulfate, K⁺ repletion, and defibrillation if the arrhythmia persists (Gupta et al., 2007) (see Chapter 29).

Mechanism of Action. Vorinostat is a hydroxamic acid modeled after hybrid polar compounds, such as hexamethylene bisacetamide (HMBA); as a class, these compounds cause differentiation of malignant cells in vitro, as do other classes of compounds with HDAC-inhibitory activity, including cyclic tetrapeptides, benzamides, and short-chain aliphatic acids. These compounds bind to a critical Zn⁺⁺ ion in the active site of HDAC enzymes. Vorinostat inhibits the enzymatic activity of HDACs at micromolar concentrations. An important distinction between vorinostat and other HDAC inhibitors is that vorinostat and the hydroxymates are pan-HDAC inhibitors, whereas other compounds have selectivity for HDAC isoenzyme subsets. The biological and clinical implications of this specificity are not clear, and the specific mechanism by which HDAC inhibitors exert their antitumor activity is uncertain. They induce cell-cycle arrest, differentiation, and apoptosis of cancer cells; nonmalignant cells are relatively resistant to these effects. They increase transcription of cell-cycle regulators, affect levels of nuclear transcription factors, and induce pro-apoptotic genes. HDAC inhibition directly blocks function of the chaperone HSP90 and stabilizes the tumor suppressor p53 (Bolden et al., 2006).

Absorption, Fate, and Excretion. Vorinostat is administered as a once-daily oral dose of 400 mg. It is inactivated by glucuronidation of the hydroxyl amine group, followed by hydrolysis of the terminal carboxamide bond and further oxidation of the aliphatic side chain (Figure 61–15). The metabolites are pharmacologically inactive. The terminal t_1/2 of vorinostat in plasma is ~2 hours. Interestingly, histones remain hyperacetylated up to 10 hours after an oral dose of vorinostat, suggesting that its effects persist beyond drug metabolism and elimination.

Therapeutic Uses. Vorinostat is approved for use in refractory cutaneous T-cell lymphoma (CTCL). In patients with refractory CTCL, vorinostat produced an overall response rate of 30%, with a median time to progression of 5 months (Duvic et al., 2007). Vorinostat and other HDAC inhibitors, including romidepsin (depsipeptide; FK228) and MGCD 0103, have shown activity in CTCL, other B and T-cell lymphomas, and myeloid leukemia.

Toxicity. The most common side effects of vorinostat are fatigue, nausea, diarrhea, and thrombocytopenia. Deep venous thrombosis and pulmonary embolism were infrequent but serious adverse events in CTCL patients receiving vorinostat. Most HDAC inhibitors in development cause QTc prolongation, although no serious cardiac toxicity has been reported with vorinostat. A small number of patients receiving infusional depsipeptide romidepsin (see below) and the hydroxamate dacinostat (NVP-LAQ 824) have developed ventricular arrhythmias while on treatment, but the causal relationship to the drugs has not been clearly established, and the cardiac risk may be lower with orally administered and/or less potent HDAC inhibitors (Piekarz et al., 2006). Caution is advised when using HDAC inhibitors in patients with underlying cardiac abnormalities, and careful monitoring of the QTc interval and correction of electrolyte (K⁺, Mg⁺⁺) abnormalities is necessary.

Romidepsin. Romidepsin, a bicyclic polypeptide derived from a soil bacterium, inhibits HDAC at low nanomolar concentrations and is approved for treatment of CTCL and for peripheral T-cell lymphomas. In a Phase II trial it produced complete responses in 4 patients with CTCL, and partial responses in 20, from a total of 71 patients treated. Its primary toxities include GI complaints (nausea vomiting) and transient myelosuppression. Its administration leads to T-wave flattening, but without clear cardiac toxicity (Piekarz et al., 2009).