Section IX - Chemotherapy of Neoplastic Diseases ppsx

The same drugs used for cytotoxic antitumor therapy have become important components of immunosuppressive regimens for rheumatoid arthritis methotrexate and cyclophosphamide, organ trans

Section IX Chemotherapy of Neoplastic Diseases

Introduction

Among the subspecialties of internal medicine, medical oncology may have had the greatest impact

in changing the practice of medicine in the past four decades, as curative treatments have been identified for a number of previously fatal malignancies such as testicular cancer, lymphomas, and leukemia New drugs have entered clinical use for disease presentations previously either

untreatable or amenable to only local means of therapy, such as surgery and irradiation At present, adjuvant chemotherapy routinely follows local treatment of breast cancer, colon cancer, and rectal cancer, and chemotherapy is employed as part of a multimodality approach to the initial treatment

of many other tumors, including locally advanced stages of head and neck, lung, cervical, and esophageal cancer, soft tissue sarcomas, and pediatric solid tumors The basic approaches to cancer treatment are constantly changing Clinical protocols are now exploring genetic therapies,

manipulations of the immune system, stimulation of normal hematopoietic elements, induction of differentiation in tumor tissues, and inhibition of angiogenesis Research in each of these new areas has led to experimental or, in some cases, routine applications for both malignant and nonmalignantdisease The same drugs used for cytotoxic antitumor therapy have become important components

of immunosuppressive regimens for rheumatoid arthritis (methotrexate and cyclophosphamide), organ transplantation (methotrexate and azathioprine), sickle cell anemia (hydroxyurea),

antiinfective chemotherapy (trimetrexate and leucovorin), and psoriasis (methotrexate) Thus, a broad spectrum of medical, surgical, and pediatric specialists employ these drugs for both

neoplastic and nonneoplastic disease

At the same time, few categories of medication in common use have a narrower therapeutic index and a greater potential for causing harmful side effects than do the antineoplastic drugs A thorough understanding of their pharmacology, drug interactions, and clinical pharmacokinetics is essential for safe and effective use in human beings

Traditionally, cancer drugs were discovered through large-scale screening of synthetic chemicals and natural products against animal tumor systems, primarily murine leukemias The agents

discovered in the first two decades of cancer chemotherapy (1950 to 1970) largely interacted with DNA or its precursors, inhibiting the synthesis of new genetic material or causing irreparable damage to DNA itself An overview of such agents is given in Figure IX–1 In recent years, the discovery of new agents has extended from the more conventional natural products such as

paclitaxel and semisynthetic agents such as etoposide, both of which target the proliferative

process, to entirely new fields of investigation that represent the harvest of new knowledge about cancer biology The first successful applications of this knowledge include diverse drugs One

agent, interleukin-2, regulates the proliferation of tumor-killing T lymphocytes and so-called natural

killer cells; this agent has proven able to induce remissions in a fraction of patients with malignant melanoma and renal cell carcinoma, diseases unresponsive to conventional drugs Another agent,

all-trans-retinoic acid, elicits differentiation and can be used to promote remission in acute

promyelocytic leukemia, even after failure of standard chemotherapy The related compound

13-cis-retinoic acid prevents occurrence of second primary tumors in patients with head and neck

cancer Initial success in characterizing unique tumor antigens and oncogenes has introduced new

possible therapeutic opportunities based on an understanding of tumor biology Thus the bcr-abl

translocation in chronic myelocytic leukemia codes for a tyrosine kinase essential to cell

proliferation and survival Inhibition of the kinase by imatinib (STI-571), a new molecularly

targeted drug, has produced a high response rate in chronic-phase patients resistant to standard therapy In a similar, though immunological, approach tumor-associated antigens, such as the her-2/neu receptor in breast cancer cells, have become the target for monoclonal antibody therapy that

has shown activity in patients These examples emphasize that the care of cancer patients is likely toundergo revolutionary changes as entirely new treatment approaches are identified, based on new knowledge of cancer biology (Kaelin, 1999) The diversity of agents useful in treatment of

neoplastic disease is summarized in Table IX–1 The classification used in Chapter 52:

Antineoplastic Agents, which follows, is a convenient framework for describing various types of agents

Figure IX–1 Summary of the Mechanisms and Sites of Action of Chemotherapeutic Agents Useful in Neoplastic Disease PALA =N-phosphonoacetyl-L-aspartate; TMP = thymidine monophosphate

It is unlikely that new therapies will totally replace existing drugs, as these drugs have become increasingly effective and their toxicities have become more manageable and predictable in recent years Improvements in their use are the result of a number of factors, including the following:

1 Drugs now are routinely used earlier in the course of the patient's management, often in conjunction with radiation or surgery, to treat malignancy when it is most curable and whenthe patient is best able to tolerate treatment Thus, adjuvant therapy and neoadjuvant

chemotherapy are used in conjunction with irradiation and surgery in the treatment of head and neck, esophageal, lung, and breast cancer patients

2 The availability of granulocyte colony-stimulating factor (G-CSF; see Chapter 54:

Hematopoietic Agents: Growth Factors, Minerals, and Vitamins) has shortened the period

of leukopenia after high-dose chemotherapy, increasing the safety of bone marrow–ablativeregimens and decreasing the incidence of life-threatening infection A similar

megakaryocyte growth and development factor has been cloned but has not yet achieved a useful place as an adjunct to chemotherapy

3 A greater insight into the mechanisms of tumor cell resistance to chemotherapy has led tothe more rational construction of drug regimens and the earlier use of intensive therapies

Drug-resistant cells may be selected from the larger tumor population by exposure to low-dose, single-agent chemotherapy The resistance that arises may be specific for the selecting agent, such

as the deletion of a necessary activating enzyme (deoxycytidine kinase for cytosine arabinoside), or more general, such as the overexpression of a general drug-efflux pump such as the P-glycoprotein,

a product of the MDR gene This membrane protein is one of several ATP-dependent transporters

that confer resistance to a broad range of natural products used in cancer treatment More recently,

it has become appreciated that mutations underlying malignant transformation, such as the loss of the p53 suppressor oncogene, may lead to drug resistance (A suppressor gene is essential for normal control of cell proliferation; its loss or mutation allows cells to undergo malignant

transformation.) Mutation of p53, or its loss, or the overexpression of the bcl-2 gene that is

translocated in nodular non-Hodgkin's lymphomas, inactivates a key pathway of programmed cell death (apoptosis) and leads to survival of highly mutated tumor cells that have the capacity to survive DNA damage Drug discovery efforts are now directed toward restoring apoptosis in tumor cells, as this process, or its absence, seems to have profound influence on tumor cell sensitivity to drugs Each of these topics concerning drug resistance is covered in greater detail in Chapter 52: Antineoplastic Agents

In designing specific regimens for clinical use, a number of factors must be taken into account Drugs are generally more effective in combination and may be synergistic through biochemical interactions These interactions are useful in designing new regimens It is more effective to use drugs that do not share common mechanisms of resistance and that do not overlap in their major toxicities Drugs should be used as close as possible to their maximum individual doses and should

be given as frequently as possible to discourage tumor regrowth and to maximize dose intensity (thedose given per unit time, a key parameter in the success of chemotherapy) Since the tumor cell population in patients with visible disease exceeds 1 g, or 109 cells, and since each cycle of therapy kills less than 99% of the cells, it is necessary to repeat treatments in multiple cycles to kill all the tumor cells

The Cell Cycle

An understanding of cell-cycle kinetics is essential for the proper use of the current generation of antineoplastic agents Many of the most potent cytotoxic agents act by damaging DNA Their toxicity is greater during the S, or DNA synthetic, phase of the cell cycle, while others, such as the vinca alkaloids and taxanes, block the formation of the mitotic spindle in M phase These agents have activity only against cells that are in the process of division Accordingly, human neoplasms that are currently most susceptible to chemotherapeutic measures are those with a high percentage

of cells undergoing division Similarly, normal tissues that proliferate rapidly (bone marrow, hair follicles, and intestinal epithelium) are subject to damage by most antineoplastic drugs, and such toxicity often limits the usefulness of drugs On the other hand, slowly growing tumors with a smallgrowth fraction (for example, carcinomas of the colon or lung) often are unresponsive to cytotoxic drugs Although differences in the duration of the cell cycle occur between cells of various types, all

cells display a similar pattern during the division process This cell cycle may be characterized as

follows (see Figure IX–2): (1) There is a presynthetic phase (G1); (2) the synthesis of DNA occurs (S); (3) an interval follows the termination of DNA synthesis, the postsynthetic phase (G2); and (4) mitosis (M) ensues—the G2 cell, containing a double complement of DNA, divides into two

daughter G1 cells Each of these cells may immediately reenter the cell cycle or pass into a

nonproliferative stage, referred to as G0 The G0 cells of certain specialized tissues may differentiateinto functional cells that no longer are capable of division On the other hand, many cells, especiallythose in slow-growing tumors, may remain in the G0 state for prolonged periods, only to reenter the division cycle at a later time Damaged cells that reach the G1/S boundary undergo apoptosis, or programmed cell death, if the p53 gene is intact and if it exerts its normal checkpoint function If the p53 gene is mutated and the checkpoint function fails, damaged cells will not be diverted to the apoptotic pathway These cells will proceed through S phase and some will emerge as a drug-resistant population Thus, an understanding of cell-cycle kinetics and the controls of normal and malignant cell growth is crucial to the design of current therapy regimens and the search for new drugs

Figure IX–2 The Cell Cycle and the Relationship of Antitumor Drug Action to the Cycle G1 is the period between mitosis and the beginning of DNA synthesis Resting cells (cells that are not preparing for cell division) are said to be in a subphase of G1, G0 S is the period of DNA synthesis; G2 the premitotic interval; and M the period of mitosis Examples of cell-cycle–dependent anticancer drugs are listed in blue below the phase in which they act Drugs that are cytotoxic for

cells at any point in the cycle are called cycle-phase-nonspecific drugs (Modified

from Pratt et al , 1994 with permission.)

Achieving Therapeutic Balance and Efficacy

While not the subject of this chapter, it must be emphasized that the treatment of most cancer patients requires a skillful interdigitation of multiple modalities of treatment, including surgery, irradiation, and drugs Each of these forms of treatment carries its own risks and benefits It is obvious that not all drugs and not all regimens are safe or appropriate for all patients Numerous factors must be considered, such as renal and hepatic function, bone marrow reserve, and the status

of general performance and accessory medical problems Beyond those considerations, however, are less quantifiable factors such as the likely natural history of the tumor being treated, the patient'swillingness to undergo harsh treatments, the patient's physical and emotional tolerance for side effects, and the likely long-term gains and risks involved

The emphasis in Chapter 52: Antineoplastic Agents is placed upon the drugs, but it is essential to point out the importance of the role played by the patient It is generally agreed that patients in goodnutritional state and without severe metabolic disturbances, infections, or other complications have better tolerance for chemotherapy and have a better chance for significant improvement than do severely debilitated individuals Ideally, the patient should have adequate renal, hepatic, and bone marrow function, the latter uncompromised by tumor invasion, previous chemotherapy, or

irradiation (particularly of the spine or pelvis) Nevertheless, even patients with advanced disease have improved dramatically with chemotherapy Although methods that would enable accurate prediction of the responsiveness of a particular tumor to a given agent are still investigational, in thefuture, molecular studies of tumor specimens may allow prediction of response and the rational

selection of patients for specific drugs Despite efforts to anticipate the development of

complications, anticancer agents have variable pharmacokinetics and toxicity in individual patients The causes of this variability are not always clear and often may be related to interindividual

differences in drug metabolism, drug interactions, or bone marrow reserves In dealing with

toxicity, the physician must provide vigorous supportive care, including, where indicated, platelet

transfusions, antibiotics, and hematopoietic growth factors (see Chapter 54: Hematopoietic Agents:

Growth Factors, Minerals, and Vitamins) Other delayed toxicities affecting the heart, lungs, or kidneys may not be reversible and may lead to permanent organ damage or death Fortunately, such toxicities will be uncommon if the physician adheres to standard protocols and respects the

guidelines for drug usage detailed in the following discussion

Chapter 52 Antineoplastic Agents

Alkylating Agents

History

Although synthesized in 1854, the vesicant properties of sulfur mustard were not described until

1887 During World War I, medical attention was first focused on the vesicant action of sulfur mustard on the skin, eyes, and respiratory tract It was appreciated later, however, that serious systemic toxicity also follows exposure In 1919, Krumbhaar and Krumbhaar made the pertinent observation that the poisoning caused by sulfur mustard is characterized by leukopenia and, in casesthat came to autopsy, by aplasia of the bone marrow, dissolution of lymphoid tissue, and ulceration

of the gastrointestinal tract

In the interval between World Wars I and II, extensive studies of the biological and chemical

actions of the nitrogen mustards were conducted The marked cytotoxic action on lymphoid tissue

prompted Gilman, Goodman, and T.F Dougherty to study the effect of nitrogen mustards on transplanted lymphosarcoma in mice, and in 1942 clinical studies were initiated This launched the era of modern cancer chemotherapy (Gilman, 1963)

In their early phases, all these investigations were conducted under secrecy restrictions imposed by the use of classified chemical-warfare agents At the termination of World War II, however, the nitrogen mustards were declassified; a general review was presented by Gilman and Philips (1946)

A more recent review is provided by Ludlum and Tong (1985)

Thousands of variants of the basic chemical structure of the nitrogen mustards have been prepared, but only a few of these agents have proven more useful than the original compound in specific

clinical circumstances (see below) At present five major types of alkylating agents are used in the

chemotherapy of neoplastic diseases: (1) the nitrogen mustards, (2) the ethylenimines, (3) the alkyl sulfonates, (4) the nitrosoureas, and (5) the triazenes

Chemistry

The chemotherapeutic alkylating agents have in common the property of becoming strong

electrophiles through the formation of carbonium ion intermediates or of transition complexes with the target molecules These reactions result in the formation of covalent linkages by alkylation of various nucleophilic moieties such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and

imidazole groups The chemotherapeutic and cytotoxic effects are directly related to the alkylation

of DNA The 7 nitrogen atom of guanine is particularly susceptible to the formation of a covalent

bond with bifunctional alkylating agents and may well represent the key target that determines their biological effects It must be appreciated, however, that other atoms in the purine and pyrimidine bases of DNA—particularly, the 1 and 3 nitrogens of adenine, the 3 nitrogen of cytosine, and the 6 oxygen of guanine—also may be alkylated, as will be the phosphate atoms of the DNA chains and amino and sulfhydryl groups of proteins.

To illustrate the actions of alkylating agents, possible consequences of the reaction of

mechlorethamine (nitrogen mustard) with guanine residues in DNA chains are shown in Figure 52–

1 First, one 2-chloroethyl side chain undergoes a first-order (SN1) intramolecular cyclization, with release of Cl– and formation of a highly reactive ethyleniminium intermediate (Figure 52– 1A) By

this reaction, the tertiary amine is converted to an unstable quaternary ammonium compound, whichcan react avidly, through formation of a carbonium ion or transition complex intermediate, with a variety of sites that possess high electron density This reaction proceeds as a second-order (SN2) nucleophilic substitution Alkylation of the 7 nitrogen of guanine residues in DNA (Figure 52– 1B),

a highly favored reaction, may exert several effects of considerable biological importance

Normally, 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 7

nitrogen 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 an adenine–thymine base pair for a guanine–cytosine base pair Second, alkylation of the 7 nitrogen labilizes the imidazole ring,

making possible the opening of the imidazole ring or depurination by excision of guanine residues Either of these seriously damages the DNA molecule and must be repaired Third, with bifunctionalalkylating 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 adequately explain both the mutagenic and the cytotoxic effects of alkylating agents However, cytotoxicity of bifunctional alkylators correlates very closely with interstrand cross-linkage of DNA(Garcia et al , 1988).

Figure 52–1 Mechanism of Action of Alkylating Agents

The ultimate cause of cell death related to DNA damage is not known Specific cellular responses

include cell-cycle arrest, DNA repair, and apoptosis, a specific form of nuclear fragmentation termed programmed cell death (Fisher, 1994) The p53 gene product senses DNA damage and

initiates apoptosis in response to DNA alkylation Mutations of p53 lead to alkylating-agent

resistance (Kastan, 1999)

All nitrogen mustards are chemically unstable but vary greatly in their degree of instability

Therefore, the specific chemical properties of each member of this class of drugs must be

considered individually in therapeutic applications For example, mechlorethamine is very unstable,and it reacts almost completely in the body within a few minutes of its administration By contrast, agents such as chlorambucil are sufficiently stable to permit oral administration Cyclophosphamiderequires biochemical activation by the cytochrome P450 system of the liver before its cytotoxicity becomes evident

The ethylenimine derivatives such as chlorambucil and melphalan react by an SN2 reaction; since the opening of the ethylenimine intermediate is acid-catalyzed, they are more reactive at acidic pH.Structure–Activity Relationship

The alkylating agents used in chemotherapy encompass a diverse group of chemicals that have in common the capacity to contribute, under physiological conditions, alkyl groups to biologically vital macromolecules such as DNA In most instances, physical and chemical parameters, such as lipophilicity, capacity to cross biological membranes, acid dissociation constants, stability in

aqueous solution, and sites of macromolecular attack, determine drug activity in vivo With several

of the most valuable agents (e.g., cyclophosphamide and the nitrosoureas), the active alkylating

moieties are generated in vivo after complex metabolic reactions.

The nitrogen mustards may be regarded as nitrogen analogs of sulfur mustard The biological

activity of both types of compounds is based upon the presence of the bis-(2-chloroethyl) grouping

While mechlorethamine has been widely used in the past, various structural modifications have

resulted in compounds with greater selectivity and stability and therefore less toxicity

Bis-(2-chloroethyl) groups have been linked to amino acids (phenylalanine), substituted phenyl groups (aminophenyl butyric acid, as in chlorambucil), pyrimidine bases (uracil), and other chemical entities in an effort to make a more stable and orally available form Although none of these

modifications has produced an agent highly selective for malignant cells, some have unique

pharmacological properties and are more useful clinically than is mechlorethamine Their structuresare shown in Figure 52–2

Figure 52–2 Nitrogen Mustards Employed in Therapy

The addition of substituted phenyl groups has produced a series of relatively stable derivatives that retain the ability to form reactive charged intermediates; the electron-withdrawing capacity of the aromatic ring greatly reduces the rate of cyclization and carbonium ion formation, and these

compounds therefore can reach distant sites in the body before reacting with components of blood and other tissues Chlorambucil and melphalan are the most successful examples of such aromatic mustards These compounds can be administered orally if desired

A classical example of the role of host metabolism in the activation of an alkylating agent is seen with cyclophosphamide—now the most widely used agent of this class The design of this molecule

was based on two considerations First, if a cyclic phosphamide group replaced the N-methyl of

mechlorethamine, the compound might be relatively inert, presumably because the

bis-(2-chloroethyl) group of the molecule could not ionize until the cyclic phosphamide was cleaved at thephosphorus–nitrogen linkage Second, it was hoped that neoplastic tissues might possess high phosphatase or phosphamidase activity capable of accomplishing this cleavage, thus resulting in theselective production of an activated nitrogen mustard in the malignant cells In accord with these predictions, the parent cyclophosphamide displays only weak cytotoxic, mutagenic, or alkylating

activity in vitro and is relatively stable in aqueous solution However, when administered to

experimental animals or patients bearing susceptible tumors, it causes marked chemotherapeutic effects, as well as mutagenicity and carcinogenicity The postulated role for phosphatases or

phosphamidases in the mechanism of action of cyclophosphamide has proven incorrect Rather, the drug undergoes metabolic activation (hydroxylation) by the cytochrome P450 mixed-function oxidase system of the liver (Figure 52–3), with subsequent transport of the activated intermediate tosites of action, as discussed below The selectivity of cyclophosphamide against certain malignant

tissues may result in part from the capacity of normal tissues, such as liver, to protect themselves

against cytotoxicity by further degrading the activated intermediates via aldehyde dehydrogenase

and other pathways

Figure 52–3 Metabolism of Cyclophosphamide

Ifosfamide is an oxazaphosphorine, similar to cyclophosphamide Cyclophosphamide has two

chloroethyl groups on the exocyclic nitrogen atom, whereas one of the two chloroethyl groups of ifosfamide is on the cyclic phosphamide nitrogen of the oxazaphosphorine ring Like

cyclophosphamide, ifosfamide is activated in the liver by hydroxylation However, 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 and the possible differences in antitumor spectrum of the two agents

Although initially considered an antimetabolite, the triazene derivative imidazole-4-carboxamide, usually referred to as dacarbazine or DTIC, functions through alkylation.Its structural formula is shown below:

5-(3,3-dimethyl-1-triazeno)-Dacarbazine requires initial activation by the cytochrome P450 system of the liver through an demethylation reaction In the target cell, spontaneous cleavage of the metabolite yields an

N-alkylating moiety, diazomethane A related triazene, temozolomide undergoes spontaneous

activation, and has significant activity against gliomas and melanoma in human beings (Agarwala and Kirkwood, 2000) It has the same profile of toxicity as DTIC, and is active against malignant gliomas and melanoma Its structure is shown below:

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 alkylating and carbamoylating moieties The structural formula of carmustine is as follows:

The antineoplastic nitrosoureas have in common the capacity to undergo spontaneous,

nonenzymatic degradation with the formation of the 2-chloroethyl carbonium ion (from CNU compounds) This strong electrophile can alkylate a variety of substances; guanine, cytidine, and adenine adducts have been identified (Ludlum, 1990) Displacement of the halogen atom can then lead to interstrand or intrastrand cross-linking of the DNA The formation of the cross-links after the initial alkylation reaction is relatively slow and can be interrupted by the DNA repair enzyme

guanine O6-alkyl transferase (Dolan et al , 1990) The same enzyme, when overexpressed in

gliomas, produces resistance to nitrosoureas and various methylating agents, including DTIC, temozolomide, and procarbazine As with the nitrogen mustards, it is generally agreed that

interstrand cross-linking is associated with the cytotoxicity of nitrosoureas (Hemminki and Ludlum,1984) In addition to the generation of carbonium ions, the spontaneous degradation of BCNU,

CCNU, and methyl-CCNU liberates organic isocyanates that attach carbamoyl groups to lysine residues of proteins, a reaction that apparently can inactivate certain DNA repair enzymes The reactions of the nitrosoureas with macromolecules are shown in Figure 52–4.

Figure 52–4 Degradation of Carmustine (BCNU) with Generation of Alkylating and Carbamoylating Intermediates

Since the formation of the ethyleniminium ion constitutes the initial reaction of the nitrogen

mustards, it is not surprising that stable ethylenimine derivatives have antitumor activity Several

compounds of this type, including triethylenemelamine (TEM) and triethylene thiophosphoramide

(thiotepa), have been used clinically In standard doses, thiotepa produces little toxicity other than myelosuppression and is thus increasingly used for high-dose chemotherapy regimens 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 relationship between the degree of the demethylation and their activity against murinetumors Altretamine requires microsomal activation to display cytotoxicity (Friedman, 2001).Several interesting compounds have emerged from a large group of esters of alkanesulfonic acids One of these, busulfan, is of value in the treatment of chronic granulocytic leukemia and in high-dose chemotherapy; its structural formula is as follows:

Busulfan is a member of a series of symmetrical bis-substituted methanesulfonic acid esters in which the length of a bridge of methylene varies from 2 to 10 The compounds of intermediate

length (n= 4 or 5) possess the highest activities and therapeutic indices Cross-linked guanine residues have been identified in DNA incubated in vitro with busulfan (Tong and Ludlum, 1980).

Pharmacological Actions

The pharmacological actions of the various groups of alkylating agents are considered together in the following discussion Although there are many similarities, some notable differences also are evident

In contrast to many other antineoplastic agents, the effects of the alkylating drugs, although

dependent on proliferation, are not cell-cycle–specific, and the drugs may act on cells at any stage

of the cycle However, the toxicity is usually expressed when the cell enters the S phase and

progression through the cycle is blocked While not strictly cell-cycle–specific, quantitative

differences may be detected when nitrogen mustards are applied to synchronized cells at different phases of the cycle Cells appear more sensitive in late G1 or S than in G2, mitosis, or early G1 Polynucleotides are more susceptible to alkylation in the unpaired state than in the helical form; during replication of DNA, portions of the molecule are unpaired

The actual mechanism(s) of cell death related to DNA alkylation are not well understood There is evidence that, in normal cells of the bone marrow and intestinal epithelium, DNA damage activates

a checkpoint dependent 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 and do not undergo apoptosis (Fisher, 1994)

The great preponderance of evidence indicates that the primary target of pharmacological doses of alkylating agents is DNA, as illustrated in Figure 52–1 A crucial distinction that must be

emphasized is between the bifunctional agents, in which cytotoxic effects predominate, and the monofunctional methylating agents (procarbazine, temozolomide), which, although cytotoxic, 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 chain scission On the other hand, the latter reactions may cause permanent modifications in DNA structure and sequence that are compatible with continued life of the cell and are transmissible to subsequent generations; such modifications may result in mutagenesis or carcinogenesis.

The remarkable DNA repair systems found in most cells likely play an important but as yet poorly defined role in the relative resistance of nonproliferating tissues, the selectivity of action against particular cell types, and acquired resistance to alkylating agents Although alkylation of a single strand of DNA often may be repaired with relative ease, interstrand cross-linkages, such as those produced by the bifunctional alkylating agents, require more complex mechanisms for repair Many

of the cross-links formed in DNA by these agents at low doses also may be corrected; higher doses cause extensive cross-linkage, and DNA breakdown occurs Specific repair enzymes for removing

alkyl groups from the O-6 of guanine (guanine O6-alkyl transferase) and the N-3 of adenine and N-7

of guanine (3-methyladenine-DNA glycosylase) have been identified (Matijasevic et al , 1993) The presence of sufficient levels of guanine O6-alkyl transferase protects cells from cytotoxic effects of nitrosoureas and methylating agents (Pegg, 1990) and confers drug resistance

Detailed information is lacking on mechanisms of cellular uptake of alkylating agents

Mechlorethamine appears to enter murine tumor cells by means of an active transport system, the natural substrate of which is choline Melphalan, an analog of phenylalanine, is taken up by at least two active transport systems that normally react with leucine and other neutral amino acids The highly lipophilic drugs, including nitrosoureas, carmustine, and lomustine, diffuse into cells

passively

Mechanisms of Resistance to Alkylating Agents

Acquired resistance to alkylating agents is a common event, and the acquisition of resistance to one alkylating agent often but not always imparts cross-resistance to others; thus, there are at least theoretical reasons to combine alkylating agents in high-dose therapy While definitive information

on the biochemical mechanisms of clinical resistance is lacking, specific biochemical changes have been implicated in the development of such resistance by tumor cells Among these changes are (1) decreased permeation of actively transported drugs (mechlorethamine and melphalan); (2) increasedproduction of nucleophilic substances, principally thiols such as glutathione, that can conjugate withand detoxify electrophilic intermediates; (3) increased activity of the DNA repair enzymes, such as

the guanine O6-alkyl transferase, that repair nitrosourea-produced alkylation; and (4) increased rates

of metabolism of the activated forms of cyclophosphamide to its inactive keto and carboxy

metabolites by aldehyde dehydrogenase (see Figure 52–3; Tew et al , 2001).

To reverse cellular changes that lead to resistance, strategies have been devised and appear to be effective in selected experimental tumors These include the use of compounds that deplete

glutathione, such as L-buthionine-sulfoximine; sulfhydryl compounds, such as WR-2721, that

selectively detoxify alkylating species in normal cells and thereby prevent toxicity; compounds such

as O6-benzylguanine that inactivate the guanine O6-alkyl transferase DNA repair enzyme; and compounds such as ethacrynic acid that inhibit the enzymes (glutathione transferases) that

conjugate thiols with alkylating agents While each of these modalities has experimental evidence to

support its use, the clinical efficacy has not yet been proven for these strategies Of these, O6benzylguanine has advanced to phase II trials used in conjunction with carmustine (BCNU) or procarbazine against malignant gliomas (Schilsky et al , 2000).

-Toxicities of Alkylating Agents

The alkylating agents differ in their patterns of antitumor activity and in the sites and severity of their side effects Most cause dose-limiting toxicity to bone marrow elements and, to a lesser extent,intestinal mucosa Most alkylating agents, including nitrogen mustard, melphalan, chlorambucil, cyclophosphamide, and ifosfamide, produce an acute myelosuppression, with a nadir of the

peripheral blood granulocyte count at 6 to 10 days and recovery in 14 to 21 days

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 For this reason, it is used as a preparative regimen in allogenic bone marrow transplantation BCNU and other chloroethylnitrosoureas cause delayed and prolonged suppression of both platelets and granulocytes, reaching a nadir 4 to 6 weeks after drug administration and reversing slowly thereafter

Both cellular and humoral immunity are suppressed by alkylating agents, which have been used to treat various autoimmune diseases Immunosuppression is reversible at doses used in most

anticancer protocols

In addition to effects on the hematopoietic system, alkylating agents are highly toxic to dividing mucosal cells, leading to oral mucosal ulceration and intestinal denudation The mucosal effects are particularly significant in high-dose chemotherapy protocols associated with bone marrow

reconstitution, as they predispose to bacterial sepsis arising from the gastrointestinal tract In these protocols, melphalan and thiotepa have the advantage of causing less mucosal damage than the other agents In high-dose protocols, a number of toxicities not seen at conventional doses become dose-limiting They are listed in Table 52–1

While mucosal and bone marrow toxicities occur predictably with conventional doses of these drugs, other organ toxicities, although less common, can be irreversible and at times lethal All alkylating agents have caused pulmonary fibrosis, and in high-dose regimens, endothelial damage that may precipitate venoocclusive disease of the liver; the nitrosoureas, after multiple cycles of therapy, may lead to renal failure; ifosfamide in high-dose regimens frequently causes a central neurotoxicity, with seizures, coma, and at times death; and all such agents are leukemogenic, particularly procarbazine (a methylating agent) and the nitrosoureas Cyclophosphamide and

ifosfamide release a nephrotoxic and urotoxic metabolite, acrolein, which causes a severe

hemorrhagic cystitis, a side effect that in high-dose regimens can be prevented by coadministration

of the sulfhydryl-releasing agent mesna (2-mercaptoethanesulfonate) Mesna, when administered with the offending agent at 60% of the drug dosage, conjugates toxic metabolites in urine

The more unstable alkylating agents (particularly nitrogen mustard and the nitrosoureas) have strong vesicant properties, damage veins with repeated use, and, if extravasated, produce ulceration.Topical application of nitrogen mustard is an effective treatment for cutaneous neoplasms such as mycosis fungoides Most alkylating agents cause alopecia

Central nervous system (CNS) toxicity is manifest in the form of nausea and vomiting, particularly after intravenous administration of nitrogen mustard or BCNU Ifosfamide is the most neurotoxic ofthis class of agents, producing altered mental status, coma, generalized seizures, and paralysis These side effects have been linked to the release of chloroacetaldehyde from the phosphate-linked chloroethyl side chain of ifosfamide High-dose busulfan may cause seizures; in addition, it

accelerates the clearance of phenytoin, an antiseizure medication (see Chapter 21: Drugs Effective

in the Therapy of the Epilepsies)

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 about four years after therapy and may affect up to 5% of patients treated on regimens containing alkylating drugs (Levine and Bloomfield, 1992) Melphalan, the nitrosoureas, and the methylating agent procarbazine have the greatest propensity to cause leukemia, while cyclophosphamide is less potent in this regard.

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

Nitrogen Mustards

The chemistry and the pharmacological actions of the alkylating agents as a group, and of the nitrogen mustards, have been presented above Only the unique pharmacological characteristics of the individual agents are considered below

Mechlorethamine

Mechlorethamine, the first nitrogen mustard to be introduced into clinical medicine, is the most reactive of the drugs in this class

Absorption and Fate

Severe local reactions of exposed tissues necessitate intravenous injection of mechlorethamine for most clinical uses In either water or body fluids, at rates affected markedly by pH,

mechlorethamine rapidly undergoes chemical transformation and combines with either water or nucleophilic molecules of cells, so that the parent drug has an extremely short mean residence time

in the body

Therapeutic Uses

Mechlorethamine HCl (MUSTARGEN) is used primarily in the combination chemotherapy regimen MOPP [mechlorethamine, ONCOVIN (vincristine), procarbazine, and prednisone] in patients with Hodgkin's disease (DeVita et al , 1972) 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 in other regimens by cyclophosphamide, melphalan, and other, more stable, alkylating agents

Clinical Toxicity

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

Like other alkylating agents, nitrogen mustard blocks reproductive function and may produce menstrual irregularities or premature menopause in women and oligospermia in men Since fetal abnormalities can be induced, this drug as well as other alkylating agents should not be used in the first trimester of pregnancy and should be used with caution in later stages of pregnancy Breast-feeding should be terminated before therapy with mechlorethamine is initiated

Local reactions to extravasation of mechlorethamine into the subcutaneous tissue result in a severe, brawny, tender induration that may persist for a long time If the local reaction is unusually severe,

a slough may result If it is obvious that extravasation has occurred, the involved area should be promptly infiltrated with a sterile isotonic solution of sodium thiosulfate (1/6 M); an ice compress then should be applied intermittently for 6 to 12 hours Thiosulfate provides an ion that reacts avidly with the nitrogen mustard and thereby protects tissue constituents

Cyclophosphamide

Pharmacological and Cytotoxic Actions

Although the general cytotoxic action of this drug is similar to that of other alkylating agents, there are notable differences Thrombocytopenia is less severe, while alopecia is marked There are no severe acute or delayed central nervous system (CNS) manifestations either in conventional doses

or in high-dose regimens Nausea and vomiting, however, may occur The drug is not a vesicant, and there is no local irritation

Absorption, Fate, and Excretion

Cyclophosphamide is well absorbed orally As mentioned above, the drug is activated by the

hepatic cytochrome P450 system (see Figure 52–3) Cyclophosphamide is first converted to

4-hydroxycyclophosphamide, which is in a steady state with the acyclic tautomer aldophosphamide

In vitro studies with human liver microsomes and cloned P450 isoenzymes have shown that

cyclophosphamide is activated by the CYP2B group of P450 isoenzymes, while a closely related oxazaphosphorine, ifosfamide, is hydroxylated by the CYP3A system (Chang et al , 1993) This

difference may account for the somewhat different patterns of antitumor activity, the slower

activation of ifosfamide in vivo, and the interpatient variability in toxicity of these two closely

related molecules 4-Hydroxycyclophosphamide may be oxidized further by aldehyde oxidase either in liver or in tumor tissue and perhaps by other enzymes, yielding the metabolites

carboxyphosphamide and 4-ketocyclophosphamide, neither of which possesses significant

biological activity It appears that hepatic damage is minimized by these secondary reactions, whereas significant amounts of the active metabolites, such as 4-hydroxycyclophosphamide and its tautomer, aldophosphamide, are transported to the target sites by the circulatory system In tumor cells, the aldophosphamide cleaves spontaneously, generating stoichiometric amounts of

phosphoramide mustard and acrolein The former is believed to be responsible for antitumor effects.The latter compound may be responsible for the hemorrhagic cystitis seen during therapy with cyclophosphamide Cystitis can be reduced in intensity or prevented by the parenteral

administration of mesna (MESNEX), a sulfhydryl compound that reacts readily with acrolein in the acid environment of the urinary tract (Tew et al , 2001).

Pretreatment with P450 inducers such as phenobarbital enhances the rate of drug activation but doesnot alter toxicity or therapeutic activity in human beings

Urinary and fecal recovery of unchanged cyclophosphamide is minimal after intravenous

administration Maximal concentrations in plasma are achieved 1 hour after oral administration, andthe half-life in plasma is about 7 hours

Therapeutic Uses

Cyclophosphamide (CYTOXAN, NEOSAR) is administered orally or intravenously Recommended

doses vary widely, and published protocols for the dosage of cyclophosphamide and other

chemotherapeutic agents and for the method and sequence of administration should be consulted

As a single agent, a daily dose of 100 mg/m2 orally for 14 days has been recommended for patients with more susceptible neoplasms, such as lymphomas and chronic leukemias A higher dosage of

500 mg/m2 intravenously every 3 to 4 weeks in combination with other drugs often is employed in the treatment of breast cancer and lymphomas The leukocyte count generally serves as a guide to dosage adjustments in prolonged therapy An absolute neutrophil count between 500 and 1000 cells per cubic millimeter is recommended as the desired target In regimens associated with bone

marrow or peripheral stem cell rescue, cyclophosphamide may be given in doses of 5 to 7 g/m2 over

a 3-day period Gastrointestinal ulceration, cystitis (counteracted by mesna and diuresis), and, less commonly, pulmonary, renal, hepatic, and cardiac toxicities may occur after high-dose therapy

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 Complete remissions and presumed cures have been reported when cyclophosphamide was given as a single agent for

Burkitt's lymphoma It is frequently used in combination with methotrexate (or doxorubicin) and fluorouracil as adjuvant therapy after surgery for carcinoma of the breast

Notable advantages of this drug are the availability of the oral route of administration and the possibility of giving fractionated doses over prolonged periods For these reasons it possesses a versatility of action that allows an intermediate range of use, between that of the highly reactive intravenous mechlorethamine and that of oral chlorambucil Beneficial results have been obtained

in multiple myeloma; chronic lymphocytic leukemia; carcinomas of the lung, breast, cervix, and ovary; and neuroblastoma, retinoblastoma, and other neoplasms of childhood

Because of its potent immunosuppressive properties, cyclophosphamide has received considerable attention for the control of organ rejection after transplantation and in nonneoplastic disorders associated with altered immune reactivity, including Wegener's granulomatosis, rheumatoid

arthritis, and the nephrotic syndrome in children Caution is advised when the drug is considered foruse in these conditions, not only because of its acute toxic effects but also because of its potential for inducing sterility, teratogenic effects, and leukemia

inappropriate secretion of antidiuretic hormone (ADH) has been observed in patients receiving cyclophosphamide, usually at doses higher than 50 mg/kg (DeFronzo et al , 1973) It is important to

be aware of the possibility of water intoxication, since these patients usually are vigorously

hydrated

Ifosfamide

Ifosfamide, an analog of cyclophosphamide, also is activated by ring hydroxylation in the liver Severe urinary tract toxicity limited the use of ifosfamide when it was first introduced in the early 1970s However, adequate hydration and coadministration of mesna now permit effective use of ifosfamide.

Therapeutic Uses

Ifosfamide currently is approved for use in combination with other drugs for germ cell testicular cancer and is widely used to treat pediatric and adult sarcomas Clinical trials also have shown ifosfamide to be active against carcinomas of the cervix and lung and against lymphomas 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 to 14 g/m2, it may cause severe neurological toxicity, including coma and death This toxicity is thought to result from a metabolite, chloracetaldehyde (Colvin, 1982) In addition to hemorrhagic cystitis, ifosfamide causes nausea, vomiting, anorexia,

leukopenia, nephrotoxicity, and CNS disturbances (especially somnolence or confusion) (see Brade

et al , 1987).

Ifosfamide (IFEX) is infused intravenously over at least 30 minutes at a dose of 1.2 g/m2 per day for

5 days Intravenous mesna is given as bolus injections in a dosage equal to 20% of the ifosfamide dosage concomitantly and again 4 and 8 hours later, for a total mesna dose of 60% of the ifosfamidedose Alternatively, mesna may be given in a single dose equal to the ifosfamide dose

concomitantly Patients also should receive at least 2 liters of oral or intravenous fluid daily

Treatment cycles are usually repeated every 3 to 4 weeks

Pharmacological and Cytotoxic Actions

The general pharmacological and cytotoxic actions of melphalan, the phenylalanine derivative of nitrogen mustard, are similar to those of other nitrogen mustards The drug is not a vesicant

Absorption, Fate, and Excretion

When given orally, melphalan is absorbed in an incomplete and variable manner, and 20% to 50%

of the drug is recovered in the stool The drug has a half-life in plasma of approximately 45 to 90 minutes, and 10% to 15% of an administered dose is excreted unchanged in the urine (Alberts et al ,

1979b)

Therapeutic Uses

The usual oral melphalan (ALKERAN) dose for multiple myeloma is 6 mg daily for a period of 2 to 3weeks, during which time the blood count should be carefully observed A rest period of up to 4 weeks should then intervene When the leukocyte and platelet counts are rising, maintenance therapy, ordinarily 2 to 4 mg daily, is begun It usually is necessary to maintain a significant degree

of bone marrow depression (total leukocyte count in the range of 2500 to 3500 cells per cubic millimeter) in order to achieve optimal results The usual intravenous dose is 16 mg/m2 infused over

15 to 20 minutes Doses are repeated at 2-week intervals for four doses and then at 4-week intervalsbased on response and tolerance Dosage adjustments should be considered based on blood cell counts and in patients with renal impairment

Although the general spectrum of action of melphalan seems to resemble that of other nitrogen mustards, the advantages of administration by the oral route have made the drug useful in the treatment of multiple myeloma

Pharmacological and Cytotoxic Actions

The cytotoxic effects of chlorambucil on the bone marrow, lymphoid organs, and epithelial tissues are similar to those observed with the nitrogen mustards Although CNS side effects can occur, these have been observed only with large doses Nausea and vomiting may result from single oral doses of 20 mg or more

Absorption, Fate, and Excretion

Oral absorption of chlorambucil is adequate and reliable The drug has a half-life in plasma of approximately 1.5 hours, and it is almost completely metabolized (Alberts et al , 1979a).

Therapeutic Uses

The standard initial daily dosage of chlorambucil (LEUKERAN) is 0.1 to 0.2 mg/kg, continued for at least 3 to 6 weeks The total daily dose, usually 4 to 10 mg, is given at one time With a fall in the peripheral total leukocyte count or clinical improvement, the dosage is reduced; maintenance therapy (usually 2 mg daily) is feasible and may be required, depending on the nature of the disease.Other dosage schedules also are used

At the recommended dosages, chlorambucil is the slowest-acting nitrogen mustard in clinical use It

is a standard agent for patients with chronic lymphocytic leukemia and primary (Waldenström's) macroglobulinemia

Clinical Toxicity

In chronic lymphocytic leukemia, chlorambucil may be given orally for months or years, achieving its effects gradually and often without toxicity to a precariously compromised bone marrow

Clinical improvement comparable to that with melphalan or cyclophosphamide has been observed

in some patients with plasma cell myeloma Beneficial results also have been reported in disorders with altered immune reactivity, such as vasculitis associated with rheumatoid arthritis and

autoimmune hemolytic anemia with cold agglutinins

Although it is possible to induce marked hypoplasia of the bone marrow with excessive doses of chlorambucil administered over long periods, its myelosuppressive action is usually moderate, gradual, and rapidly reversible Gastrointestinal discomfort, azoospermia, amenorrhea, pulmonary fibrosis, seizures, dermatitis, and hepatotoxicity may be rarely encountered A marked increase in the incidence of leukemia and other tumors has been noted in a large controlled study of its use for the treatment of polycythemia vera by the National Polycythemia Vera Study Group, as well as in patients with breast cancer receiving long-term adjuvant chemotherapy (Lerner, 1978)

Ethylenimines and Methylmelamines

Triethylenemelamine (TEM), Thiotepa (Triethylene Thiophosphoramide), and Altretamine

(Hexamethylmelamine; HMM)

Pharmacological and Cytotoxic Effects

Although nitrogen mustards have largely replaced ethylenimines in general clinical practice, this class of agents continues to have specific use Thiotepa (THIOPLEX) is active as an intravesicular agent in bladder cancer and is used as a component of experimental high-dose chemotherapy regimens (Kletzel et al , 1992), and altretamine (HEXALEN), formerly known as

hexamethylmelamine, is used in patients with advanced ovarian cancer after failure of first-line

therapies

Both thiotepa and its primary metabolite, triethylenephosphoramide (TEPA), to which it is rapidly converted by hepatic mixed-function oxygenases (Ng and Waxman, 1991), are capable of forming DNA cross-links The aziridine rings open after protonation of the ring-nitrogen, leading to a reactive molecule

Absorption, Fate, and Excretion

TEPA becomes the predominant form of the drug present in plasma within 5 minutes of thiotepa administration The parent compound has a plasma half-life of 1.2 to 2 hours, as compared to a half-life of 3 to 24 hours for TEPA Thiotepa pharmacokinetics are essentially the same in children as in adults at conventional doses (up to 80 mg/m2), and drug and metabolite half-lives are unchanged in children receiving high-dose therapy of 300 mg/m2 per day for 3 days (Kletzel et al , 1992) Less

than 10% of the administered drug appears in urine as the parent drug or the primary metabolite The remainder is metabolized, interacts with biological molecules, or undergoes spontaneous chemical degradation

Clinical Toxicities

The toxicities of thiotepa are essentially 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.Alkyl Sulfonates

Pharmacological and Cytotoxic Actions

Busulfan is unique in that, in conventional doses, it exerts few pharmacological actions other than myelosuppression At low doses, selective depression of granulocytopoiesis is evident, leading to itsprimary use in the chronic phase of chronic myelogenous leukemia (CML) However, platelets and erythroid elements also may be suppressed as the dosage is raised, and in some patients a severe andprolonged pancytopenia results In low doses, cytotoxic action does not appear to extend to either the lymphoid tissues or the gastrointestinal epithelium In high-dose regimens, new toxicities, including pulmonary fibrosis and venoocclusive disease of the liver, become apparent

Absorption, Fate, and Excretion

Busulfan is well absorbed after oral administration in doses of 2 to 6 mg/day, and it disappears fromthe blood with a half-life of 2 to 3 hours Almost all of the drug is excreted in the urine as

methanesulfonic acid In high doses, children under 18 years of age clear the drug faster than do adults, and tolerate higher doses (Vassal et al , 1993).

Therapeutic Uses

In treating chronic granulocytic leukemia, the initial oral dose of busulfan (MYLERAN, BUSULFEX) varies with the total leukocyte count and the severity of the disease; daily doses from 2 to 8 mg are recommended to initiate therapy and are adjusted appropriately to subsequent hematological and clinical responses, with the aim of reduction of the total leukocyte count to 10,000 cells per cubic millimeter Maintenance doses of 1 to 3 mg may be given daily

The beneficial effects of busulfan in chronic granulocytic leukemia are well established, and clinicalremissions may be expected in 85% to 90% of patients after the initial course of therapy, but the drug has largely been replaced by interferon-alfa and hydroxyurea

In CML, reduction of the leukocyte count is noted during the second or third week, and regression

of splenomegaly follows Beneficial results have been reported in other myeloproliferative

disorders, including polycythemia vera and myelofibrosis with myeloid metaplasia High doses of busulfan (640 mg/m2) have been used effectively in combination with high doses of

cyclophosphamide to prepare patients with acute myelogenous leukemia for bone marrow

transplantation (Santos et al , 1983) High-dose regimens are given in multiple doses over 3 to 4

days to reduce the incidence of acute CNS toxicities, including tonic-clonic seizures, which may occur several hours after each dose As mentioned earlier, busulfan induces the metabolism of phenytoin

Clinical Toxicity

The major toxic effects of busulfan are related to its myelosuppressive properties, and prolonged thrombocytopenia may be a hazard Occasional instances of nausea, vomiting, diarrhea, impotence, sterility, amenorrhea, and fetal malformation have been reported The drug is leukemogenic In the initial phase of chronic granulocytic leukemia treatment, hyperuricemia, resulting from extensive purine catabolism accompanying the rapid cellular destruction, and renal damage from precipitation

of urates have been noted The concurrent use of allopurinol is recommended to avoid this

complication A number of unusual complications have been observed in patients receiving

busulfan, but their relation to the drug is poorly understood; these include a syndrome resembling Addison's disease (but without steroid deficiency), cataracts, gynecomastia, cheilosis, glossitis, anhidrosis, and pulmonary fibrosis (Tew et al , 2001).

Nitrosoureas

The nitrosoureas have an important role in the treatment of brain tumors and gastrointestinal

neoplasms They appear to function as bifunctional alkylating agents but differ in both

pharmacological and toxicological properties from conventional nitrogen mustards Carmustine (BCNU) and lomustine (CCNU) have attracted special interest because of their high lipophilicity and, thus, their capacity to cross the blood–brain barrier, an important property in the treatment of brain tumors Unfortunately, with the exception of streptozocin, the nitrosoureas used in the clinic

to date cause profound, cumulative myelosuppression that restricts their therapeutic value In addition, 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

Streptozocin, originally discovered as an antibiotic, is of special interest This compound has a methylnitrosourea (MNU) moiety attached to the 2 carbon of glucose It has a high affinity for cells of the islets of Langerhans and causes diabetes in experimental animals Streptozocin is useful

in the treatment of human pancreatic islet cell carcinoma and malignant carcinoid tumors

Unmodified MNU, the active moiety of streptozocin, is cytotoxic to selected human tumors and produces delayed myelosuppression Furthermore, MNU is particularly prone to cause

carbamoylation of lysine residues of proteins (see Figure 52–4) Unlike MNU, streptozocin is not

myelosuppressive and displays little carbamoylating activity Thus, the nitrosourea-type moiety has been attached to various carrier molecules, with alterations in crucial properties such as tissue

specificity, distribution, and toxicity Chlorozotocin, an agent in which the 2 carbon of glucose is

substituted by the chloronitrosourea group (CNU), is not diabetogenic and, unlike many other nitrosoureas, causes little myelosuppression or carbamoylation However, it has no clear therapeuticadvantage over the other members of its class

Carmustine (BCNU)

Pharmacological and Cytotoxic Actions

Carmustine's major action is its alkylation of DNA at the O6-guanine position It kills cells in all phases of the cell cycle This drug characteristically causes an unusually delayed myelosuppression,with a nadir of the leukocyte and platelet counts at 4 to 6 weeks In high doses with bone marrow rescue, it produces hepatic venoocclusive disease, pulmonary fibrosis, renal failure, and secondary leukemia (Tew et al , 2001).

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 half-life of from 15 to 90 minutes or longer (see

Levin et al , 1978) Approximately 30% to 80% of the drug appears in the urine within 24 hours as

degradation products The entry of alkylating metabolites into the cerebrospinal fluid (CSF) is rapid, and their concentrations in the CSF are 15% to 30% of the concurrent plasma values

(Oliverio, 1976)

Therapeutic Uses

Carmustine (BICNU) usually is administered intravenously at doses of 150 to 200 mg/m2, given by infusion over 1 to 2 hours, and it is not repeated for 6 weeks When used in combination with other chemotherapeutic agents, the dose is usually reduced by 25% to 50%

The spectrum of activity of carmustine is similar to that of other alkylating agents, with significant responses observed in Hodgkin's disease and a lower response rate in other lymphomas and

myeloma Because of its ability to cross the blood–brain barrier, carmustine is used as a component

of multimodality treatment of malignant astrocytomas and metastatic tumors of the brain Beneficialresponses have been reported in patients with melanoma and gastrointestinal tumors

Streptozocin

This naturally occurring nitrosourea is an antibiotic derived from Streptomyces acromogenes It has

been particularly useful in treating functional, malignant pancreatic islet cell tumors It affects cells

in all stages of the mammalian cell cycle

Absorption, Fate, and Excretion

Streptozocin is administered parenterally After intravenous infusions of 200 to 1600 mg/m2, peak concentrations in the plasma are 30 to 40 g/ml; the half-life of the drug is approximately 15 minutes Only 10% to 20% of a dose is recovered in the urine (Schein et al , 1973).

Therapeutic Uses

Streptozocin (ZANOSAR) 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 can then be increased to a maximum of 1500 mg/m2

Streptozocin has been used primarily in patients with metastatic pancreatic islet cell carcinoma, and beneficial responses are translated into a significant increase in 1-year survival rate and a doubling

of median survival time for the responders

Clinical Toxicity

Nausea is a frequent side effect Renal or hepatic toxicity occurs in approximately two-thirds of cases; although usually reversible, renal toxicity is dose-related and cumulative and may be fatal, and proximal tubular damage is the most important toxic effect Serial determinations of urinary protein are most valuable in detecting early renal effects Streptozocin should not be given with other nephrotoxic drugs Hematological toxicity—anemia, leukopenia, or thrombocytopenia—occurs in 20% of patients

Triazenes

Dacarbazine (DTIC)

Dacarbazine functions as a methylating agent after metabolic activation in the liver Its active metabolite is a monomethyl triazino derivative, the same metabolite formed spontaneously by its analog, temozolomide It kills cells in all phases of the cell cycle Dacarbazine resistance has been

ascribed to the repair of methylated guanine bases in DNA by guanine O6-alkyl transferase.

Absorption, Fate, and Excretion

Dacarbazine is administered intravenously; after an initial rapid phase of disappearance (t1/2 of about

20 minutes), the drug is removed from plasma with a terminal half-life of about 5 hours (Loo et al ,

1976) The half-life is prolonged in the presence of hepatic or renal disease Almost one-half of the compound is excreted intact in the urine by tubular secretion Elevated urinary concentrations of 5-aminoimidazole-4-carboxamide (AIC) are derived from the catabolism of dacarbazine, rather than

by inhibition of de novo purine biosynthesis Concentrations of dacarbazine in CSF are

approximately 14% of those in plasma (Friedman, 2001)

Therapeutic Uses

Dacarbazine (DTIC -DOME) is administered intravenously The recommended regimen for malignantmelanoma is to give 3.5 mg/kg per day, intravenously, for a 10-day period; this is repeated every 28days 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

At present, dacarbazine is employed in combination regimens for the treatment of malignant

melanoma, Hodgkin's disease, and adult sarcomas Temozolomide (TEMODAL), the spontaneously activated analog, has shown activity in patients with malignant gliomas (Newlands et al , 1992;

Agarwala and Kirkwood, 2000)

solid tumor, choriocarcinoma (Hertz, 1963) The consistent cure of choriocarcinoma by

methotrexate provided great impetus to investigations into the chemotherapy of cancer Interest in folate antagonists further increased with the introduction of high-dose regimens with "rescue" of host toxicity by the reduced folate, leucovorin (folinic acid, citrovorum factor) These methods extend the usefulness of methotrexate to tumors such as osteogenic sarcoma that do not respond to lower doses

Recognition that methotrexate, an inhibitor of dihydrofolate reductase, also directly inhibits the

folate-dependent enzymes of de novo purine and thymidylate synthesis focused attention on the

development of antifolate analogs that specifically target these other folate-dependent enzyme

targets of methotrexate (see Figure 52–5) Replacement of the 5, 8, and/or 10 nitrogens of the

pteridine ring of folate, as well as various side-chain substitutions, has generated a series of new inhibitors that preserve the common folate potential to form long-lived, intracellular

polyglutamates These new agents, however, have greater capacity for transport into tumor cells (Messmann and Allegra, 2001), and exert their primary inhibitory effect on thymidylate synthesis (raltitrexed, TOMUDEX), purine biosynthesis (lometrexol) or both [the multitargeted antifolate

permefrexed (MTA)] (Calvete et al , 1994; Beardsley et al , 1986; Chen et al , 1999).

Figure 52–5 Sites of Action of Methotrexate and Its Polyglutamates AICAR, aminoimidazole carboxamide; TMP, thymidine monophosphate; dUMP, deoxyuridine monophosphate; FH2Glun, dihydrofolate polyglutamate; FH4Glun, tetrahydrofolate polyglutamate; GAR, glycinamide ribonucleotide; IMP, inosine monophosphate; PRPP, 5-phosphoribosyl-1-pyrophosphate

Aside from its antineoplastic activity, methotrexate also has been used with benefit in the therapy ofthe common skin disease psoriasis (McDonald, 1981; see Chapter 65: Dermatological

Pharmacology) Additionally, methotrexate inhibits cell-mediated immune reactions and is

employed as an immunosuppressive agent, for example, in allogeneic bone marrow and organ transplantation and for the treatment of dermatomyositis, rheumatoid arthritis, Wegener's

granulomatosis, and Crohn's disease (Messmann and Allegra, 2001; Feagan et al , 1995; see

Chapter 53: Immunomodulators: Immunosuppressive Agents, Tolerogens, and Immunostimulants).Structure–Activity Relationship

Folic acid is an essential dietary factor from which is derived a series of tetrahydrofolate cofactors that provide single carbon groups for the synthesis of precursors of DNA (thymidylate and purines) and RNA (purines) A detailed description of the biological functions and therapeutic applications

of folic acid appears in Chapter 54: Hematopoietic Agents: Growth Factors, Minerals, and

Vitamins

The enzyme dihydrofolate reductase (DHFR) is the primary site of action of most folate analogs

studied to date (see Figures 52–5 and 52–6) Inhibition of DHFR leads to toxic effects through

partial depletion of the tetrahydrofolate cofactors that are required for the synthesis of purines and thymidylate (Messmann and Allegra, 2001) and through direct inhibition of the folate-dependent enzymes of purine and thymidylate metabolism by the polyglutamates of methotrexate and the dihydrofolate polyglutamates that accumulate with DHFR inhibition (Figure 52–5) (Allegra et al ,

1986, 1987b) Inhibitors of DHFR differ in their relative potency for blocking enzyme 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

44: Antimicrobial Agents: Sulfonamides, Trimethoprim-Sulfamethoxazole, Quinolones, and Agentsfor Urinary Tract Infections; pyrimethamine, Chapter 40: Drugs Used in the Chemotherapy of Protozoal Infections: Malaria) By contrast, methotrexate is an effective inhibitor of DHFR in all species investigated Crystallographic studies have revealed the atomic basis for the high affinity of methotrexate for DHFR (Kraut and Matthews, 1987; Schweitzer et al , 1989; Bystroff and Kraut,

1991; Blakley and Sorrentino, 1998) and the species specificity of the various DHFR inhibitors (Matthews et al , 1985; Stone and Morrison, 1986).

Figure 52–6 The Structure–Activity Bases for Antifolate Action

Because folic acid and many of its analogs are very polar, they cross the blood–brain barrier poorly and require specific transport mechanisms to enter mammalian cells (Elwood, 1989; Dixon et al ,

1994) Two inward folate transport systems are found on mammalian cells: (1) a folate receptor, which has high affinity for folic acid but lesser ability to transport methotrexate and other analogs (Elwood, 1989); and (2) the reduced folate transporter, the major transit protein for methotrexate, raltitrexed, and most analogs (Westerhof et al , 1995) Once in the cell, additional glutamyl residues

are added to the molecule by the enzyme folylpolyglutamate synthetase (Cichowicz and Shane, 1987) Intracellular methotrexate polyglutamates have been identified with up to six glutamyl residues Since these higher polyglutamates cross cellular membranes poorly, if at all, this serves as

a mechanism of entrapment and may account for the prolonged retention of methotrexate in tumors and normal tissues 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, but not for DHFR

Novel folate antagonists have been identified that exploit differences between the folate influx

system in certain tumors and that in normal tissues (e.g., bone marrow) The analog deaza aminopterin (edatrexate) is transported into some tumor cells much more efficiently than into

10-ethyl,10-normal tissues and is an excellent inhibitor of DHFR This promising compound is undergoing clinical evaluation (Grant et al , 1993) In efforts to bypass the obligatory membrane transport

system and facilitate penetration of the blood–brain barrier, lipid-soluble folate antagonists also have been synthesized Trimetrexate (Figure 52–6) was one of the first to be tested for clinical activity The analog was found to have modest antitumor activity, primarily in combination with leucovorin (5-formyl tetrahydrofolate) rescue However, it has proven to be beneficial in the

treatment of Pneumocystis carinii pneumonia (Allegra et al , 1987a).

The other important new folate analog, MTA or pemetrexed (ALTIMA) (Figure 52–6), is a

tetrahydrofolate analog It readily converts to polyglutamates that inhibit thymidylate and purine biosynthesis, as well as dihydrofolate reductase In early trials, it has shown activity against colon cancer, mesothelioma, and non-small cell lung cancer (Rusthoven et al , 1999).

To function again as a cofactor, FH2 must be reduced to FH4 by DHFR Inhibitors, such as

methotrexate, with a high affinity for DHFR (Ki 0.01 to 0.2 nM) prevent the formation of FH4, producing an acute intracellular deficiency of certain folate coenzymes and a vast accumulation of the toxic inhibitory substrate, FH2 polyglutamate The one-carbon transfer reactions crucial for the

de novo synthesis of purine nucleotides and thymidylate cease, with the subsequent interruption of

the synthesis of DNA and RNA (as well as other vital metabolic reactions) The toxic effects of methotrexate may be terminated by administering leucovorin (N5-formyl FH4; folinic acid)

Leucovorin, a fully reduced folate coenzyme, enters cells via a specific carrier-mediated transport

system and is converted to other active folate cofactors (Boarman et al , 1990).

As with most antimetabolites, methotrexate is only partially selective for tumor cells and is toxic to all rapidly dividing normal cells, such as those of the intestinal epithelium and bone marrow Folate antagonists kill cells during the S phase of the cell cycle and are most effective when cells are in thelogarithmic phase of growth

Mechanisms of Resistance to Antifolates

In experimental systems, a vast array of biochemical mechanisms of acquired resistance to

methotrexate have been demonstrated (Figure 52–7) affecting each known step in methotrexate action, including: (1) impaired transport of methotrexate into cells (Assaraf and Schimke, 1987; Trippett et al , 1992); (2) production of altered forms of DHFR that have decreased affinity for the

inhibitor (Srimatkandada et al , 1989); (3) increased concentrations of intracellular DHFR through

gene amplification or altered gene regulation (Pauletti et al , 1990; Matherley et al , 1997); (4)

decreased ability to synthesize methotrexate polyglutamates (Li et al , 1992); and (5) decreased

thymidylate synthase activity (Curt et al , 1985) DHFR levels in leukemic cells increase within 24

hours after treatment of patients with methotrexate; this likely reflects induction of new enzyme synthesis Recent investigations have demonstrated that the intracellular level of DHFR protein is controlled at the level of mRNA translational efficiency through an autoregulatory mechanism whereby the DHFR protein may bind to and control the translational efficiency of its own

messenger RNA (Chu et al , 1993) Over longer periods of treatment, tumor cell populations

emerge that contain markedly increased levels of DHFR These cells contain multiple gene copies

of DHFR either in mitotically unstable double-minute chromosomes or in stable, homogeneously staining regions or amplisomes of the tumor cell chromosomes First identified as an explanation for resistance to methotrexate (Schimke et al , 1978), gene amplification has since been implicated

in the resistance to many antitumor agents, including fluorouracil and pentostatin

(2'-deoxycoformycin) (Stark and Wahl, 1984) Evidence supports the conclusion that DHFR gene amplification is clinically significant in patients with lung cancer (Curt et al , 1983) and leukemia

(Goker et al , 1995).

Figure 52–7 Mechanisms of Tumor Cell Resistance to Methotrexate TMP, thymidine monophosphate; dUMP, deoxyuridine monophosphate; FH2, dihydrofolate; FH4, tetrahydrofolate; Glun, polyglutamate

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

General Toxicity and Cytotoxic Action

The primary toxic effects of methotrexate and other folate antagonists used in cancer chemotherapy are exerted against rapidly dividing cells of the bone marrow and gastrointestinal epithelium Mucositis, myelosuppression, and thrombocytopenia reach their maximum in 5 to 10 days after drug administration and—except in instances of altered drug excretion—reverse rapidly thereafter

In addition to its acute toxicities, methotrexate can cause pneumonitis, characterized by patchy inflammatory infiltrates that rapidly regress upon discontinuation of drug In some cases, patients

can be rechallenged with drug without toxicity The etiology is not clearly allergic.

A second toxicity of particular significance in its chronic administration in patients with psoriasis orrheumatoid arthritis is hepatic fibrosis and cirrhosis Increased hepatic portal fibrosis is detected with higher frequency than in control patients after 6 months or longer of continuous oral

methotrexate treatment of psoriasis Its presence should lead to discontinuation of methotrexate Acute, reversible elevation of hepatic enzymes is detected in serum after high-dose administration but is rarely associated with permanent changes

Folic acid antagonists are toxic to developing embryos In preliminary trials, methotrexate has been highly effective when used with the prostaglandin analog, misoprostol, in inducing abortion in first trimester pregnancy (Hausknecht, 1995)

Absorption, Fate, and Excretion

Methotrexate is readily absorbed from the gastrointestinal tract at doses of less than 25 mg/m2, but larger doses are absorbed incompletely and are routinely administered intravenously Peak

concentrations in the plasma of 1 to 10 M are obtained after doses of 25 to 100 mg/m2, and

concentrations of 0.1 to 1 mM are achieved after high-dose infusions of 1.5 g/m2 or more After intravenous administration, the drug disappears from plasma in a triphasic fashion (Sonneveld et al ,

1986) The rapid distributive phase is followed by a second phase, which reflects renal clearance

(t1/2 of about 2 to 3 hours) A third phase has a half-life of approximately 8 to 10 hours This phase half-life, if unduly prolonged by renal failure, may be responsible for major toxic effects of the drug on the marrow and gastrointestinal tract Distribution of methotrexate into body spaces,

third-such as the pleural or peritoneal cavity, occurs slowly However, if third-such spaces are expanded (e.g.,

by ascites or pleural effusion), they may act as a site of storage and release of drug, with resultant prolonged elevation of plasma concentrations and more severe toxicity

Approximately 50% of methotrexate is bound to plasma proteins and may be displaced from plasmaalbumin by a number of drugs, including sulfonamides, salicylates, tetracycline, chloramphenicol, and phenytoin; caution should be used if these are given concomitantly Of the drug absorbed, about90% is excreted unchanged in the urine within 48 hours, mostly within the first 8 to 12 hours A small amount of methotrexate also is excreted in the stool, probably through the biliary tract

Metabolism of methotrexate in human beings is usually minimal After high doses, however,

metabolites do accumulate; these include 7-hydroxy-methotrexate, which is potentially nephrotoxic (Messmann and Allegra, 2001) 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 antiinflammatory agents), that are nephrotoxic (e.g., cisplatin),

or that are weak organic acids (e.g., aspirin or piperacillin) can delay drug excretion and lead to

severe myelosuppression (Stoller et al , 1977; Iven and Brasch, 1988; Thyss et al , 1986) Particular

caution must be exercised in treating patients with renal insufficiency, and the dose should be adjusted in these patients in proportion to decreases in renal function

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 There also is evidence for enterohepatic

recirculation

It is important to emphasize that concentrations of methotrexate in cerebrospinal fluid are only 3%

of those in the systemic circulation at steady state; hence, neoplastic cells in the CNS are probably not killed by standard dosage regimens When high doses of methotrexate are given (>1.5 g/m2),

followed by leucovorin rescue (see below), cytotoxic concentrations of methotrexate may be

attained in the CNS

Therapeutic Uses

Methotrexate (methotrexate sodium; amethopterin; FOLEX, MEXATE, RHEUMATREX, 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 to 25 mg intravenously weekly An initial

parenteral test dose of 5 to 10 mg is recommended to detect any possible idiosyncrasy It also is used intermittently at low dosage to induce remission in refractory rheumatoid arthritis

(Hoffmeister, 1983) Complete awareness of the pharmacology and toxic potential of methotrexate

is a prerequisite for its use in these nonneoplastic disorders (Weinstein, 1977)

Methotrexate is a useful drug in the management of acute lymphoblastic leukemia in children It is

of great value in remission induction and consolidation, used in high doses, and in the maintenance

of remissions in leukemia For maintenance therapy, it is administered intermittently at doses of 30 mg/m2 intramuscularly weekly in two divided doses or in 2-day "pulses" of 175 to 525 mg/m2 at monthly intervals 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 (Borsi and Moe, 1987) Methotrexate is of very limited value in the types of leukemia seen in adults, except for treatment and prevention of leukemic meningitis The intrathecaladministration 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 is effective also in patients whose systemic disease has become resistant to methotrexate, since the leukemic cells in the CNS beyond the blood–brain barrier have survived in a pharmacological sanctuary and may retain their original degree of sensitivity to the drug The recommended intrathecal dose in all patients over 3 years of age is 12 mg (Bleyer, 1978) The dose is repeated every 4 days until malignant cells are no longer evident in the CSF Leucovorin may be administered to counteract the toxicity of methotrexate that escapes into the systemic circulation, although this is generally not necessary Since methotrexate administered into the lumbar space distributes poorly over the cerebral convexities, the drug may bemore effectively distributed through the use of an intraventricular Ommaya reservoir The use of 1-

mg doses of methotrexate at intervals of 12 to 24 hours yields an effective regimen with reduced neurotoxicity

Methotrexate is of established value in choriocarcinoma and related trophoblastic tumors of women;cure is achieved in approximately 75% of advanced cases treated sequentially with methotrexate and dactinomycin, and in over 90% when early diagnosis is made In the treatment of

choriocarcinoma with methotrexate, 1 mg/kg 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 gonadotropin titers are used as a guide for persistence of disease

Beneficial effects also are observed in patients with osteosarcoma and mycosis fungoides and when methotrexate is used as part of the combination therapy of Burkitt's and other non-Hodgkin's

lymphomas and carcinomas of the breast, head and neck, ovary, and bladder High-dose

methotrexate, with leucovorin rescue, can cause substantial tumor regression in osteosarcoma and incombination therapy of leukemias and non-Hodgkin's lymphomas A 6- to 72-hour infusion of relatively large amounts of methotrexate may be employed intermittently (from 250 mg to 7.5 g/m2

or more), but only when leucovorin rescue is used Such regimens produce cytotoxic concentrations

of drug in the cerebrospinal fluid (CSF) and protect against leukemic meningitis A typical regimen includes the infusion of methotrexate for 6 hours followed by leucovorin at a dose of 15 mg/m2

every 6 hours for seven doses, with the goal of rescuing normal cells and thereby preventing

toxicity Other dosage regimens also are used The administration of methotrexate in high dosage has the potential for serious toxicity and should be performed only by experienced chemotherapists who are able to monitor concentrations of methotrexate in plasma If methotrexate values measured

48 hours after drug administration are 1 M or higher, higher doses (100 mg/m2) of leucovorin must

be given until the plasma concentration of methotrexate falls below the toxic threshold of 2 x 10–8 M(Stoller et al , 1977) With appropriate precautions, these schedules are relatively free of toxicity It

is imperative to maintain the output of a large volume of alkaline urine, since methotrexate

precipitates in the renal tubules in acidic urine In the presence of malignant effusions, delayed clearance may cause severe toxicity In patients who become oliguric, isolated reports suggest that continuous-flow hemodialysis can eliminate methotrexate at a rate approximating 50% of the clearance rate in patients with intact renal function (Wall et al , 1996) Methotrexate in high doses

with leucovorin rescue has been studied clinically for many years with promising results in

osteosarcoma, childhood leukemia, and non-Hodgkin's lymphoma, although the optimal timing and dose of leucovorin required and the optimal schedule of methotrexate administration remain to be established (Ackland and Schilsky, 1987)

Clinical Toxicities

As previously stated, the primary toxicities of methotrexate affect the bone marrow and the

intestinal epithelium Such patients may be at risk for spontaneous hemorrhage or life-threatening infection, and they may require prophylactic transfusion of platelets and broad-spectrum antibiotics

if febrile Side effects usually disappear within 2 weeks, but prolonged suppression of the bone marrow may occur in patients with compromised renal function who have delayed excretion of the drug The dosage of methotrexate must be reduced in proportion to any reduction in creatinine clearance

Additional toxicities of methotrexate include alopecia, dermatitis, interstitial pneumonitis,

nephrotoxicity, defective oogenesis or spermatogenesis, abortion, and teratogenesis Hepatic

dysfunction is usually reversible but sometimes leads 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

Pyrimidine Analogs

This class of agents encompasses a diverse and interesting group of drugs that have in common the capacity to inhibit the biosynthesis of pyrimidine nucleotides or to mimic these natural metabolites

to such an extent that the analogs interfere with the synthesis or function of nucleic acids Analogs

of deoxycytidine and thymidine have been synthesized as inhibitors of DNA synthesis, and an analog of uracil, 5-fluorouracil, effectively inhibits both RNA function and/or processing and

synthesis of thymidylate (see Figure 52–8) Drugs in this group have been employed in the

treatment of diverse afflictions, including neoplastic diseases, psoriasis, and infections caused by fungi and DNA-containing viruses The pathways for metabolic activation and degradation of these compounds during systemic administration present opportunities for the development of synergistic combination therapies with other clinically effective drugs

Figure 52–8 Structures of Available Pyrimidine Analogs

General Mechanism of Action

The best-characterized agents in this class are the halogenated pyrimidines, a group that includes

fluorouracil (5-fluorouracil, or 5-FU), floxuridine (5-fluoro-2'-deoxyuridine, or 5-FUdR), and

idoxuridine (5-iodode-oxyuridine; see Chapter 50: Antimicrobial Agents: Antiviral Agents

(Nonretroviral)) If one compares the van der Waals radii of the various 5-position substituents, the dimension of the fluorine atom resembles that of hydrogen, whereas the bromine and iodine atoms are larger and close in size to the methyl group Thus, idoxuridine behaves as an analog of

thymidine, and its primary biological action results from its phosphorylation and ultimate

incorporation into DNA in place of thymidylate In 5-FU, the smaller fluorine at position 5 allows the molecule to mimic uracil biochemically However, the fluorine–carbon bond is much tighter than that of C—H and prevents the methylation of the 5 position of 5-FU by thymidylate synthase Instead, in the presence of the physiological cofactor 5,10-methylene tetrahydrofolate, the

fluoropyrimidine locks the enzyme in an inhibited state Thus, substitution of a halogen atom of the correct dimensions can produce a molecule that sufficiently resembles a natural pyrimidine to interact with enzymes of pyrimidine metabolism but at the same time interferes drastically with certain other aspects of pyrimidine action

A number of 5-FU analogs have reached the clinic The most important of these is capecitabine (N4-pentoxycarbonyl-5'-deoxy-5-fluorocytidine), a drug with proven activity against colon and breast cancers This orally administered agent is converted to 5'-deoxy-5-fluorocytidine by

carboxylesterase activity in liver and other normal and malignant tissues From that point, it is

converted to 5'-deoxy-fluorodeoxyuridine by cytidine deaminase The final step in its activation occurs when thymidine phosphorylase cleaves off the 5'-deoxy sugar, leaving intracellular 5-FU Tumors with elevated thymidine phosphorylase activity seem particularly susceptible to this drug (Ishikawa et al , 1998).

Nucleotides in RNA and DNA contain ribose and 2'-deoxyribose, respectively Among the various modifications of the sugar moiety that have been attempted, the replacement of the ribose of

cytidine with arabinose has yielded a useful chemotherapeutic agent, cytarabine (AraC) As may be

seen in Figure 52–8, the hydroxyl group in this molecule is attached to the 2'-carbon in the , or upward, configuration, as compared with the , or downward, position of the 2'-hydroxyl in ribose The arabinose analog is recognized enzymatically as a 2'-deoxyriboside; it is phosphorylated to a nucleoside triphosphate that competes with dCTP for incorporation into DNA (Chabner et al ,

2001), where it blocks elongation of the DNA strand and its template function

Two other cytidine analogs have received extensive clinical evaluation 5-Azacytidine, an inhibitor

of DNA methylation as well as a cytidine antimetabolite, becomes incorporated predominantly into

RNA and has antileukemic as well as differentiating actions in vitro A newer analog,

2',2'-difluorodeoxycytidine (gemcitabine), becomes incorporated into DNA and inhibits the elongation

of nascent DNA strands (see Figure 52–8) It has promising activity in various human solid tumors,

including pancreatic, lung, and ovarian cancer

Fluorouracil and Floxuridine (Fluorodeoxyuridine)

Mechanism of Action

5-FU requires enzymatic conversion to the nucleotide (ribosylation and phosphorylation) in order toexert its cytotoxic activity (Figure 52–9) Several routes are available for the formation of the 5'-monophosphate nucleotide (F-UMP) in animal cells 5-FU may be converted to fluorouridine by uridine phosphorylase and then to F-UMP by uridine kinase, or it may react directly with 5-

phosphoribosyl-1-pyrophosphate (PRPP), in a reaction catalyzed by the enzyme orotate

phosphoribosyl transferase, to form F-UMP Many metabolic pathways are available to F-UMP, including incorporation into RNA A reaction sequence crucial for antineoplastic activity involves reduction of the diphosphate nucleotide by the enzyme ribonucleotide diphosphate reductase to the deoxynucleotide level and the eventual formation of 5-fluoro-2'-deoxyuridine-5'-phosphate (F-dUMP) 5-FU also may be converted directly to the deoxyriboside 5-FUdR by the enzyme

thymidine phosphorylase and further to F-dUMP, a potent inhibitor of thymidylate synthesis, by thymidine kinase This complex metabolic pathway for the generation of F-dUMP may be bypassedthrough use of the deoxyribonucleoside of fluoro–uracil—floxuridine (fluorodeoxyuridine, FUdR)

—which is converted directly to F-dUMP by thymidine kinase

Figure 52–9 Activation Pathways for 5-Fluorouracil (5-FU) and 5-Floxuridine (FUR) FUDP, floxuridine diphosphate; FUMP, floxuridine monophosphate; FUTP, floxuridine triphosphate; FUdR, fluorodeoxyuridine; FdUDP,

fluorodeoxyuridine diphosphate; FdUMP, fluorodeoxyuridine monophosphate; FdUTP, fluorodeoxyuridine triphosphate; PRPP, 5-phosphoribosyl-1-

pyrophosphate

The interaction between F-dUMP and the enzyme thymidylate synthase leads to deletion of TTP, a necessary constituent of DNA (Figure 52–10) The folate cofactor, 5,10-methylenetetra-hydrofolate,and F-dUMP form a covalently bound ternary complex with the enzyme This inhibitory complex resembles the transition state formed during the normal enzymatic reaction when dUMP is

converted to thymidylate Although the physiological complex progresses to the synthesis of

thymidylate by transfer of the methylene group and two hydrogen atoms from folate to dUMP, this reaction is blocked in the inhibitory complex by the stability of the fluorine carbon bond on F-dUMP; sustained inhibition of the enzyme results (Santi et al , 1974).

Figure 52–10 Site of Action of 5-Fluoro-2'-Deoxyuridine-5'-Phosphate FdUMP) 5-FU, 5-fluorouracil; dUMP, deoxyuridine monophosphate; TMP, thymidine monophosphate; TTP, thymidine triphosphate; FdUMP,

(5-fluorodeoxyuridine monophosphate; FH2Glun, dihydrofolate polyglutamate;

FH4Glun, tetrahydrofolate polyglutamate

5-FU also is incorporated into both RNA and DNA In 5-FU–treated cells, both F-dUTP and dUTP (the substrate that accumulates behind the blocked thymidylate synthase reaction) incorporate into DNA in place of the depleted physiological TTP The significance of the incorporation of F-dUTP and dUTP into DNA is unclear (Canman et al , 1993) 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 thymidylate synthase inhibition (Mauro et al , 1993) 5-FU

incorporation into RNA also causes toxicity as the result of major effects on both the processing and

functions of RNA (Armstrong, 1989; Danenberg et al , 1990).

A number of biochemical mechanisms have been identified that are associated with resistance to thecytotoxic effects of 5-FU or floxuridine These mechanisms include loss or decreased activity of theenzymes necessary for activation of 5-FU, decreased pyrimidine monophosphate kinase (which decreases incorporation into RNA), amplification of thymidylate synthase (Washtein, 1982), and altered thymidylate synthase that is not inhibited by F-dUMP (Barbour et al , 1990) Both

experimental studies and clinical trials support the position that the response to 5-FU correlates significantly with low levels of the degradative enzymes, dihydrouracil dehydrogenase and

thymidine phosphorylase, and a low level of expression of the target enzyme, thymidylate synthase (van Triest et al , 2000) Recent investigations have demonstrated that the level of thymidylate

synthase is finely controlled by an autoregulatory feedback mechanism wherein the thymidylate synthase protein interacts with and controls the translational efficiency of its own messenger RNA This mechanism provides for the rapid modulation of the level of thymidylate synthase necessary for cellular division and also may be an important mechanism by which malignant cells become rapidly insensitive to the effects of 5-fluorouracil (Chu et al , 1991; Swain et al , 1989) Some

malignant cells appear to have insufficient concentrations of 5,10-methylene tetrahydrofolate and, thus, cannot form maximal levels of the inhibited ternary complex with thymidylate synthase Addition of exogenous folate in the form of 5-formyl-tetrahydrofolate (leucovorin) increases formation of the complex in both laboratory and clinical experiments and has enhanced responses to5-FU in clinical trials (Ullman et al , 1978; Grogan et al , 1993) Except for inadequate intracellular

folate pools, it is not established which (if any) of the other mechanisms is associated with clinical resistance to 5-FU and its derivatives (Grem et al , 1987).

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 These agents, along with their proposed mechanisms of interaction, are shown in Table 52-2 The most clinically interesting combinations with 5-FU include methotrexate, interferon, leucovorin, or cisplatin, all of which are currently under investigation to define their ultimate clinical roles Agents that inhibit early steps in

pyrimidine biosynthesis, such as PALA (N-phosphonoacetyl-L-aspartate), an inhibitor of aspartate transcarbamylase, provide synergistic interaction with 5-FU in experimental systems, but these combinations have no proven clinical value (Grem et al , 1988) 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 (Grem, 2001).Absorption, Fate, and Excretion

5-FU and floxuridine are administered parenterally, since absorption after ingestion of the drugs is unpredictable and incomplete Metabolic degradation occurs in many tissues, particularly the liver Floxuridine is converted by thymidine or deoxyuridine phosphorylases into 5-FU 5-FU is

inactivated by reduction of the pyrimidine ring; this reaction is 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 (Lu et al ,

1993; 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, which is ultimately degraded to

-fluoro- -alanine (Heidelberger, 1975; Zhang et al , 1992).

Rapid intravenous administration of 5-FU produces plasma concentrations of 0.1 to 1.0 mM;

plasma clearance is rapid (t1/2 10 to 20 minutes) Urinary excretion of a single dose of 5-FU given intravenously amounts to only 5% to 10% in 24 hours Although the liver contains high

concentrations of DPD, dosage does not have to be modified in patients with hepatic dysfunction, presumably because of degradation of the drug at extrahepatic sites or by vast excess of this enzyme

in the liver Given by continuous intravenous infusion for 24 to 120 hours, 5-FU achieves plasma concentrations in the range of 0.5 to 8.0 M 5-FU readily enters the CSF, and concentrations greater than 0.01 M are sustained for up to 12 hours following conventional doses (Grem, 2001).Capecitabine is well absorbed orally, yielding high plasma concentrations of 5'-deoxy-

fluorodeoxyuridine (5'-dFdU), which disappears with a half-life of about 1 hour 5-FU levels are less than 10% of those of 5'-dFdU 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).

Therapeutic Uses

5-Fluorouracil

Accumulated experience with 5-FU (ADRUCIL) indicates that the drug produces partial responses in 10% to 20% of patients with metastatic carcinomas of the breast and the gastrointestinal tract; beneficial effects also have been reported in carcinoma of the ovary, cervix, urinary bladder,

prostate, pancreas, and oropharyngeal areas For average-risk patients in good nutritional status withadequate hematopoietic function, the weekly dosage regimen employs 750 mg/m2 alone or 500 to

600 mg/m2 with leucovorin once each week for 6 of 8 weeks Other regimens use daily doses of 500mg/m2 for 5 days, repeated in monthly cycles When used with leucovorin, daily doses of 5-FU must be reduced to 375 to 425 mg/m2 for 5 days because of mucositis and diarrhea It also has been given as a continuous infusion for up to 21 days (300 mg/m2 per day), or as a biweekly 48-hour continuous infusion (de Gramont et al , 1998).

Floxuridine (FUdR)

FUdR (fluorodeoxyuridine; FUDR) is used primarily by continuous infusion into the hepatic artery for treatment of metastatic carcinoma of the colon or following resection of hepatic metastases (Kemeny et al , 1999); the response rate to such infusion is 40% to 50%, or double that observed

with intravenous administration Intrahepatic arterial infusion for 14 to 21 days may be used with minimal systemic toxicity However, there is a significant risk of biliary sclerosis if this route is used for multiple cycles of therapy (Kemeny et al , 1987; Hohn et al , 1986) Continuous infusion

of floxuridine into the arterial blood supply of tumors at other sites, such as in the head and neck region, may provide beneficial clinical effects With any of these regimens, 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 to 14.Capecitabine (XELODA)

Capecitabine is approved by the United States Food and Drug Administration (FDA) for the

treatment of metastatic breast cancer in patients who have not responded to a regimen of paclitaxel

and an anthracycline antibiotic (see below) The recommended dose is 2500 mg/m2 daily, given orally in two divided doses with food, for 2 weeks followed by a rest period of 1 week This cycle is

then repeated two more times.

Combination Therapy

Higher response rates are seen when 5-FU is used in combination with other agents, such as

cyclophosphamide and methotrexate (breast cancer), cisplatin (head and neck cancer), and with leucovorin in colon cancer (see Table 52-2) The use of 5-FU in combination regimens has

improved survival in the adjuvant treatment for breast cancer (Early Breast Cancer Trialists

Collaborative Group, 1988) and, with leucovorin, for colorectal cancer (Wolmark et al , 1993)

5-FU is a potent radiation sensitizer and is being used with concurrent radiotherapy for primary therapy of locally advanced tumors of the head and neck, esophagus, lung, and rectum 5-FU is usedwidely with very favorable results for the topical treatment of premalignant keratoses of the skin and multiple superficial basal cell carcinomas It also is effective in severe recalcitrant psoriasis (Alper et al , 1985).

progressing to total alopecia, nail changes, dermatitis, and increased pigmentation and atrophy of the skin may be encountered Neurological manifestations, including an acute cerebellar syndrome, have been reported, and myelopathy has been observed after the intrathecal administration of 5-FU Cardiac toxicity, particularly acute chest pain with evidence of ischemia in the electrocardiogram, also may occur The low therapeutic indices of these agents emphasize the need for very skillful supervision by physicians familiar with the action of the fluorinated pyrimidines and the possible hazards of chemotherapy

Capecitabine causes much the same spectrum of toxicities as 5-FU (diarrhea, myelosuppression), but also a progressive hand-foot syndrome consisting of erythema, desquamation, pain, and

sensitivity to touch of the palms and soles

Cytarabine (Cytosine Arabinoside; AraC)

Cytarabine (1- -D-arabinofuranosylcytosine; AraC) is the most important antimetabolite used in thetherapy of acute myelocytic leukemia It is the single most effective agent for induction of

remission in this disease (for review, see Garcia-Carbonero et al , 2001).

Mechanism of Action

This compound is an analog of 2'-deoxycytidine with the 2'-hydroxyl in a position trans to the

3'-hydroxyl of the sugar, as shown in Figure 52–8 The 2'-3'-hydroxyl causes steric hindrance to the rotation of the pyrimidine base around the nucleosidic bond The bases of polyarabinonucleotides

cannot stack normally, as do the bases of polydeoxynucleotides.

AraC penetrates cells by a carrier-mediated process shared by physiological nucleosides As with most purine and pyrimidine antimetabolites, cytarabine must be "activated" by conversion to the 5'-monophosphate nucleotide (AraCMP), a reaction catalyzed by deoxycytidine kinase AraCMP can then react with appropriate nucleotide kinases to form the diphosphate and triphosphate nucleotides (AraCDP and AraCTP) AraC competes with the physiological substrate deoxycytidine 5'-

triphosphate (dCTP) for incorporation into DNA by DNA polymerases The incorporated AraCMP residue is a potent inhibitor of DNA polymerase The effects of AraC on DNA polymerase activity extend not only to DNA chain elongation during semiconservative DNA replication, but also to DNA repair There is a significant relationship between inhibition of DNA synthesis and the total amount of AraC incorporated into DNA Thus, incorporation of about five molecules of AraC per

104 bases of DNA decreases cellular clonogenicity by about 50%

AraC also causes an unusual reiteration of DNA segments, thus increasing the possibility of

recombination, crossover, and gene amplification In addition, AraC is converted intracellularly to AraCDP-choline, an analog of the physiological CDP-choline, which inhibits the synthesis of membrane glycoproteins and glycolipids Furthermore, AraCMP inhibits the transfer of galactose,

N-acetylglucosamine, and sialic acid to cell-surface glycoproteins, and AraCTP inhibits the

synthesis of CMP-acetylneuraminic acid Thus, AraC may alter membrane structure, antigenicity, and function

AraC induces terminal differentiation of leukemic cells in tissue culture, an effect that is

accompanied by decreased c-myc oncogene expression (Bianchi Scarra et al , 1986) These changes

in morphology and oncogene expression occur at concentrations above the threshold for

cytotoxicity and may simply represent terminal injury of cells However, molecular analysis of bonemarrow specimens from some leukemic patients in remission after AraC therapy has revealed persistence of leukemic markers, suggesting that differentiation may have occurred

The precise mechanism of cellular death caused by AraC is not fully understood Fragmentation of DNA is observed in AraC-treated cells, and there is cytological and biochemical evidence for apoptosis in both tumor and normal tissues (Smets, 1994) A complex system of interacting

transduction signals ultimately determines whether or not a cell exposed to a cytotoxic agent is destined to die Exposure of leukemic cells to AraC stimulates the formation of ceramide, a potent inducer of apoptosis On the other hand, an increase in protein kinase C (PKC) activity is observed

in leukemic cells in response to AraC in vitro Because PKC activation is known to oppose

apoptosis in hematopoietic cells, the lethal actions of AraC may depend, at least partially, on its relative effects on the PKC and sphingomyelin pathways (Strum et al , 1994) Transcriptional

regulation of gene expression may be another key mechanism through which malignant cell growth and differentiation are regulated The induction of some transcription factors, such as AP-1 (a dimer

of jun-fos or jun-jun proteins) and NF- B, has been temporarily associated with AraC-induced apoptosis (Kharbanda et al , 1990) It also has been reported that induction of pRb phosphatase

activity by AraC leads to a hypophosphorylated pRb that binds to and inactivates the E2F

transcription factor, inhibiting the transcription of numerous genes involved in cell-cycle

progression (Ikeda et al , 1996).

In addition to biochemical factors that determine response, cell kinetic properties exert an importantinfluence on the results of AraC treatment It is likely that continued inhibition of DNA synthesis for a duration equivalent to at least one cell cycle is necessary to expose cells during the S, or DNA-synthetic, phase of the cycle This mechanism may thus be important when AraC is administered by