Section IX - Chemotherapy of Neoplastic Diseases ppsx

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 nonmalignant
disease. 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 to
undergo 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 when
the 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–ablative
regimens 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 to
the 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 (the
dose 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 10
9
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 small
growth 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 (G
1
); (2) the synthesis of DNA occurs
(S); (3) an interval follows the termination of DNA synthesis, the postsynthetic phase (G

2
); and (4)
mitosis (M) ensues—the G
2
cell, containing a double complement of DNA, divides into two
daughter G
1
cells. Each of these cells may immediately reenter the cell cycle or pass into a
nonproliferative stage, referred to as G
0
. The G
0
cells of certain specialized tissues may differentiate
into functional cells that no longer are capable of division. On the other hand, many cells, especially
those in slow-growing tumors, may remain in the G
0
state for prolonged periods, only to reenter the
division cycle at a later time. Damaged cells that reach the G
1
/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. G
1

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 G
1
, G
0
. S is the period of DNA synthesis; G
2
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's
willingness 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 good
nutritional 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 the
future, 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 cases
that 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 (S
N
1) 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, which
can 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 (S

N
2)
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 bifunctional
alkylating agents, such as nitrogen mustard, the second 2-chloroethyl side chain can undergo a
similar cyclization reaction and alkylate a second guanine residue or another nucleophilic moiety,
resulting in the cross-linking of two nucleic acid chains or the linking of a nucleic acid to a protein,
alterations that would cause a major disruption in nucleic acid function. Any of these effects could
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. Cyclophosphamide
requires 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 S
N
2 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 structures
are 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 the
phosphorus–nitrogen linkage. Second, it was hoped that neoplastic tissues might possess high
phosphatase or phosphamidase activity capable of accomplishing this cleavage, thus resulting in the
selective 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 to
sites 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 5-(3,3-dimethyl-1-triazeno)-
imidazole-4-carboxamide, usually referred to as dacarbazine or DTIC, functions through alkylation.
Its structural formula is shown below:
Dacarbazine requires initial activation by the cytochrome P450 system of the liver through an N-
demethylation reaction. In the target cell, spontaneous cleavage of the metabolite yields an
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 O
6
-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 murine
tumors. 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.
Cytotoxic Actions
The most important pharmacological actions of the alkylating agents are those that disturb DNA
synthesis and cell division. The capacity of these drugs to interfere with DNA integrity and function
in rapidly proliferating tissues provides the basis for their therapeutic applications and for many of
their toxic properties. Whereas certain alkylating agents may have damaging effects on tissues with

normally low mitotic indices—for example, liver, kidney, and mature lymphocytes—they are most
cytotoxic to rapidly proliferating tissues in which a large proportion of the cells are in division.
These compounds may readily alkylate nondividing cells, but cytotoxicity is markedly enhanced if
DNA is damaged in cells programmed to divide. Thus, DNA alkylation itself may not be a lethal
event if DNA repair enzymes can correct the lesions in DNA prior to the next cellular division.
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 G
1
or S than in G
2
, mitosis, or early G
1
.
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 G
1
/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 O
6
-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 O
6
-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) increased
production of nucleophilic substances, principally thiols such as glutathione, that can conjugate with
and detoxify electrophilic intermediates; (3) increased activity of the DNA repair enzymes, such as
the guanine O
6
-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 O
6
-benzylguanine that inactivate the guanine O
6
-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, O
6
-
benzylguanine 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 of
this 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/m
2
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 does
not 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, and
the 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/m
2
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/m
2
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/m
2
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 for
use in these conditions, not only because of its acute toxic effects but also because of its potential
for inducing sterility, teratogenic effects, and leukemia.
Clinical Toxicity
Nausea and vomiting, myelosuppression with platelet sparing, and alopecia are common to virtually
all regimens using cyclophosphamide. Mucosal ulcerations and, less frequently, interstitial
pulmonary fibrosis also may result from cyclophosphamide treatment. Extravasation of the drug
into subcutaneous tissues does not produce local reactions, and thrombophlebitis does not
complicate intravenous administration. The occurrence of sterile hemorrhagic cystitis has been
reported in 5% to 10% of patients. As noted above, this has been attributed to chemical irritation
produced by acrolein. Its incidence is significantly reduced by coadministration of mesna (Brock
and Pohl, 1986). For routine clinical use, ample fluid intake is recommended. Administration of the
drug should be interrupted at the first indication of dysuria or hematuria. The syndrome of
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/m
2
, 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/m
2
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 ifosfamide
dose. 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.
Pharmacokinetics
Ifosfamide has a half-life in plasma of approximately 15 hours after doses of 3.8 to 5.0 g/m
2
and a
somewhat shorter half-life at lower doses.
Toxicity
Ifosfamide has virtually the same toxicity profile as does cyclophosphamide, with perhaps greater
platelet suppression, neurotoxicity, and, in the absence of mesna, urothelial damage.
Melphalan
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 3
weeks, 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/m
2
infused over
15 to 20 minutes. Doses are repeated at 2-week intervals for four doses and then at 4-week intervals
based 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.
Clinical Toxicity
The clinical toxicity of melphalan is mostly hematological and is similar to that of other alkylating
agents. Nausea and vomiting are infrequent. Alopecia does not occur at standard doses, and changes
in renal or hepatic function have not been observed.
Chlorambucil
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/m
2
), and drug and metabolite half-lives are unchanged in
children receiving high-dose therapy of 300 mg/m
2
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
Busulfan
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 its
primary 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 and
prolonged 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 from
the 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 clinical
remissions 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/m
2
) 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 therapeutic
advantage over the other members of its class.
Carmustine (BCNU)
Pharmacological and Cytotoxic Actions
Carmustine's major action is its alkylation of DNA at the O
6
-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/m
2
, 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. Beneficial

responses 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/m
2
, 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/m
2
once daily for 5 days; this
course is repeated every 6 weeks. Alternatively, 1000 mg/m
2
can be given weekly for 2 weeks, and
the weekly dose can then be increased to a maximum of 1500 mg/m
2
.
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 O
6
-alkyl transferase.
Absorption, Fate, and Excretion
Dacarbazine is administered intravenously; after an initial rapid phase of disappearance (t
1/2
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 malignant
melanoma is to give 3.5 mg/kg per day, intravenously, for a 10-day period; this is repeated every 28
days. Alternatively, 250 mg/m
2
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).

Clinical Toxicity
The toxicity of both DTIC and temozolomide includes nausea and vomiting in more than 90% of
patients; this usually develops 1 to 3 hours after treatment and may last up to 12 hours.
Myelosuppression, with both leukopenia and thrombocytopenia, is usually mild to moderate. A
flulike syndrome, consisting of chills, fever, malaise, and myalgias, may occur during treatment
with DTIC. Hepatotoxicity, alopecia, facial flushing, neurotoxicity, and dermatological reactions
also have been reported.
Antimetabolites
Folic Acid Analogs
Methotrexate
Antifolates occupy a special place in antineoplastic chemotherapy, in that they produced the first
striking, although temporary, remissions in leukemia (Farber et al. , 1948) and the first cure of a
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