19_03

SECTION 19.3
Antitumor Alkylating Agents

OLIVER MICHAEL COLVIN

HISTORY OF THE ALKYLATING AGENTS

A nitrogen mustard alkylating agent was the first nonhormonal chemical that demonstrated significant clinical antitumor activity. The clinical evaluation of nitrogen
mustards as antitumor agents evolved from the observed clinical effects of sulfur mustard gas used as a weapon in World War I. This gas was used because of its
vesicant effect on the skin and mucous membranes, especially the eyes and respiratory tract. 1 However, in addition to this deadly effect, depression of the
hematopoietic and lymphoid systems was observed in victims and experimental animals.2 These observations led to further studies that used the less volatile nitrogen
mustards (Fig. 19.3-1). Studies published in 1946 demonstrated regression of tumors, especially lymphomas 3,4 and 5 and led to the introduction of the compound
nitrogen mustard (mechlorethamine, Mustargen) into clinical practice. Subsequently, less toxic and more clinically effective nitrogen mustard derivatives and other
types of alkylating agents have been developed.

CHEMISTRY AND CYTOTOXICITY OF ALKYLATING AGENTS

The alkylating agents react with (or “alkylate”) many electron-rich atoms in cells to form covalent bonds. The most important reactions with regard to their antitumor
activities are reactions with DNA bases. Some alkylating agents are monofunctional and react with only one strand of DNA. Others are bifunctional and react with an
atom on each of the two strands of DNA to produce a “cross-link” that covalently links the two strands of the DNA double helix. Unless repaired, this lesion will prevent
the cell from replicating effectively. The lethality of the monofunctional alkylating agents results from the recognition of the DNA lesion by the cell and the response of
the cell to that lesion. Analogous cellular reactions may occur to the interstrand cross-links, but such reactions have not been definitively established.

CLASSES OF ALKYLATING AGENTS AND THEIR PROPERTIES

NITROGEN MUSTARDS

Mustargen

Mustargen is currently used in the MOPP [Mustargen, vincristine (Oncovin), procarbazine, prednisone] regimen for the treatment of Hodgkin's disease 6 but rarely for
other purposes. The other nitrogen mustards in significant clinical use are cyclophosphamide, ifosfamide, melphalan, and chlorambucil ( Fig. 19.3-2). All these
compounds produce cytotoxicity by forming covalent interstrand cross-links in DNA (as shown in Fig. 19.3-3 for Mustargen). The nitrogen mustard cross-link has been
demonstrated to occur in the G-X-C/C-Y-G configuration, 7 as opposed to the G-C/C-G cross-link that had previously been predicted. 8 The formation of the
G-X-C/C-X-G cross-link has been postulated to occur on the basis of the greater frequency of approximation of the N7 atoms of the two guanylates in the
G-X-C/C-X-G configuration, as opposed to the G-C/C-G configuration. 9

Mustargen is available only as an intravenous preparation that can also be used topically for cutaneous malignancies. In the MOPP regimen, Mustargen is used at a
dose of 6 mg/m2 on days 1 and 8 of the monthly schedule. Toxicities unique to the agent are topical irritation and pain on injection if given too rapidly. The clearance
of the drug is very rapid, but pharmacokinetics have not been performed with modern techniques.

Cyclophosphamide

The most frequently used alkylating agent, cyclophosphamide, is used for the treatment of breast cancer in combination with doxorubicin (Adriamycin) 10 or with
methotrexate and 5-fluorouracil11 and for the treatment of lymphomas,12,13 childhood tumors,14,15 and many solid tumors.16 High doses of cyclophosphamide are
frequently used in conjunction with bone marrow transplantation 17,18 and 19 and for the treatment of autoimmune diseases.20,21

Cyclophosphamide is inactive in vitro and is metabolized by P-450 enzymes in the liver to active species, as shown in Figure 19.3-4. The initial product is
4-hydroxycyclophosphamide (4-HC), which is released from the liver into the circulation. 22 This compound is in equilibrium with an open-ring tautomer,
aldophosphamide. Aldophosphamide spontaneously eliminates acrolein to produce phosphoramide mustard, 23 which is an active bifunctional alkylating species. 24
Phosphoramide mustard is zwitterionic at physiologic pH 25 and enters cells poorly. 4-HC-aldophosphamide is not charged and enters cells facilely. While
phosphoramide mustard is toxic to cells in vitro at concentrations of 100 μM and higher, 4-HC is cytotoxic in the range of 10 μM. 26 Thus, 4-HC-aldophosphamide
serves as an efficient delivery system for phosphoramide mustard, which has been demonstrated to produce an interstrand DNA cross-link analogous to the cross-link
produced by mechlorethamine.7 Recent studies by Shulman-Roskes et al.27 have demonstrated that phosphoramide mustard readily eliminates chloroethylaziridine, 27
which probably also plays a role in the cross-linking of DNA in cells exposed to 4-HC.

As shown in Figure 19.3-4, 4-HC is a substrate for the enzyme aldehyde dehydrogenase. 28 In cells that contain this enzyme, the bulk of the 4-HC is oxidized to
carboxyphosphamide, which is not an active alkylating agent. Consequently, cells with high aldehyde dehydrogenase (ALDH) content are resistant to the metabolites
of cyclophosphamide.29,30 Early hematopoietic stem cells and megakaryocytes contain high levels, as do the epithelial stem cells in the small intestine and mucous
membranes.30,31 These observations explain why cyclophosphamide administration produces a shorter period of hematopoietic depression, 32 is relatively sparing of
platelets, and is associated with less gastrointestinal toxicity and mucositis than other alkylating agents. 33

4-HC is too unstable to be used as a reagent, but the compound 4-hydroperoxycyclophosphamide (see Fig. 19.3-4) is spontaneously converted in aqueous solution to
4-HC and can be used for invitro studies of cell sensitivity. 34,35 This compound has also been used for the invitro treatment of autologous bone marrow to reduce the
number of tumor cells returned to the patient. 36

Cyclophosphamide is available as tablets for oral administration or as an intravenous preparation. The drug is used at a variety of doses and schedules. Oral
administration is particularly used for autoimmune diseases at a daily dose of approximately 100 mg. Because of its rapid absorption and high bioavailability, even
very high doses can be given orally, but high intermittent doses are usually given intravenously. In moderate-dose combination chemotherapy, doses of
cyclophosphamide in the range of 750 mg are usually used. For high-dose therapy in conjunction with hematopoietic cell transplantation, doses of up to 50 mg/kg for
2 or 4 days in combination with other agents are used.

The bulk (nearly 70%) of a dose of cyclophosphamide is excreted in the urine as the inactive carboxyphosphamide. 37,38 At high doses (approximately 50 mg/kg),
plasma concentrations of up to 400 μM of cyclophosphamide are achieved, 38 and clearance depends on the renal clearance and the rate of microsomal metabolism in
the liver. With improved and more facile techniques to measure 4-HC concentrations accurately, the clinical pharmacology of cyclophosphamide and this critical
transport intermediate are being more carefully defined. Studies in patients receiving high-dose therapy have demonstrated considerable variation in the rates of
clearance of cyclophosphamide between patients, with consequent differences in the peak concentrations (1 to 15 μM) and total exposure of the patient to 4-HC (60
to 140 μM.hours).39,40 The total exposure to 4-HC is probably the major determinant of therapeutic effect. Currently, several programs are evaluating dose adjustment
regimens based on the initial pharmacokinetics of cyclophosphamide and 4-HC. While it is known that substantial concentrations of phosphoramide mustard are
present in plasma (up to 10 μM after 60 mg/kg of cyclophosphamide 38), this concentration is well below the concentrations needed for invitro cytotoxicity of
phosphoramide mustard.26

A unique toxicity of cyclophosphamide and other oxazophosphorines is a characteristic hemorrhagic cystitis 41,42 due to irritation of the bladder mucosa from urinary
metabolites. Acrolein has been identified as the metabolite most responsible for this effect, 43 but phosphoramide mustard and chloracetaldehyde may contribute to
this toxicity. Careful hydration and emptying of the bladder are crucial to avoiding this toxicity, which has produced massive and even fatal hemorrhage. Another
toxicity that has been associated with cyclophosphamide is an antidiuretic effect, especially at high doses. 44 This effect may produce marked fluid retention and
electrolyte abnormalities, particularly low sodium, and seizures and fatalities have been seen. 45 It is important to avoid low-sodium-containing fluids after high-dose
cyclophosphamide, and the fluid retention syndrome has been treated with furosemide to promote free water clearance. 46 The most severe dose-limiting toxicity of
cyclophosphamide is a fulminant cardiac toxicity,47 which is often fatal when seen clinically. This toxicity is seen only after the high doses used in bone marrow
transplantation. It was initially seen in patients receiving 60 mg/kg/d of cyclophosphamide for 4 days, and the incidence has decreased since lower doses have been
used. The syndrome usually presents with severe cardiac failure, beginning approximately 10 days after drug administration, with a dilated heart and low
electrocardiogram voltage. There is a characteristic pathologic picture of edema, interstitial hemorrhage, and cardiac necrosis. 47

Ifosfamide

Ifosfamide is a structural isomer of cyclophosphamide that is often used in the treatment of sarcomas and pediatric tumors (see Fig. 19.3-2). There is more chloroethyl
side chain oxidation of ifosfamide (up to 50%) than of cyclophosphamide (<10%), and the degree of such metabolism is more variable than with cyclophosphamide. 48
Oxidation of the chloroethyl groups produces chloroacetaldehyde, which is probably responsible for the neurotoxicity 49 and renal toxicity50 that have been seen with
ifosfamide therapy. Since the oxidation of a chloroethyl side chain produces a much less toxic monofunctional agent, higher doses of ifosfamide than
cyclophosphamide must be used clinically. The studies of the clinical pharmacology of ifosfamide have been more limited than those of cyclophosphamide but have
demonstrated large intrapatient variability in the pharmacokinetics and metabolism of the agent during repeated administrations. 51,52

Melphalan

Melphalan is now used principally for the treatment of multiple myeloma, 53 for high-dose myeloablative therapy in conjunction with bone marrow transplantation, 54 and
for the isolated limb perfusion of localized tumors, 55 especially malignant melanoma and sarcomas (see Fig. 19.3-2). Melphalan is an amino acid analogue and is
actively transported into cells by amino acid transport systems. 56,57 It has been demonstrated that cellular uptake 58 and transport into the central nervous system
(CNS)59 of melphalan can be modulated by the amino acid content in the extracellular fluid.
Melphalan is available both as tablets and as an intravenous preparation. For the treatment of multiple myeloma, melphalan is usually used orally at a dose of 0.25
mg/kg for 4 days, with prednisone on the same schedule every 4 to 6 weeks. At these doses, peak plasma concentrations of 0.625 μM are found, but absorption is
variable.60 For bone marrow transplantation, doses of melphalan of 100 to 140 mg/m2 are used.61 At these doses, peak concentrations of melphalan of 40 to 50 μM
are reached.61,62

Chlorambucil

Chlorambucil is used for the treatment of B-cell chronic lymphocytic leukemia 63 and lymphomas64 and for the immunosuppressive therapy of autoimmune diseases.65
It is administered orally and is well tolerated when given either by daily administration or intermittent high-pulse doses. 64 Chlorambucil is well tolerated by most
patients and can be used successfully for patients who have severe nausea and vomiting with cyclophosphamide or melphalan.

Chlorambucil is available only in an oral formulation. For chronic leukemia and immunosuppression, daily doses of 3 to 6 mg are given for a number of weeks, or 12
mg/m2 may be given monthly. Pulsed dose pulse chlorambucil for lymphoma is given orally at a dose of 16 mg/m 2 daily for 5 consecutive days each month.64
Chlorambucil is metabolized to a less active derivative—phenylacetic acid mustard—and the clinical pharmacology of chlorambucil is very similar to that of
melphalan.66

AZIRIDINES AND EPOXIDES

The aziridine agents are related to the nitrogen mustards but contain uncharged aziridine rings that are less reactive than the aziridinium rings formed by most of the
nitrogen mustards. The two aziridine agents that are frequently used clinically are thiotepa and mitomycin C ( Fig. 19.3-5). The diepoxide dianhydrogalactitol reacts
with DNA in a similar fashion to the aziridines but has been succeeded in clinical use by dibromodulcitol, which spontaneously generates dianhydrogalactitol insitu
(Fig. 19.3-6).

Thiotepa

Thiotepa is now used most frequently in combination with other alkylating agents in high-dose therapy with stem cell support. 39,67 Thiotepa has been demonstrated to
react with the N7 position of guanylic acid in DNA 68 and to cross-link DNA,69 indicating that it is acting similarly to the nitrogen mustards. Thiotepa is desulfurated by
cytochrome P-450 enzymes70 to produce tepa. Tepa is less toxic than thiotepa and has been demonstrated to produce alkali-labile sites in DNA, rather than
cross-links.69 These findings suggest that tepa reacts differently from thiotepa and produces monofunctional alkylation of DNA.

In combination with cyclophosphamide for high-dose therapy, thiotepa has been given as a continuous infusion for 4 days, at a daily dose of 200 mg/m 2. Under these
conditions, steady-state levels of 2 to 6 μM of thiotepa are rapidly achieved. 71 Thiotepa is also used at a dose of 900 mg/m2 in combination with high-dose
cyclophosphamide and cisplatin.72

Mitomycin C

Mitomycin C is an antibiotic extracted from a Streptomyces species and is used for the treatment of breast cancer,73 esophageal cancer,74 and gastrointestinal
tumors.75 As seen in Figure 19.3-5, this compound contains an aziridine ring. Particularly under hypoxic conditions, mitomycin C is reduced, with activation of the C1
position of the aziridine ring. This carbon then reacts in the minor groove with the extracyclic N2 amino group of a guanylic acid, 76,77 positioning the 10 carbon of the
carbamate moiety to react with the N2 of a guanylic acid residue in an adjacent base pair in the complementary DNA strand. Mitomycin C and its reduced metabolites
can also produce intrastrand guanylic acid–guanylic acid cross-links that produce bending of the DNA. 78

In combination regimens, mitomycin C is given at doses of 10 to 15 mg/m2 every 4 to 6 weeks. After a dose of 15 mg/m2, peak plasma concentrations of 3 μM are
seen.79

Dianhydrogalactitol

Dianhydrogalactitol (see Fig. 19.3-6) is a hexitol derivative that contains two epoxide groups and cross-links DNA through the N7 atoms of guanylic acid, 80
presumably through the nucleophilic attack of the N7 atoms on the strained-ring epoxide groups. This compound was evaluated in clinical trials and demonstrated
modest antitumor activity.81,82 However, the structurally related dibromodulcitol (see Fig. 19.3-6) has demonstrated more antitumor activity83,84 and is still being used in
combination chemotherapy of breast cancer, cervical cancer, and brain tumors. Dibromodulcitol is hydrolyzed to dianhydrogalactitol, and its better antitumor activity is
presumably due to more effective localization of the reactive agent in tumor cells. 85 Dibromodulcitol is usually administered at a dose of 1 g/m 2, which produces a
maximum plasma concentration of approximately 50 μM.86

ALKYL SULFONATES: BUSULFAN

Busulfan (Myeleran), other alkyl sulfonates, and the related sulfamates react with DNA by a direct displacement reaction (as shown in Fig. 19.3-7). Busulfan has been
demonstrated to cross-link DNA,87 but the structure of the cross-link has not been established. A chemically related agent, hepsulfam, with seven methylene units
between the reactive groups, has been demonstrated to form a DNA G-X-C/C-X-G interstrand cross-link analogous to those formed by the nitrogen mustards. 88
Haddow and Timmis89 reported in 1953 that busulfan was active against chronic myelogenous leukemia. Busulfan was for many years the principal agent used to treat
this disease, before being replaced by the use of hydroxyurea 90 and interferon-a,91 both of which have proved to be more effective than busulfan. The most frequent
use of busulfan in cancer therapy today is in high-dose therapy for many tumors, including chronic myelogenous leukemia, in conjunction with bone marrow or stem
cell transplantation. For this application, high doses of busulfan are combined with cyclophosphamide, total body irradiation, or other agents. 18,92,93 and 94 The
effectiveness of busulfan for this purpose is undoubtedly related to its marked myeloablative properties, 95 the mechanistic bases of which are not understood.

Until recently, busulfan was available only as an oral preparation, but intravenous preparations are now available. For hematopoietic transplantation, busulfan is
usually given as 1 mg/kg every 6 hours for 4 days, for a total dose of 16 mg/kg. Peak concentrations of busulfan after each dose are approximately 10 μM. 96 High
doses of busulfan have been associated with venoocclusive disease of the liver. This syndrome consists of hepatomegaly, jaundice, ascites, and hepatic failure with a
high mortality rate.97 Grochow et al.96 have demonstrated that pharmacokinetic monitoring and dose adjustment of the busulfan can markedly reduce the incidence of
venoocclusive disease.

NITROSOUREAS

The members of the nitrosourea group of therapeutic alkylating agents are related to the alkylnitrosoamines and similar compounds that have long been known to be
carcinogenic. Methylnitrosoguanidine and methylnitrosourea are monofunctional alkylating agents and were found to have modest antitumor activity. 98,99
Montgomery100 and others101,102 evaluated a number of analogues of these compounds and demonstrated remarkable antitumor effects of bischloroethylnitrosourea
(BCNU; Fig. 19.3-8) against mouse tumors, and particularly against intracerebral tumors, which had been refractory to most agents because of the blood–brain
barrier.100,101 and 102 BCNU was found to produce interstrand cross-linking of DNA,103 which has been demonstrated to occur through the spontaneous generation of a
chloroethyldiazonium species104 and the series of reactions illustrated in Figure 19.3-9.105 As illustrated, this interstrand cross-link occurs between a guanylate in DNA
and the base-paired cytidylate in the other strand of the DNA. 106

Bischloroethylnitrosourea

BCNU (carmustine; see Fig. 19.3-8) demonstrated activity against brain tumors clinically 107 and has continued to be used in the treatment of gliomas and other brain
tumors. BCNU has also been used in the treatment of multiple myeloma108 and in high-dose therapy in conjunction with bone marrow and stem cell transplantation. 109
BCNU can also be administered to brain tumors by direct injection 110 and by the implantation of biodegradable polymers containing BCNU into the brain. 111

Cyclohexylchloroethylnitrosourea

Cyclohexylchloroethylnitrosourea (CCNU, lomustine; see Fig. 19.3-8) is a more lipid-soluble nitrosourea. It is administered orally and is used in the treatment of brain
tumors.112,113

Methylcyclohexylchloroethylnitrosourea

Methylcyclohexylchloroethylnitrosourea (semustine; see Fig. 19.3-8) is an oral investigational drug that has been used in the treatment of gastrointestinal tumors. 114
N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosourea
N'-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N-(2-chloroethyl)-N-nitrosourea (nimustine; see Fig. 19.3-8) is more water-soluble than the other chloroethylnitrosoureas
and has been used for the treatment of CNS tumors by the intraarterial 115 and intrathecal routes.116

Clinical Pharmacology

As a single agent, BCNU is usually used in a dose of 125 to 200 mg/m 2 every 6 to 8 weeks. In combination with doxorubicin for multiple myeloma, a dose of 30 mg/m2
every 3 to 4 weeks has been used.117 After doses in the range of 100 mg/m2, peak plasma concentrations are in the range of 5 μM. 118 For high-dose therapy of breast
cancer, BCNU is given at a dose of 600 mg/m2 in combination with cyclophosphamide and cisplatin. 119 After this dose of BCNU, the peak plasma levels of BCNU have
been shown to be approximately 5 μM.120 Phenobarbital has been demonstrated to increase the clearance of BCNU 121 and to decrease the toxic and therapeutic
effects. CCNU is administered in doses similar to those of BCNU. The parent CCNU has not been detected, but the peak concentrations of the ring hydroxylated
metabolites are approximately 3 μM after doses of 130 mg/m2.122

Specific Toxicities

Hematopoietic toxicity of the nitrosoureas is severe and is delayed, with the nadir of the granulocytes occurring approximately 5 to 6 weeks after administration. 123
This finding indicates that these agents selectively damage a very primitive hematopoietic precursor.

HYDRAZINE AND TRIAZINE DERIVATIVES

The hydrazine and triazene derivative compounds are analogous to the nitrosoureas in that they decompose spontaneously or are metabolized to produce an alkyl
carbonium ion, which alkylates DNA. Hydrazine and its substituted analogues are known carcinogens 124 that inhibit gluconeogenesis in cells 125 and have been
promoted as antitumor agents.126 However, objective preclinical and clinical studies have not supported a significant antitumor effect 127,128 for hydrazine analogues in
general.

Procarbazine

Procarbazine is a phenylhydrazine derivative that was initially developed as an inhibitor of monoamine oxidase but was found to have significant antitumor activity in
preclinical models and clinically ( Fig. 19.3-10).129 Procarbazine was one of the components of the first effective combination chemotherapy regimen, MOPP, for
Hodgkin's disease.6 The agent is currently used for the treatment of Hodgkin's disease 6,130 and for the treatment of primary brain tumors.113,131 Procarbazine has been
demonstrated to be metabolized to a DNA-methylating agent, 132,133 and 134 which is most likely methylazoxyprocarbazine.135,136 Since procarbazine is a monoamine
oxidase inhibitor, patients can experience CNS depression 137 or stimulation138 and acute hypertension, especially after the ingestion of tyramine-rich foods.

Dacarbazine

Dacarbazine , or DTIC [(dimethyltriazeno)imidazole-carboxamide], is a triazene derivative that is metabolized by microsomal N-demethylation, predominantly in the
liver, to an intermediate that spontaneously decomposes to release a methyldiazonium that methylates DNA ( Fig. 19.3-11).139,140 and 141 Dacarbazine is used in the
regimen of doxorubicin, bleomycin, vinblastine, and dacarbazine for the treatment of Hodgkin's disease 130,142 and for the treatment of malignant melanoma.119,143
FIGURE 19.3-11. Generation of methyl diazonium from the triazenes dacarbazine and temozolomide.

Temozolomide

Temozolomide is a triazene analogue that spontaneously decomposes to produce a methyl diazonium ion, as illustrated in Figure 19.3-11.144,145 This compound may
produce a more homogeneous distribution of the short-lived MITC [(methyltriazeno)- imidazole-carboxamide], which is spontaneously generated from temozolomide at
all sites, than does dacarbazine, which is metabolized to MITC in the liver. The principal toxicities seen in phase I trials have been neutropenia and thrombocytopenia,
and tumor responses were seen in those trials 146,147 in patients with glioma and melanoma. Phase II trials in patients with gliomas have shown response rates of 20%
to 30%,148,149 but phase II trials in patients with sarcomas 150 and pancreatic cancer151 did not demonstrate significant responses.

These agents exert their toxicity predominantly through the methylation of the O 6 position of guanylic acid in DNA. Therefore, cells that contain significant
O6-alkyltransferase or are deficient in mismatch repair will be resistant to them (as discussed in the section Mechanisms of Toxicity and Drug Resistance).

Procarbazine is an oral preparation and used in the MOPP regimen for Hodgkin's disease at a dose of 100 mg/m 2/d for 14 days.142 Because of its complex
metabolism, pharmacokinetic studies have been limited. Dacarbazine is an intravenous preparation and is used in the regimen of doxorubicin, bleomycin, vinblastine,
dacarbazine for Hodgkin's disease at a dose of 375 mg/m2/d for 15 days.142 For the treatment of malignant melanoma, a dose of 200 to 250 mg/m2/d for 5 days is used
and, at this dose, peak plasma concentrations of dacarbazine are approximately 30 μM. 152 This agent has been used as a single agent with bone marrow
transplantation at a dose of 2000 mg/m2.153 At this dose, the maximum plasma concentration of dacarbazine was 800 μM.153 Temozolomide is usually given orally at
150 to 250 mg/m2/d for 5 days. Reid et al. 154 measured peak concentrations of MTIC of 0.5 to 5 μM after administration of these doses of temozolomide. 154 Baker et
al.155 studied the pharmacokinetics of 14C-labeled temozolomide and found peak concentrations of temozolomide of approximately 30 μM and peak concentrations of
MTIC of approximately 1 μM.

MECHANISMS OF TOXICITY AND DRUG RESISTANCE
REACTION WITH CELLULAR MOLECULES

The alkylating agents are potent electrophiles and react with many electron-rich molecules within the cell to be inactivated. The principal such molecule is glutathione
(GSH), a tripeptide with a free cysteine sulfhydryl that is present at millimolar concentrations in cells ( Fig. 19.3-12). This small nucleophile is known to react with and
inactivate virtually all the therapeutic alkylating agents, and a correlation between elevated cellular GSH concentrations and resistance to nitrogen mustards has been
demonstrated.156,157 The GSH S-transferase enzymes catalyze the conjugation of GSH with electrophiles, and increased activity of this class of enzymes enhances
GSH-mediated resistance.158,159 and 160 The GSH conjugates of specific alkylating agents have been characterized, 161,162 and 163 and the specific isoenzymes of GST that
catalyze their formation have been characterized. 164,165,166,167 and 168

Buthionine sulfoximine is an inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the GSH synthesis pathway, and decreases the GSH
concentration in cells. 169 Exposure to this compound sensitizes both normal and tumor cells to alkylating agents. 156,170,171 In a phase I clinical trial, buthionine
sulfoxime has been shown to increase the hematologic toxicity of melphalan 172 and is currently in further clinical trials to determine whether this agent can increase
the clinical antitumor efficacy of melphalan.

Cells can also be sensitized to alkylating agents by exposure to inhibitors of GSH S-transferases, 173,174 and a clinical trial of the GSH S-transferase inhibitor
sulfasalazine with melphalan demonstrated increased nausea and vomiting but no increase in hematopoietic toxicity. 175 The membrane transporter multidrug
resistance protein is known to mediate the efflux of GSH conjugates from the cell, 176 and Barnouin et al.177 have demonstrated that this system can transport the GSH
conjugates of chlorambucil and melphalan from cells. The observations suggest that modulation of these systems could enhance the efficacy of alkylating agents.

Kelley et al.178 demonstrated that transfection of metallothionein into cells produced increased resistance to chlorambucil and melphalan. Subsequently, Yu et al. 179
have demonstrated that the thiol groups of metallothionein will bind melphalan and phosphoramide mustard. 180 It has also been demonstrated that exposure of cells to
zinc will increase metallothionein concentration in the cell and increase resistance of the cells to melphalan, doxorubicin, and cisplatin. 181

ENHANCED DNA REPAIR: O6 ALKYLATION

Another mechanism of cellular resistance to alkylating agents is repair of the DNA damage that the agents produce. The most defined mechanism of cellular repair of
alkylating agent damage is that of the enzyme O6-alkylguanine-alkyltransferase. As illustrated in Figure 19.3-13, this enzyme can remove an alkyl group from the O6
position of guanine, and the alkylated enzyme is then rapidly degraded. 182 This mechanism has been shown to be effective in protecting normal and tumor cells from
the carcinogenic and toxic effects of DNA methylating agents, such as temozolomide and procarbazine. 183 Erickson et al.184 demonstrated that this enzyme would also
remove the 6-chloroethyl lesion produced by the alkylation of guanine by the chloroethylnitrosoureas and produce resistance to these compounds, and this
observation has been confirmed and extended.185

It has been shown that such compounds as O6-benzylguanine will be acted on by O 6-alkylguanine-DNA alkyltransferase (see Fig. 19.3-13) to remove the benzyl
group186 and that the enzyme will be rapidly degraded and depleted. Such compounds have been demonstrated to reverse tumor resistance due to O 6AT to the O6
alkylating agents invitro and invivo,187,188 and clinical trials of the combination of such agents and O 6-methylguanine are currently in progress. 189,190

However, inhibitors of O6AT enhance the hematopoietic toxicity of O6 alkylating therapeutic agents. Hematopoietic stem cells have been successfully transfected with
O6AT variants that are resistant to O 6-benzylguanine and related compounds. 191 The hematopoietic systems of animals populated with these cells are resistant to the
combination of O6-benzylguanine and BCNU,192 and clinical trials of this approach to improve the efficacy of chloroethylnitrosoureas and methylating agents are
planned.

CROSS-LINK REPAIR

The use of alkaline elution and other techniques ( Fig. 19.3-14) has demonstrated that DNA interstrand cross-links produced by nitrogen mustards can be removed in
bacteria193 and mammalian cells.194 The mechanism of such repair has not been elucidated, but nucleotide excision repair 195 and poly(adenosine diphosphate–ribose)
polymerase196 appear to play a role.

Caffeine and related compounds have been demonstrated to enhance the cytotoxicity of nitrogen mustard. 197 This effect was associated with abrogation of G2 arrest.
O'Connor et al.198,199 demonstrated that the G2 arrest associated with nitrogen mustard resistance was associated with decreased activity of cdc2 kinase in the
resistant cells. Caffeine has also been shown to inhibit nucleotide excision repair by binding to the subunit that recognizes the damage and helps to mediate this
repair activity.200 Elevated Bcl-2 has also been associated with nitrogen mustard resistance. 201

A medulloblastoma cell line has been demonstrated to be resistant to activated cyclophosphamide (4-hydroperoxycyclophosphamide) on the basis of increased
removal of DNA interstrand cross-links.34,202 This cell does not appear to repair cross-links produced by BCNU and busulfan, indicating that the recognition of the
nitrogen mustard cross-link is fairly specific.

IN VIVO RESISTANCE

Kobayashi et al.203 and St. Croix et al.204 have described resistance to alkylating agents and other antitumor agents that is associated with aggregation of tumor cells.
This resistance is present when the tumor cells are growing invivo or in three-dimensional invitro culture with adherence between the cells but is not present when the
cells are dispersed in two-dimensional culture. This type of resistance has also been associated with increased metastatic potential. 205

COMMON TOXICITIES

Toxicities that are associated with specific alkylating agents are described in the discussions of the individual agents. The toxicities common to the alkylating agents
as a class are described here.

HEMATOPOIETIC TOXICITY

The usual dose-limiting toxicity for an alkylating agent is hematopoietic toxicity. As described, cyclophosphamide usually produces a relatively rapid nadir of the
granulocytes, with recovery within 3 weeks after a single dose or short course. 32,33,206 Cyclophosphamide is also relatively platelet-sparing. The reason for the relative
hematopoietic sparing properties of cyclophosphamide is the high concentrations of the enzyme aldehyde dehydrogenase in hematopoietic stem cells and
megakaryocytes.30,31

The nitrosoureas produce an unusual delayed hematopoietic toxicity, with nadirs of both granulocytes and platelets at 5 to 6 weeks after administration. 123 Severe
granulocytopenia and thrombocytopenia are also characteristic of busulfan. 207 An interesting characteristic of busulfan is its relative sparing of lymphocytes. The
different hematopoietic effects of alkylating agents, except for the characteristics of cyclophosphamide, are not explained but suggest significant differences in
selectivity of the agents for hematopoietic precursors.

GASTROINTESTINAL TOXICITY

The alkylating agents frequently produce nausea and vomiting, although this effect is usually not as severe as with the platinum agents. Cyclophosphamide produces
severe nausea and vomiting in some patients, but these patients usually tolerate chlorambucil, which is clinically less emetogenic. The nausea and vomiting produced
by alkylating agents are known to be mediated significantly through the CNS. 208,209 With the higher doses of alkylating agents used in bone marrow transplantation,
increased nausea and vomiting are seen but can usually be controlled by corticosteroids and the newer antiserotonin antiemetics. 210,211 and 212 The alkylating agents
can cause significant toxicity to the gastrointestinal mucosa and produce mucositis, stomatitis, and diarrhea, especially with the high doses of melphalan and thiotepa
used in bone marrow transplantation.213

GONADAL TOXICITY

The alkylating agents can produce significant gonadal toxicity. The characteristic testicular lesion in men is depletion of germ cells without damage to the Sertoli cells,
which was first described with nitrogen mustard in 1948. 214 This lesion is also seen, often in association with oligospermia or aspermia, after treatment with other
alkylating agents.215,216 Spermatogenic dysfunction is reversible in some patients. 217,218
Women treated with alkylating agents may develop amenorrhea associated with a marked decrease in ovarian follicles. 215,219,220 This complication and its irreversibility
increase with the age of the woman.221

PULMONARY TOXICITY

Interstitial pneumonitis and fibrosis were initially reported as a consequence of busulfan therapy but have subsequently been reported to occur after therapy with
melphalan,222 but have subsequently been reported to occur after therapy with melphalan, 223 chlorambucil,224 cyclophosphamide,225,226 mitomycin C,227 and
BCNU.228,229 The clinical manifestations of this toxicity are dyspnea and a nonproductive cough, which can progress to cyanosis, pulmonary insufficiency, and death.
The syndrome has particularly been associated in frequency and severity with high doses of BCNU. 230,231 The greater pulmonary toxicity of BCNU may be due to the
spontaneous decomposition of BCNU, which produces chloroethyl isocyanate in addition to the alkylating chloroethyl diazonium moiety described. 232 Chloroethyl
isocyanate is an analogue of methyl isocyanate, a known pulmonary toxin that produced many deaths when released in an industrial accident in Bhopal, India. 233

ALOPECIA

Alopecia from chemotherapy was first described after administration of dimethylmyeleran, an analogue of busulfan. 234 The alkylating agents now most associated with
alopecia are cyclophosphamide and ifosfamide. Feil and Lamoureux 235 examined the alopecia-producing effects of metabolites and analogues of cyclophosphamide
and proposed that the alopecic effect was due to the facile entry of a lipophilic metabolite (now known to be 4-HC) into the hair follicles. This hypothesis is consistent
with the fact that vincristine, doxorubicin, and the taxanes, all associated with alopecia, are fairly lipophilic.

TERATOGENICITY

All the therapeutically used alkylating agents are teratogenic in animal studies. 236,237,238 and 239 A review of the literature in 1968 found that 4 of 25 children born to
mothers who received alkylating agents during the first trimester of pregnancy had fetal malformations. 240 On the basis of the limited information available, women
treated with an alkylating agent during the first trimester of pregnancy may have a risk as high as 15% of having a malformed infant. Administration of alkylating
agents during the second and third trimesters has not been associated with increased fetal malformations. 241,242 More recent reviews support the lack of malformations
produced by treatment during the second and third trimesters, 243,244 and one review cites 19 women treated during the first trimester with no infant malformations. 244

CARCINOGENESIS

In the 1970s, there were reports of acute leukemia occurring in patients who had been treated with alkylating agents, 245,246,247,248 and 249 and subsequent experience has
confirmed the occurrence of this complication. The incidence of leukemia is difficult to estimate because of the variety of agents, doses, and combinations used but is
probably approximately 5%. In one group of 12 ovarian cancer patients receiving a high dose of melphalan, 4 developed acute leukemia. 248 In one report, the
incidence of leukemia was found to be higher after melphalan treatment than after cyclophosphamide therapy. 250 This observation may be related to the stem
cell–sparing properties of cyclophosphamide. 30 An increased frequency of solid tumors also occurs after alkylating agent therapy. 251,252

IMMUNOSUPPRESSION

In 1921, Hektoen and Corper253 reported an inhibitory effect of sulfur mustard on antibody production. While all the alkylating agents produce some degree of
immunosuppression, cyclophosphamide is the most immunosuppressive.254 Cyclophosphamide and chlorambucil are the alkylating agents most commonly used for
the treatment of autoimmune diseases.255,256,257,258 and 259
Selective inhibition of immunosuppressor cells with low doses of an activated analogue of cyclophosphamide and with melphalan has been demonstrated in
vitro260,261,262 and 263 and in vivo263,264 and enhancement of the immune response has been shown in vivo.263 For this reason, low doses of cyclophosphamide have been
used in conjunction with immunotherapy.265,266 Because of its potent immunosuppressive properties, cyclophosphamide has long been used in preparative regimens
for allogeneic stem cell transplantation for malignancy 267 and more recently for the autologous transplantation of autoimmune disease. 268,269 The use of high doses of
cyclophosphamide without stem cell support has now been reported to produce complete remissions in autoimmune diseases. 21,270,271