Folic acid antagonists, often called antifols, are cytotoxic drugs used as antineoplastic, antimicrobial, anti-inflammatory, and immune-suppressive agents. While several folate antagonists have been developed, and several are now in clinical trial, methotrexate (MTX) is the antifol with the most extensive history and widest spectrum of use. MTX is an essential drug in the chemotherapy regimens used to treat patients with acute lymphoblastic leukemia, lymphoma, osteosarcoma, breast cancer, choriocarcinoma, and head and neck cancer, as well as being an important agent in the therapy of patients with nonmalignant diseases, such as rheumatoid arthritis, psoriasis, and graft-versus-host disease.
Folate antagonists are thought to act as cytotoxic drugs by interfering with one or more biosynthetic steps involving folate coenzymes. Theoretically, a folate analogue might function in one of several ways, for example, by competing with folates for uptake into cells, by inhibiting the formation of folate coenzymes, or by inhibiting one or more reactions that are mediated by folate coenzymes. Thus far, however, all the clinically important antineoplastic folate analogues that have been developed appear to work primarily by inhibiting dihydrofolate reductase (DHFR). This blocks the reduction of dihydrofolate to tetrahydrofolate. The former is generated from the latter, which is the methyl donor and is partially oxidized during the de novo synthesis of thymidine monophosphate from deoxyuridine monophosphate.
In addition to the clinical usefulness of MTX and other folate antagonists,
knowledge of the mechanism of action and the pharmacology of these agents
has yielded additional dividends in terms of information on important
principles of cancer chemotherapy and mechanisms of drug resistance of
general applicability to all types of antineoplastic agents. MTX, the
prototype antifol, has probably been studied as intensively as any drug
employed in present-day clinical medicine; a Medline search revealed more
than 23,000 citations from 1966 to the present that include methotrexate
as a title word.
This chapter will primarily review the clinical use and metabolism of
MTX and discuss several analogues which have been developed to overcome
resistance or have a target other than DHFR. For the reader needing additional,
specific information, several recent complete texts are available.1 Finally,
due to limitation in space, the references in this chapter have been selected
to highlight key “classic” papers and representative clinical and basic
manuscripts. The authors apologize to their friends and colleagues in
the field since only 1% of the 23,000 citations will be reflected in this
bibliography.
Historical overview
The observation of serum folate deficiency among patients with acute leukemia prompted some investigators in the early 1940s to postulate that acute leukemia might be a result of a deficiency of this vitamin, as in the case of pernicious anemia, in which megaloblasts, resembling leukemic blasts, predominate in the bone marrow. The availability of crystalline pteroylglutamic acid (PGA, folic acid), first isolated from spinach in 1941 and synthesized in 1945,2 prompted investigators to test this compound for possible antineoplastic activity. It was soon recognized that administration of these substances not only was ineffective, but possibly even accelerated the course of the disease in patients with chronic myelocytic leukemia and acute leukemia.3
Efforts to treat these leukemias, thus, turned to creating a folate deficiency. Some encouraging results were obtained through use of folate-deficient diets, either alone or in combination with a weak folate antagonist, probably 7-methyl PGA. Soon after, aminopterin (4-amino-4-deoxy PGA) was synthesized and found by Farber and his co-investigators to be effective in producing temporary remissions in more than half his patients with acute leukemia.4 This demonstration was a landmark in cancer chemotherapy; it provided the first demonstration that an antimetabolite could be an effective antineoplastic agent, and provided the stimulus for the development of other antimetabolites as possible antitumor agents.
Since the initial study demonstrating the usefulness of aminopterin in the treatment of acute leukemia of childhood, there has been a sustained interest in and a continued re-evaluation of this and other folate antagonists. In studies with mice bearing the L1210 leukemia, MTX (4-amino-4-deoxy-10-methyl PGA; amethopterin) was found to have a more favorable therapeutic index than aminopterin, and, thus, for the last 40 years, MTX has supplanted aminopterin in the clinic. A randomized comparison of the relative efficacy and morbidity of these two drugs has never been done in the clinic.
In recent years, an ever-broadening use for MTX has evolved. Although details of its therapeutic use are given elsewhere in this text, the broad spectrum of use of this drug deserves emphasis. The drug has been used not only for the treatment of neoplastic diseases but also for the treatment of certain non-neoplastic conditions, such as rheumatoid arthritis, asthma, and generalized psoriasis, and as an immunosuppressive agent.5,6 MTX is the drug of choice in the treatment of choriocarcinoma, where its use provided the first demonstration of drug cure of cancer. Approximately 50% of these patients appear to be cured with the use of MTX alone. MTX is used in curative combination regimens to treat patients with acute lymphocytic leukemia (ALL) and lymphoma, and in combination regimens to treat advanced breast cancer, bladder cancer, and cancer of the head and neck. The drug is also used in high doses with leucovorin (LV) rescue as a component of adjuvant therapies for breast cancer and osteosarcoma.
The newer antifols are rationally designed analogues of folates or MTX. They have been synthesized either in an effort to overcome cellular resistance to MTX or to inhibit the metabolism of folate or folate-mediated reactions instead of or in addition to DHFR. Some of these have been approved as antimicrobials or antineoplastic agents, and others are still in clinical trial
Mechanism of Actions
The prototypic antifol DHFR inhibitor is a 4-amino–substituted pterin compound. The substitution of an amino moiety for 4-hydroxyl results in a folate analogue with several thousand-fold increase in affinity for DHFR. The Ki of MTX for DHFR is below 10-10 M, well below the Km of the natural substrate, dihydrofolate, which is in the micromolar range. It is this remarkable increase in affinity compared with the natural substrate that resulted in MTX being considered a “stoichiometric inhibitor” of DHFR.7,8
By stoichiometrically inhibiting DHFR, a key enzyme in the thymidylate cycle (Fig. 46.1), MTX disrupts a critical step in the synthesis of DNA.9 In rapidly dividing cells, the inhibition of thymidylate biosynthesis leads to a decrease in thymidine triphosphate pools, a decrease in DNA synthesis, and eventually cell death.10,11 Inhibition of tetrahydrofolate formation leading to the inhibition of purine synthesis and rapid cell death has also been described as occurring in lymphoblasts treated with high doses of MTX.12
Although the Kd of the DHFR-NADPH complex for MTX is on the order of 10-11 L/M at mildly acid pH, MTX is required in molar excess to the target enzyme DHFR in order to completely inhibit tetrahydrofolate synthesis.13,14 This is because the pH is higher in the intact cell, and blockade of the enzyme results in increased amounts of dihydrofolate and its polyglutamates, which compete with the inhibitor for binding to the enzyme.7,8 Inasmuch as DHFR is in excess in most cells and is much more efficient in terms of enzyme turnover than thymidylate synthase, only a small fraction of the total enzyme need remain catalytically functional to maintain the intracellular reduced folate pool.15
In recent years, the important role of polyglutamylation as a determinant of MTX sensitivity has been elucidated (Fig. 46.2).16,17 A single enzyme, folylpolyglutamate synthetase (FPGS), appears to be responsible for adding glutamate residues in ?-carboxyl linkage to both folate coenzyme and MTX and other analogues with a glutamate moiety.18–21 This enzyme process, by which up to seven or eight additional glutamate molecules are added to folate coenzymes or MTX, serves to add additional negative charges to these molecules, thus markedly reducing efflux.22 In addition, MTX polyglutamates bind as tightly to DHFR as does MTX and may dissociate less rapidly from DHFR than MTX.23,24 MTX polyglutamates are also potent inhibitors of other folate-requiring enzymes, including glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases and thymidylate synthase (TS).25 Dihydrofolate polyglutamates and the formylated form of this coenzyme (10-formyl dihydrofolate), which increase after MTX blockage of DHFR, are also potent inhibitors of TS and GAR transformylase.26
In light of recent interest in angiogenesis as an important target for antineoplastic therapy, it is worthwhile to note that MTX has long been used as an anti-inflammatory agent at doses well below those thought to be cytotoxic.27 It may be that this anti-inflammatory effect and some degree of its antineoplastic activity are due to its ability to inhibit endothelial cell proliferation at physiologically attainable concentrations, as was first documented in 1989
Biological Chemistry
Mammalian cells require reduced-folate coenzymes for replication. Folate, as a 1-carbon carrier, is central to the de novo synthesis of both purines and pyrimidines. Folate deficiency can result in apoptosis and/or increased mutagenesis.29 Inhibition of DNA precursor synthesis by MTX and its polyglutamate forms results in inhibition of DNA synthesis in both normal and neoplastic cells. RNA and protein synthesis continue, leading to unbalanced cell growth and “megaloblast” or giant cell formation, followed by cell death unless the folate pool is restored.
Given the important role of folates in the synthesis of DNA, it is not surprising that the intracellular content of many of the enzymes involved in folate metabolism and function fluctuate with the cell cycle and are under the same controls as other proteins important to DNA synthesis and mitosis. TS, DHFR, and FPGS, for example, are known to increase as cells move from the G1- into the S-phase.
A relative lack of FPGS may explain the observation that a cell population with a large number of G0 cells would be less affected by the same concentration and time of exposure to MTX than a population with relatively few resting cells. Thus, MTX, like other inhibitors of DNA synthesis, is most effective when it is employed in the treatment of neoplastic disease characterized by rapidly growing populations with a small percentage of cells in the resting or G0 phase.30
Some selectivity and, thus, the successful use of this drug in certain cancers have attended the use of high intermittent pulses of the drug, which has little effect on bone marrow and the gastrointestinal tract, organs which, if normal, are characterized by having a substantial number of stem cells “out of cycle” or in the so-called G0 state. This relative kinetic selectivity may be lost when either the bone marrow or the gastrointestinal mucosa is compromised by previous radiographic or drug therapy, or by infiltration with tumor cells or infection.
On the other hand, the same data concerning the cell-cycle dependence of the antineoplastic function of antifolates argue against high-dose intermittent pulsing and in favor of more prolonged, lower-dose administration, as is used in the successful treatment of patients with acute lymphoblastic leukemia and those with non-neoplastic disorders, such as arthritis. With more prolonged dosing schedules, it will more often be the case that sufficient antifolate will be available to inhibit DNA synthesis as each fraction of the asynchronously growing tumor enters the cell cycle. The theoretical advantages of emphasizing prolonged dosage schedules over total dose have been borne out by clinical data,31 although the optimal schedule clearly may be different for different classes of tumors.
The effectiveness of MTX against certain tumors (e.g., carcinoma of breast, osteogenic sarcoma) is difficult to explain on the basis of a rapid growth rate. In the latter condition, high-dose therapy appears to be necessary, with folinic acid (citrovorum, LV) rescue. High plasma levels of MTX may lead, by passive diffusion, to a greater intracellular concentration of the drug, resulting in polyglutamate formation. The process may result in retention of a high concentration of MTX polyglutamates, thus leading to prolonged inhibition of DHFR. In contrast, normal gut and marrow progenitor cells appear to have a limited capacity to polyglutamylate and thus retain MTX.
Pharmacokinetics of MTX
Because interpatient variation in MTX pharmacokinetic parameters can explain much of the variation in MTX toxicity and efficacy, MTX is one of few anticancer agents for which pharmacokinetic data are routinely used in clinical practice.
Plasma concentrations of MTX have been well correlated with toxicity.33 In multivariate analyses, high-risk concentrations have been associated with poor urinary alkalinization and emesis, suggesting that understanding the basis for variation in pharmacokinetics should help clinicians tailor supportive care to avoid excess toxicity.
Similarly, clinical use of pharmacokinetic data is clearly related to improved MTX activity. Retrospective analysis of children with ALL shows that lower MTX clearance 34 and higher MTX concentrations35 are associated with lower risk of relapse. Even more intriguing are data from a prospective randomized trial in patients with ALL comparing dosing of BSA with individualized dosing based on pharmacokinetic data, which showed significantly improved complete continuous remission rates in the individualized therapy arm.36 It is possible, however, that these results are protocol specific and need to be replicated using other protocols.
Absorption
In contrast to older studies that showed good absorption at low doses of MTX,37,38 recent studies have emphasized the relatively poor and unpredictable nature of the absorption of this drug after oral administration.39–41 The extent of absorption may be less than 50%, even at low doses (< 15 mg/m2) and further decreases with increasing oral doses. Following oral administration, peak plasma concentrations may occur 1 to 5 hours after a dose of 15 to 30 mg/m2. It has also been found that a dose and schedule of 25 mg/m2 given every 6 hours results in a plasma concentration greater than 0.5 ?M in more than 85% of pediatric patients with ALL.42 Moreover, over an 18-month time period, there was no malabsorption as a result of therapy, which also included daily 6-mercapopurine. Food, nonabsorbable antibiotics, bile salts, and a shortened intestine transit time may decrease the rate and extent of MTX absorption.43 Therefore, it is suggested that the drug be taken with clear liquids on an empty stomach.
Distribution
After intravenous (IV) administration, MTX distributes within an initial volume of approximately 18% (0.18 L/kg of body weight), with a variable steady-state volume of 40 to 80% of body weight.44 The initial distribution phase has a T1/2 of 30 to 45 minutes; the beta T1/2 is 3 to 4 hours. Elimination and clearance from third-space and enterohepatic recirculation is 6 to 20 hours.45 MTX binding to plasma proteins, especially to albumin, is approximately 50%.46 The 7-hydroxymetabolite of MTX is 90% bound to plasma proteins but apparently does not interfere with MTX binding to plasma proteins at the concentrations found in patients. The highest tissue-to-plasma concentrations found in humans are in the liver and kidney, followed by the gastrointestinal tract. Greater plasma levels in humans, as compared with those in mice, are attributed to less rapid excretion in the bile and by the kidney and a longer residue time in the small intestine.47,48 Prolonged plasma levels after high-dose MTX infusions in humans have been attributed to decreased transit rate secondary to gastrointestinal obstruction.
The variability in kinetics after high-dose MTX may also be a function of the canalicular multi-organic acid transporter (cMOAT), which is also known as MRP2. Like mdr, MRP2 is a member of the ATP-binding cassette (ABC) superfamily of transport proteins. By transporting organic acids, with co-transport of glutathione, MRP may serve a protective role in chemical toxicity and oxidative stress. Abnormal functioning of this protein results in Dubin Johnson syndrome.49 Abnormal MRP2 activity may affect the pharmacokinetics and pharmacodynamic profile of campthothecins, CDDP, and vinca alkaloids, and overexpression of MRP2 has been shown to confer resistance to MTX in vitro.50 It may be that there are pharmacogenetic polymorphisms in MRP2 that account for the large interpatient variability in pharmacokinetics of high-dose MTX.
Patients with pleural or peritoneal effusions may be at increased risk for developing toxicity to high-dose MTX as a result of “third spacing,” or MTX trapping in the infusion, and slow release leading to sustained MTX concentrations in serum.51 This phenomenon is more of a problem when high doses of the drug are administered. In these circumstances, higher doses and prolonged rescue with LV may be necessary, until the serum level of MTX decreases to less than 5 × 10-8 M.
After high doses of MTX (> 6 g/m2), serum concentrations in the range of 10-4 to 10-3 M are achieved.52,53 At these concentrations, the active transport of this drug is saturated, limiting further influx of the drug to passive diffusion. These high extracellular MTX concentrations inhibit the uptake of reduced folates, including exogenously administered LV; this explains the need for larger doses of LV to reverse MTX action caused by the competitive nature of this interaction at the transport level. Studies of MTX metabolism in lymphoblasts in vitro have also shown that too high an extracellular drug concentration can impede the metabolism of MTX to a polyglutamate54 The clinical significance is not known, but the observation further calls into view whether the use of high-dose MTX, other than to penetrate a sanctuary site such as the central nervous system (CNS) or in the therapy of osteosarcoma, is worth the expense and risks.55,56
The passage of MTX from plasma to cerebrospinal fluid (CSF) is poor, and MTX does not achieve cytocidal concentrations in the cerebrospinal fluid (CSF) after conventional doses (15 to 30 mg/m2).57,58 CSF concentrations after MTX administration are dose related, and cytocidal levels are obtained with doses of 500 mg/m2 and higher. After high-dose systemic MTX administration, lumbar CSF and ventricular CSF concentrations were similar. As recently noted, high-dose MTX may be able to replace intrathecal administration for the treatment of patients with leptomeningeal disease.59
When MTX is administered by the lumbar route into the CSF, it distributes unreliably into the ventricles, while MTX given by an indwelling ventricular shunt provides reproducible therapeutic drug concentrations (> 10-6 M) for at least 48 hours.60 An improved dose schedule utilizing the administration of multiple small doses of intrathecal MTX has been suggested.61 Following intrathecal administration, MTX slowly exits into the systemic circulation with a T1/2 of 8 to 10 hours.62 Systemic toxicity can be observed if multiple doses of intrathecal MTX are administered without LV rescue. The pharmacology of intrathecal MTX and the amount of intraventricular MTX may be altered by overt meningeal leukemia and the positioning of the patient at the time of lumbar puncture.63
Among experimental agents, metoprine (2,4-diamino-5-(3'4'-dichlorophenyl)-6 methyl pyrimidine, DDMP) is highly lipid soluble and crosses the blood–brain barrier readily giving high CNS levels.64 Other antifolates, including trimetrexate and piritrexim, despite being more lipophilic than MTX, are only poorly transported into the CNS.65 Although MTX is accumulated poorly into the CSF, even small doses of LV given orally can increase CSF folates significantly. This systemic rescue, especially if given too early after MTX, may rescue cells in the CSF compartment.66 It should also be remembered that too much LV rescue (i.e., gram for gram with MTX) may lead to serious metabolic problems since the LV is calcium LV. Increased plasma calcium and an increased plasma pH may lead to further renal damage.67
The clinical observation that irradiation followed by MTX treatment may predispose patients to neurotoxicity (see below) may be a consequence of the effect of radiation therapy on the blood-brain barrier.68
Metabolism
The major metabolite of MTX is 7-hydroxy MTX (7-OH MTX) (see Fig. 46.2).69,70 This hydroxylation process is due to hepatic aldehyde oxidase and results in a much less active form of MTX, as it is only 1% as potent an inhibitor of DHFR as is MTX.71,72 The 7-hydroxy metabolite is less water soluble than is MTX and may contribute to the renal toxicity frequently seen after high doses of the antifolate.73
A second, less important pathway of metabolism of MTX occurs in the intestine. MTX is hydrolysed by bacteria to the pteroate (4-amino-4-deoxy-N10-methyl pteroic acid, dAMPA) and glutamic acid (see Fig. 46.2).74 Like 7-OH MTX, dAMPA is also a relatively inactive metabolite with approximately 1/200th the affinity of MTX for DHFR. Excretion of dAMPA in the urine accounts for only a small percentage of the dose administered (< 5%).
As mentioned, the third metabolic product of MTX that occurs via intracellular conversion is MTX polyglutamate. MTX polyglutamates are at least as potent inhibitors of DHFR as is MTX, and have a slower rate of dissociation from DHFR than does MTX.24 MTX polyglutamates are not found in plasma or urine because of the activity of ?-glutamyl hydrolase(s) (GGH, conjugase) in plasma that convert folyl and MTX polyglutamates to monoglutamates. Like MTX, 7-OH MTX is also polyglutamylated intracellularly, and retention of these polyglutamate forms could contribute to MTX cytotoxicity.75,76
Biliary Excretion
Following IV administration of doses of 30 to 80 mg/m2, 0.4 to 20% of the administered dose can be recovered in bile. Less than 10% of MTX is recovered in feces collected over 24 hours.77 The enterohepatic recycling of MTX has been estimated using the d-isomer as a reference marker for nonabsorbable drug.78
As discussed earlier, it is interesting to note that the mechanism of biliary excretion of MTX seems to be the canalicular multi-specific organic anion transporter (cMOAT), a member of the ABC family of proteins which is identical to the multi-drug resistance protein, MRP2
Inadvertent Drug Interactions
Several drugs, including antibiotics, may increase toxicity when used
with MTX and should be avoided in cancer patients, if possible.79 Obviously,
drugs such as aspirin that increase the possibility of bleeding in patients
who are at risk of thrombocytopenia should be avoided. Deleterious and
even fatal reactions have been reported due to interaction between MTX
and nonsteroidal anti-inflammatory drugs, in particular with naproxen
and ketoprofen.80–83 This increased toxicity may be due to decreased renal
elimination, possibly as a result of competition for renal secretion.84
Other commonly used organic drugs may also potentiate MTX toxicity, such
as phenylbutazone, salicylate, and probenecid.85,86 Probenecid was shown
to increase the efficacy of MTX in tumor-bearing mice, but it has not
been used clinically with this goal in mind.87
Methylxanthines, such as caffeine or aminophylline, may be useful to decrease
toxicity in the setting of delayed MTX clearance. MTX administration has
been shown to increase adenosine content,88 possibly by inhibiting AICAR
transformylase, thus allowing an increase in AICAR, which alters adenosine
metabolism. Since adenosine will decrease GFR, adenosine receptor competitive
antagonists, like the methylxanthines, will act as a specific diuretic
to increase MTX elimination.89
Increased toxicity was also reported when trimethoprim, the antibacterial agent, was used together with MTX. Presumably, this antifolate, with only weak binding affinity to mammalian DHFR, lowers folate stores, especially in patients with subclinical folate deficiency, making marrow cells more susceptible to MTX-induced toxicity.90,91 Patients receiving MTX should also avoid alcohol because of the risk of hepatic fibrosis and cirrhosis.
Adverse Effects
Hematologic Toxicity
Tissues that are self-renewing—that is, the bone marrow and epithelial cells—are at highest risk for damage by the folate antagonists. Bone marrow progenitor cells of all lineages are affected by MTX, but neutropenia usually predominates. Recovery after a single dose is usually rapid, taking place 14 to 21 days following a nadir that occurs approximately 10 days after drug administration. The effects on marrow are dose related, but there is considerable variability among patients. Subclinical folate deficiency, usually caused by poor nutrition; impaired renal function (pretreatment with cisplatin is a risk factor); a stressed marrow owing to previous radiotherapy, chemotherapy, or infection; and the use of trimethoprim-sulfa for Pneumocystis carinii prophylaxis may predispose patients to hematologic (and gastrointestinal) toxicity to MTX. Young patients usually tolerate MTX better than older individuals do, a fact presumably related to clearance of the drug by the kidneys. The administration of LV, before 42 hours have elapsed, if in an appropriate dose, may prevent or lessen MTX toxicity and allow larger doses of the antifolate to be administered.122
Gastrointestinal Toxicity
Mucositis is a common side effect of MTX treatment and usually becomes manifest 3 to 5 days following a dose or course of the drug. This is an early sign of MTX toxicity, and the drug should be discontinued when it occurs. Subsequent doses should not be increased unless the mucositis is grade 1 or less. More severe gastrointestinal toxicity is manifest by diarrhea, which may progress to severe bloody diarrhea. When this occurs in association with neutropenia, patients are at high risk of sepsis and death. Such patients should be hospitalized and managed vigorously with fluids and antibiotics. These severe side effects generally occur in a setting of renal damage, usually a consequence of high doses of MTX, but may also occur in patients treated with conventional doses. MTX blood levels and serum creatinine levels should be monitored and appropriate doses of LV administered, along with the supportive measures instituted (see below). Nausea and vomiting, even with high doses of MTX, are usually mild to moderate, and most patients do not require antinausea medication.
Renal Toxicity
Conventional-dose MTX regimens, not requiring LV, were occasionally reported to cause renal toxicity, presumably as a direct effect of MTX on the renal tubular epithelium.123 With the introduction of high-dose regimens requiring LV rescue, renal toxicity leading to delayed MTX clearance sometimes resulted in severe marrow and gastrointestinal toxicity, occasionally fatal, especially in adults.55 This toxicity is believed to be due to precipitation of MTX and its less soluble metabolite, 7-OH MTX, in the tubules, as well as to a possible direct effect of this drug on the renal tubule.73 The use of vigorous hydration, often with osmotic diuresis and alkalinization of urine to increase solubility of MTX and 7-OH MTX, has markedly ameliorated this problem. Occasional patients, even with this regimen (Table 46.3), exhibit renal impairment. Through careful monitoring of MTX and creatinine serum levels, these patients may be identified and larger doses and prolonged duration of LV employed to prevent toxicity.
Extremely high levels of MTX (> 10-3 M) are difficult to rescue, even with high doses of LV.124 Hemodialysis and peritoneal dialysis have proved ineffective in substantially lowering MTX plasma levels.124 Charcoal hemoperfusion columns have been used successfully in a small number of patients.126 Oral charcoal and cholestyramine have also been used to bind MTX in the gut, thus limiting enterohepatic recirculation and toxicity.127 Thymidine (1 to 3 g/m2/d) is also capable of rescuing patients from MTX toxicity, but this metabolite is not generally available.128 Carboxypeptidase G1 or more recently, the recombinant form, G2, an enzyme capable of cleaving the peptide bond in MTX resulting in glutamate and dAMPA (see Fig. 46.2), has also been used to rapidly lower MTX levels, but dAMPA is even less soluble than MTX.129 This enzyme has also been proposed for use as a “rescue” agent, on the basis of studies in experimental tumors.130 When given in combination with thymidine and LV, carboxypeptidase G2 was recently shown to be highly effective in 20 patients at high risk for developing life-threatening MTX toxicity after the onset of MTX-induced nephrotoxicity and delayed MTX excretion.131
Hepatotoxicity
Chronic low-dose continuous treatment with MTX has been associated with portal fibrosis and, in some patients, frank cirrhosis.132 The basis for this liver damage is not known, but it may result from interference with folate homeostasis, since acute MTX hepatotoxicity in rats is reversed by choline administration.133 Cirrhosis has been reported in patients with psoriasis, rheumatoid arthritis, and ALL treated with long-term continuous oral MTX.134 Alcohol and other hepatotoxic drugs should be avoided in this patient population. Intermittent schedules with pulse therapy appear to decrease the incidence of fibrosis and cirrhosis.135 In the absence of other complicating conditions as noted above, it may be that the hepatotoxicity has been somewhat exaggerated. Acute elevations of liver enzymes (SGOT) commonly occur several days after treatment with high-dose MTX but rapidly return to normal and do not appear to predict for chronic liver toxicity.136 On the basis of a recent study of children with ALL, it also appears that in the absence of hepatitis C, there are no significant permanent sequelae in patients with increased liver enzymes, even to 10 to 20 times the normal.137
Central Nervous System Toxicity
Although intrathecal MTX has been used extensively to treat patients
with meningeal leukemia, its use has been associated with neurotoxicity,
ranging from mild to severe. In cases of inadvertent overdosing (>
100 mg), fatalities have been reported.
The most common immediate side effect of intrathecal MTX administration,
made manifest by severe headache, fever, meningismus, vomiting, and CSF
pleocytosis, is thought to be caused by a chemical arachnoiditis directly
or perhaps by the release of adenosine, which is a potent autocoid in
the CNS. This effect of adenosine has been ameliorated by systemic administration
of low doses of methylxanthines, such as aminophyllin and theophylline,
which act as competitive antagonists at adenosine receptors.89 Dosage
adjustment or switching to cytosine arabinoside may be required if these
symptoms persist (see Chapter 175).
More serious neurotoxicity has been observed in 5 to 10% of patients receiving 12 to 15 mg/m2 of MTX intrathecally, consisting of motor paralysis of the extremities, cranial nerve palsies, seizures, and even coma. Inasmuch as these signs are seen mainly in adult patients with active meningeal disease, it is often difficult to distinguish these side effects from meningeal leukemia. However, 10 to 20% grade 3-4 neurotoxicity was also encountered in pediatric patients with ALL without CNS involvement being treated with 1 g/m2 IV in addition to intrathecal administration.138 This subacute toxicity usually arises during the second or third week of intrathecal treatment and has been attributed to slow CSF clearance of MTX.139 Recent biochemical studies based on the notion that MTX causes a “biochemical folate deficiency” have led to finding increased amounts of homocysteine in the plasma and CSF of some patients treated with MTX. Since homocysteine is an excitotoxic amino acid (glutamate analogue) that activates the N-methyl-D-aspartate receptor (NMDA), it may be that the subacute neurotoxicity of MTX can be ameliorated by an antagonist of the NMDA receptor. Dextromethorphan is such a drug. Anecdotal success has resulted in the development of a prospective trial of this antitussive agent as a means to eliminate at least some of the neurotoxicity of MTX.140
A severe chronic demyelinating encephalopathy has also been observed
in children treated prophylactically with intrathecal MTX who have also
received prophylactic cranial irradiation (> 2,000 cGy).141 These patients
develop dementia and limb spasticity, and even coma, months or years after
intrathecal MTX treatment. Computed tomography scans show cortical thinning,
ventricular enlargement, and diffuse intracerebral calcifications.142
Rarely, encephalopathy has been reported in patients treated only with
high-dose intravenous MTX. Acute transient cerebral dysfunction occurring
several days after high-dose systemic MTX treatment has also been reported;
in these patients, signs (paresis, aphasia, seizures) usually resolve
within 2 to 3 days.143,144
In patients who receive an MTX overdose intrathecally (> 100 mg), immediate
CSF removal with ventricolumbar perfusion is indicated.145 Recently, intrathecal
use of carboxypeptidase G2 was shown to markedly decrease mortality in
animals given a lethal dose of MTX intrathecally and may be the preferred
treatment for this complication when the enzyme is available.146 Intrathecal
or systemic LV is not indicated in these cases, since it is unlikely that
this toxicity is attributable to inhibition of DHFR.
Pulmonary Toxicity
Although uncommon, pulmonary toxicity due to MTX has been described and has been noted even in patients receiving low-dose oral MTX for rheumatoid arthritis.147,148 The clinical picture usually consists of cough, dyspnea, fever, and hypoxemia. Chest radiographs are nonspecific but show patchy interstitial infiltrates. Pneumocystis carinii must be ruled out, especially in patients also receiving steroids. Histologic examinations show diffuse interstitial lymphocytic infiltrates, giant cells, and noncaseating granulomas. In some patients, a peripheral eosinophilia is observed, raising the possibility that this is an allergic pneumonitis. The process may progress to fibrosis, and it is important to discontinue MTX while the pulmonary toxicity is reversible. Some patients have been retreated without recurrence of the problem.
Skin Toxicity
Skin toxicity to MTX occurs in 5 to 10% of patients, consisting of an erythematous rash, characteristically noted on the neck and upper trunk. The rash may be pruritic and relatively insignificant and usually lasts for several days. In other instances, especially when related to other signs of severe MTX toxicity, it may progress to severe bullous formation and desquamation.149 Sun-exposed areas may be more sensitive to MTX.150 A cutaneous vasculitis after intermediate-dose MTX has also been reported.151
Teratogenic and Mutagenic Effects
MTX is known to be a potent abortifacient, especially if administered during the first trimester of pregnancy. However, there is no indication of a higher than normal incidence of fetal abnormalities in women who have been successfully treated with MTX for choriocarcinoma. These women also have not had a higher-than-normal incidence of secondary malignancies. Thus far, there is no evidence that MTX has any mutagenic or carcinogenic effects.152
Miscellaneous Toxicity
Osteoporosis has been reported with chronic low-dose MTX administration.153 Fever, seizures, recall of radiation toxicity or phototoxicity, and anaphylactoid reactions have been reported with high-dose administration.154 Pleuritic and left-upper-quadrant pain, presumably attributable to splenic capsule inflammation, has been reported with a moderately high-dose regimen
Resistance to Antifolates
Although the development of effective chemotherapeutic regimens including MTX has significantly improved the therapy of a number of different malignancies (Table 46.4), achieving prolonged disease-free survival is still difficult, even in chemotherapy-sensitive diseases. The efficacy of MTX, as with other antineoplastic agents, is ultimately limited by either inherent resistance or resistance acquired during the course of therapy. Resistance to MTX has been documented to occur as a result of changes at each step of MTX transport into the cell, metabolism to MTX-polyglutamates, interaction with the target enzyme DHFR, and breakdown of polyglutamates. Additionally, because the activity of both DHFR and FPGS fluctuates with cell cycle, it is becoming increasingly clear that deregulation of cell cycle genes may have a profound effect on antimetabolite resistance.
The recent application of molecular biologic techniques, coupled with the cloning of the human genes coding for many of the proteins involved in MTX metabolism, has made it possible to study the genetic alterations underlying the phenotypic changes associated with cellular resistance to MTX.
Intrinsic Resistance to MTX
A number of lines of investigation are beginning to explain the range of intrinsic resistance to MTX seen across different tumor types both clinically and in vitro. Comparisons of leukemic blasts obtained at diagnosis from patients with AML with those obtained from patients with ALL suggest that differing abilities to form long-chain MTX polyglutamates to some degree explains the relative clinical resistance of AML to MTX, compared with ALL. AML blasts have been shown to accumulate less long-chain MTX polyglutamate than ALL blasts,155,156 with no differences in MTX transport or in DHFR. In addition, fresh human tumor cells from patients with soft tissue sarcoma as well as certain human cancer cell lines naturally resistant to MTX, especially to short-term exposures, have been similarly found to have a low capacity to form long-chain MTX polyglutamates.157,158
Recent investigations are beginning to clarify the relative contributions of alterations in the activity of the enzymes FPGS and GGH to MTX resistance due to decreased MTX-polyglutamate accumulation. Higher MTX-polyglutamate accumulation in B-lineage ALL blasts as compared with T-lineage blasts may be explained by the finding of higher FPGS activity in B-lineage blasts.159,160 The possibility that different isoforms of FPGS are expressed in different tissues, explaining differences in intrinsic sensitivity to MTX, is supported by the finding of differences in FPGS affinity for MTX between AML and ALL cell lines and blast samples161 and between resistant and sensitive sarcoma cell lines.162
The fact that the ratio of FPGS to GGH enzyme activity in leukemic blasts obtained at diagnosis correlates better with the ratio of long-chain to total MTX polyglutamate accumulation than the activity of either enzyme alone suggests that the balance of the two enzymes determines the steady-state MTX polyglutamate accumulation and intrinsic MTX sensitivity.163
Impaired ability to transport MTX into cells through the reduced folate carrier (RFC) also can cause intrinsic resistance. Decreased expression of the RFC mRNA has been documented by quantitative RT-PCR in osteosarcoma samples at initial biopsy,164 possibly explaining the clinical observation that MTX is ineffective against this disease at conventional doses but is effective in higher doses. Additionally, mutations in the RFC gene corresponding to altered transport function have been documented both in resistant cell lines165 and in leukemic blasts at diagnosis.166
Finally, lack of the retinoblastoma protein (pRB) frequently deleted or altered in many tumor types, may play a role in MTX resistance. In the absence of pRB levels of the transcription factor E2F increase, resulting in an increase in transcription of several genes involved in DNA replication, including DHFR.167,168 When a human osteosarcoma cell line lacking pRB is transfected with the cDNA encoding pRB, its intrinsic resistance to MTX is reversed.169
Acquired Resistance to MTX
Along with natural resistance, acquired drug resistance remains a major obstacle to effective chemotherapy. For example, more than 95% of pediatric patients with B-lineage ALL achieve a complete remission, but 5-year disease-free survival rates are only 75 to 80% using MTX-based continuation therapy. Re-treatment of relapsed patients with the same agents is less effective because of the development of drug resistance.
Four major mechanisms of acquired resistance to MTX have been described in experimental tumors and clinical samples: (1) an increase in DHFR activity due to amplification of this gene, (2) a decrease in the uptake of MTX due to either a decreased influx of MTX through the RFC or a decrease of long-chain polyglutamate formation, or (3) a mutation that results in an altered DHFR with decreased binding to MTX25.
Amplification of the DHFR gene, resulting in increased levels of the enzyme, has been identified as a common mechanism of acquired MTX resistance. Since the original description of the DHFR gene amplification in MTX-resistant mouse tumor cells,170 a number of mouse, hamster, and human MTX-resistant cell lines have been described, with increased DHFR and amplification of the DHFR gene as a mechanism of MTX resistance.171–173 Unstable or reversible resistance due to gene amplification has usually been associated with the presence of “double minute” or centromereless chromosomes containing the DHFR amplicon, while high-level stable resistance has been associated with an abnormal banding region, often referred to as a homogeneously staining region (HSR).174–176 It has also been demonstrated that gene amplification as a mechanism of resistance occurs in some patients treated with MTX.177–179
Some clinical studies have shown a strong correlation between the amount of MTX polyglutamates formed in blasts and disease-free survival in children with ALL.17,180 More recent studies, however, have produced contradictory data.181 Thus, it needs to be reasserted that such data are protocol-specific and need to be interpreted cautiously when extrapolating to newer protocols. Cell lines have been described that are resistant to MTX solely because of impaired polyglutamylation.93 These cells were obtained by a more clinically relevant selection schedule consisting of short-term, high-dose treatments with MTX, rather than by continuous exposure to this drug. Recent studies have indicated that the basis of the defect in these cells is an alteration in the enzyme folylpolyglutamate synthetase.182
Although defects in polyglutamylation have been described in several MTX-resistant cell lines, the resistance of these cells has usually been found to be attributable to a combination of mechanisms.183,184 Decreased levels of MTX polyglutamates in cells may also result from increased breakdown; indeed, both intrinsic and acquired resistance to MTX in cell lines have been attributed to increased levels of g-glutamyl hydrolase activity.158,185
At the point of entry into the cell, either mutations or deletions in RFC could result in decreased uptake of MTX and MTX resistance. The development of a competitive displacement flow-cytometric assay using the fluorescent lysine analogue of MTX, N?-(4-amino-4-deoxy-N10-methylpteroyl)-N?-(4'-fluoresceinthiocarbamyl)-L-lysine or PT 430 has provided a sensitive method of detecting transport resistance to MTX.186 The availability of the cDNA encoding the human RFC187and the development of quantitative RT-PCR to measure RFC mRNA expression have allowed further characterization of the molecular basis for decreased transport. Using these techniques, decreased transport of MTX through the RFC has been shown to be a common mechanism of acquired resistance to MTX in leukemic blasts from patients with relapsed ALL.188
Although several MTX-resistant cell lines have been found to possess an altered DHFR that has a decreased affinity for MTX, only few altered human DHFRs have been characterized in any detail.189–193 Point mutations in several cell lines, including human cells, have been detected that cause a change in the binding of MTX to the enzyme, and have usually involved amino acids that bind to the inhibitor by hydrophobic interaction.5 The first mutation of an amino acid in a nonactive site region (trp?gly) associated with MTX resistance in L1210 cells was recently reported.194 Evidence for mutations in the gene for DHFR as a mechanism for resistance in blast cells from patients has not yet been documented, but sensitive methodology (polymerase chain reaction, amplification of DHFR cDNA) to allow sequencing and detection of possible mutations has become available only recently.5,195It may be possible to develop antifolates with specificity for altered DHFR enzymes.5,196 These efforts will be guided by a detailed knowledge of the structure of this enzyme and its interaction with substrates and inhibitors.197–199
It is also possible to convert normal marrow to a state of resistance to MTX by transfection with an altered DHFR in a viral vector.200,201 These experimental studies open up the possibility of clinical trials with these viral constructs, with the goal of allowing increased doses of MTX to be safely administered to patients with cancer.
Strategies to overcome resistance to MTX
Understanding the molecular bases of normal folate physiology, MTX cytotoxicity, and MTX resistance is allowing and guiding the rational design of new folate antagonists and strategies to selectively target resistant cells. Thus, to overcome resistance, the newer antifolates have been designed to have one or more of the following properties: increased transport into the cell by either increased affinity for RFC or independence of RFC, independence of polyglutamylation or increased polyglutamylation by virtue of increased affinity for FPGS, increased inhibition of DHFR or TS, or increased inhibition of enzymes responsible for purine synthesis. The work will undoubtedly be guided by computer graphics, using crystallographic data from the target enzymes.
Aminopterin
Before proceeding to the second- and later-generation folate antagonists, there are data to support returning to an older antifol. After producing remarkable, though transient, responses in the 1940s, aminopterin (AMT) was abandoned because MTX had more predictable toxicity and a better therapeutic index in a murine model. No randomized comparison of the two was ever performed in the clinic.
Preclinical models show greater potency for AMT. Leukemic blasts from pediatric patients have been shown to accumulate AMT better than MTX,203 probably because AMT has higher affinity for FPGS than does MTX. Given the demonstrated importance of antifolate accumulation by malignant cells as a prognostic indicator,17 AMT has again entered clinical trials. A recent phase I and pharmacokinetic trial of AMT was performed in patients with refractory malignancies, using divided oral dosing. Good oral bioavailability was shown, with mucositis as the dose-limiting toxicity at 2.5 mg/m2 every 12 hours for two doses weekly.204 When vitamin A and delayed LV rescue were added and the dose was reduced to 2 mg/m2, mucosal toxicity was greatly diminished. Responses were observed in a patient with metastatic endometrial adenocarcinoma and another with AML. Phase II trials are currently underway in endometrial cancer and acute leukemia.
Newer Inhibitors of DHFR
MTX is an extremely potent inhibitor of DHFR, and while it may be possible to develop inhibitors that are more tightly bound or may irreversibly inactivate this enzyme, unless these compounds possess other advantages (i.e., more avid uptake and/or more efficient retention by malignant cells as compared with normal cells), selectivity may not improve.
10-ethyldeazaaminopterin (10-EDAM, Fig. 46.3), developed by Sirotnak and associates, was chosen for clinical trial after detailed structure activity studies demonstrated that hydrophobic substitutions at the N10 position of aminopterin resulted in improved uptake and retention (polyglutamylation) by tumor cells, as compared with normal cells.205 The drug is now under active clinical investigation, and encouraging response rates have been noted in patients with non–small cell lung cancer, head and neck cancer, breast cancer, and malignant fibrous histiocytoma.206–208 One limitation to its use might be that it may be relatively ineffective against MTX-resistant cells, since it utilizes the same carrier mechanism for transport and is polyglutamylated by the same enzyme as is MTX. Additionally, significant neurotoxicity was seen when it was given at high doses.
The nonclassic antifolates, trimetrexate and piritrexim (see Fig. 46.3), currently in phase II trials, are also potent inhibitors of DHFR, but cross the cell membrane by passive or facilitative diffusion rather than by the reduced-folate transport carrier.205,209 Consequently, these antifolates are still effective cytotoxic agents against MTX-resistant cells when the mechanism of resistance is impaired transport, decreased polyglutamylation, or even low-level amplification of DHFR.210–212 Cells resistant to MTX owing to a mutation in the enzyme leading to decreased binding of the inhibitor may or may not be cross-resistant to trimetrexate, depending on the nature of the mutation.5 However, trimetrexate, unlike MTX, is a substrate for the MDR efflux pump and so may show cross-resistance to other MDR substrate antineoplastic agents.213 These drugs also differ from MTX in that they are not substrates for polyglutamate synthetase; therefore, retention depends on other factors. Certain sensitive tumor cells appear to retain trimetrexate in concentrations that are in excess of that required to inhibit DHFR completely, after efflux in drug-free medium. The mechanism of this retention has not been determined.
Another intriguing possibility currently under investigation is that some human tumors, either intrinsically or after treatment, may resemble the Pneumocystis organism in that they are unable to transport reduced folates and MTX well.214 Similar to the approach currently being taken to treat Pneumocystis infections,215 the coadministration of trimetrexate and LV would be nontoxic to the host but could be cytotoxic to such tumors.216,217 Trimetrexate is also under investigation as a modulating agent. On the basis of experimental studies that showed that trimetrexate followed by 5-FU and high-dose LV led to synergistic cell kill, when MTX followed by 5-FU and LV did not. Acceptable toxicity and responses were noted even in this phase I investigation.218 Phase II studies of this combination have found activity in advanced colorectal carcinoma, with manageable toxicity.219,220 Phase III studies are in progress, comparing 5FU/LV with this combination.
Inhibitors of Other Folate-dependent Enzymes
During recent years, other targets for the development of folate antagonists have been identified, including TS, GAR, and AICAR transformylase, and methionine synthetase.25 Potent inhibitors of TS and GAR transformylase have been synthesized and are now under active investigation (see Fig. 46.3).
Inhibitors of Thymidylate Synthetase.
The potential advantages of folate inhibitors of TS over 5-FU are that these agents are not incorporated into RNA, and that the greater DUMP levels that may result as a consequence of inhibition of this enzyme might increase, rather than decrease, the inhibition of TS.221 On the basis of a series of structure-activity studies and toxicity studies in animals, the folate analogue, N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-l-glutamic acid (D1694, ralitrexed), was chosen for further clinical trials and has shown good clinical activity in colorectal carcinoma.222,223 Of interest is that ralitrexed, even more so than MTX, is a “pro-drug,” in that polyglutamylation increases cytotoxicity. Phase III studies have been completed, and activity comparable with 5-FU/LV has been demonstrated in patients with colon cancer. Ralitrexed has been licensed in Europe and Canada for use in the treatment of colon cancer.
Inhibitors of Purine Synthesis.
5–10–Dideazatetrahydrofolate (dDTHF) (Lometrexol) is also undergoing clinical trials (see Fig. 46.3). This compound is also a pro-drug; the addition of glutamates to the molecule markedly increases the inhibition of GAR transformylase.80 dDTHF is extremely potent, and low doses of this agent have produced delayed and prolonged marrow suppression in early clinical trials that was not predicted by rodent toxicity data.224 This may be due to its rapid accumulation by folate receptor positive cells, and to the relatively folate-deficient state of patients in contrast to that of rodent models. Administration of 1 to 5 mg of folic acid before Lometrexol has decreased toxicity.225
Multitargeted Antifolates.
Since much clinical resistance is related to amplification or mutation of single target enzymes, an antifol that inhibits more than one biosynthetic pathway and/or multiple steps within a single pathway has the theoretical advantage that the development of significant resistance would be less likely. The antifol analogue LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5- yl)ethyl] benzoyl]-L-glutamic acid; MTA) was initially developed as an inhibitor of GARFT, but was found to have inhibitory activity against AICAR transformylase, DHFR and TS as well. It is a substrate for RFC and can be polyglutamylated by FPGS. Polyglutamylation appears necessary for MTA to significantly inhibit TS and GARFT but not for inhibition of DHFR.
Results of phase I trials of MTA were reported in 1995.226 Toxicities were similar to those seen with other antifols, with neutropenia being the major dose-limiting toxicity. Non–dose-limiting toxicity included transient elevations in serum transaminases, mucositis, and rashes. The recently reported correlation between pretreament serum homocysteine and occurrence of grade 3 or 4 toxicity227 suggests a means of predicting excess toxicity from MTA that needs to be tested prospectively. A number of phase II studies are ongoing or completed in a wide range of tumor types and were recently reviewed.228 Responses to MTA have been seen in patients with a variety of solid tumor types
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