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New Antifolates in Clinical Development

New Antifolates in Clinical Development

ABSTRACT: Numerous new antifolate drugs have been developed in an attempt to overcome the potential mechanisms of tumor cell resistance to methotrexate, which can include decreased drug transport into cells; decreased polyglutamation, leading to increased drug efflux from cells; decreased drug affinity for folate-dependent enzymes; mutations of dihydrofolate reductase (DHFR), a key enzyme required for the maintenance of adequate intracellular reduced folate levels that is inhibited by methotrexate; and increased expression of the DHFR protein. Promising antifolate compounds undergoing clinical testing as anticancer agents include trimetrexate (which was recently approved by the FDA for the treatment of Pneumocystis carinii pneumonia), edatrexate, piritrexim, Tomudex, and lometrexol. The mechnisms of action, dosage, pharmacokinetics, clinical toxicity, and antitumor activity of these drugs are profiled. [ONCOLOGY 9(7):649-665, 1995]

Introduction

Antifolates, such as methotrexate, were one of the earliest classes
of drugs developed for clinical use in cancer chemotherapy. Today,
methotrexate is still used extensively in the treatment of human
leukemia, breast cancer, head and neck cancer, choriocarcinoma,
osteosarcoma, and lymphoma. Antifolate compounds also have important
clinical utility outside the realm of oncology, in the treatment
of such diverse diseases as rheumatoid arthritis, psoriasis, bacterial
and plasmodial infections, and opportunistic infections associated
with AIDS.

Currently, numerous promising antineoplastic antifolate drugs
are in clinical development, and many of these agents have interesting
and unique mechanisms of action. An understanding of the cellular
pharmacology of these agents will help the practicing clinician
effectively use these new drugs as they become available for the
treatment of human malignancies.

Methotrexate

Mechanism of Action

The most widely used and best understood antifolate in cancer
therapy is methotrexate, which differs from the essential vitamin,
folic acid, by having an amino group substituted for a hydroxyl
at the 4-position on the pteridine ring (Figure 1). This change
transforms the enzyme substrate into a tight-binding inhibitor
of dihydrofolate reductase (DHFR), a key enzyme required to maintain
adequate intracellular levels of reduced folates [1].

Dihydrofolate reductase is critically important because folate
molecules are biochemically active only in their fully reduced
form as tetrahydrofolates. The tetrahydrofolates are essential
cofactors that donate one-carbon groups in the enzymatic biosynthesis
of thymidylate and purine nucleotide precursors for DNA synthesis
(Figure 2). One reduced folate, 5,10-methylenetetrahydrofolate,
participates in the reaction catalyzed by the enzyme thymidylate
synthase (TS), which converts deoxyuridylate (dUMP) into thymidylate
(dTMP). As a consequence of this reaction, 5,10-methylenetetrahydrofolate
undergoes oxidation to dihydrofolate, which must then be reduced
by DHFR back to tetrahydrofolate in order to replenish the intracellular
reduced-folate pools.

Another reduced folate, 10-formyltetrahydrofolate, serves as a
one-carbon donor for the reactions catalyzed by glycinamide ribonucleotide
(GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases.
These enzymes are involved in the de novo biosynthesis of purine
nucleotides. Thus, inhibition of DHFR by antifolates can lead
ultimately to the decreased production of several essential precursors
for DNA synthesis.

Both methotrexate and naturally occurring folate compounds can
undergo intracellular metabolism to polyglutamate derivatives.
These reactions, catalyzed by the enzyme folylpolyglutamyl synthase
(FPGS), attach up to six glutamate residues to the pteridine ring,
which help trap these molecules within the cell by decreasing
their efflux. Methotrexate polyglutamates are also potent direct
inhibitors of DHFR, as well as other folate-dependent enzymes,
such as TS and GAR and AICAR transformylases. Furthermore, DHFR
inhibition leads to the accumulation of dihydrofolate polyglutamates
within the cell, which can directly inhibit the folate-dependent
enzymes involved in the synthesis of thymidylate and purine nucleotides
[2,3]. Thus, inhibition of DNA synthesis by the antifolates is
a multifactorial process, resulting from the partial depletion
of the intracellular reduced folate pool and from the direct inhibition
of folate-dependent enzymes.

Administration of exogenous reduced folates, such as leucovorin
calcium, to methotrexate-treated nonmalignant cells efficiently
replenishes the reduced folate pool and directly competes with
the drug-induced inhibition of folate-dependent enzymes. This
is the biochemical rationale for the clinical use of leucovorin
rescue to prevent severe toxicity in high-dose methotrexate chemotherapy
regimens.

Ultimately, the depletion of thymidylate and purine nucleotide
cofactors for DNA synthesis leads to a cessation of DNA synthesis,
but it is not clear whether this action alone is enough to induce
cell death. Lethal DNA damage resulting from a drug-induced lack
of essential nucleotides may occur because of ineffective DNA
repair, or because of misincorporation of uracil deoxynucleotides
into DNA. Additional studies on this important issue are clearly
necessary. Furthermore, the relative importance of the inhibition
of thymidylate or purine nucleotide synthesis in the generation
of methotrexate-induced cytotoxicity has yet to be defined.

Cellular Pharmacology

Influx into Cells--At least two distinct, energy-dependent,
carrier-mediated transport systems are responsible for the uptake
of methotrexate into mammalian cells [4]. The classic reduced-folate
carrier system, which transports reduced folates, such as 5-methyltetrahydrofolate,
and antifolates, such as methotrexate, has affinity constants
in the micromolar range, and it is a relatively less efficient
transporter of folic acid. A second carrier system that utilizes
the hydrophobic membrane-associated binding protein, the human
folate receptor (hFR), has a higher affinity (nanomolar range)
for folic acid and reduced folates than it does for methotrexate.
Some tumors, such as human ovarian cancers, overexpress the hFR
on their cell surface.

The exact contribution of these two transport pathways to the
uptake of methotrexate in clinical cancer chemotherapy is an area
of intensive research. However, methotrexate resistance resulting
from the decreased activity of one or both of these transport
systems has been demonstrated in vitro, suggesting that transport
deficiencies may be clinically important [1]. More lipophilic
antifolates, such as trimetrexate and piritrexim, are not substrates
for these carrier-mediated folate transport systems, and can enter
cells by either passive diffusion or by other transport mechanisms.
Cell lines that are resistant to methotrexate because of decreased
transport generally retain their sensitivity to these more lipophilic
antifolate agents [5].

Efflux from Cells--Efflux of methotrexate from the cell
is also mediated by several different transport systems, some
of which are clearly distinct from the influx systems. Methotrexate
efflux is not associated with the P-glycoprotein, multidrug resistance
(MDR) system that has been described for numerous other antineoplastic
agents. However, drug efflux is heavily influenced by the degree
of methotrexate polyglutamation. As mentioned previously, both
normal and malignant cells contain the enzyme FPGS, which can
add glutamyl groups in a gamma peptide linkage to the pteridine
ring in naturally occurring folates and in some antifolate drugs
as well. This reaction serves two important functions: First,
it facilitates the accumulation of intracellular folates by converting
them into large anions that are less readily transported out of
the cell. Second, polyglutamation enhances the affinity of methotrexate
for several folate-dependent enzymes, including TS and AICAR transformylase.

Polyglutamation of methotrexate occurs more slowly compared to
naturally occurring folates; however, the resulting methotrexate
polyglutamates have extremely long intracellular half-lives, and
can be detected in some tissues several months following a single
drug administration. The accumulation of methotrexate polyglutamates
in normal tissues, such as the liver, reduces the natural polyglutamation
of endogenous folates, and may account for the chronic hepatotoxicity
associated with methotrexate therapy [1]. In addition, the selective
nature of methotrexate cytotoxicity may result, in part, from
the increased polyglutamation of methotrexate in cancer cells
compared to normal tissues. The decreased ability to polyglutamate
antifolates also appears to be an important mechanism of clinical
drug resistance [6].

Binding to DHFR--Methotrexate is a competitive inhibitor
of DHFR that binds noncovalently to this enzyme at the same binding
site as the normal substrate, dihydrofolate. This interaction
also depends on the intracellular concentration of reduced nicotinamide
dinucleotide phosphate (NADPH), which is a normal cofactor
for DHFR. Point mutations both inside and outside of the enzyme's
active site have been identified that decrease the binding affinity
of DHFR for methotrexate [1]. Thus, mutations in the DHFR enzyme
are another potential mechanism of resistance to antifolates.

Sensitivity to methotrexate cytotoxicity is highly dependent on
the absolute amount of DHFR enzyme within the cell. Both human
tumors and cancer cell lines that have increased levels of DHFR
due to gene amplification are relatively resistant methotrexate
[7]. More subtle mechanisms may also exist that allow cells to
acutely increase DHFR expression in response to antifolate treatment.

The expression of DHFR appears to be controlled at least partially
by the binding of the DHFR protein to its own mRNA (Figure 3)
[8]. This binding of DHFR prevents further synthesis of the DHFR
protein, resulting in an autoregulatory negative feedback loop.
Excess normal substrate, such as dihydrofolate, or inhibitors,
such as methotrexate, interfere with the binding of the DHFR protein
to its mRNA and block this negative feedback loop, thereby allowing
the synthesis of DHFR protein to continue unimpeded. This permits
normal cellular function to be maintained, even in the presence
of inhibitors such as methotrexate or other antifolates. The importance
of this mechanism in the development of clinical antifolate resistance
must be defined.

For a detailed discussion of the pharmacokinetics of methotrexate,
including specific dosage and scheduling information, readers
are referred to reference 1.

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