An Overview of Folate Metabolism: Features Relevant to the
`Action and Toxicities of Antifolate Anticancer Agents
`
`Hilary Calvert
`
`S INC E the observation of reduced folate levels
`
`in children with leukemia made by Farber et
`all in the 1940s, the study offolic acid metabolism
`and the act ion of antifolare drugs has been inti (cid:173)
`mately linked to the development of canc er ther(cid:173)
`apeutics. Folic acid plays a role in a wide range of
`metabolic pathways in various species. In humans
`it is an essential vitamin and functions primarily in
`the processes involved in cellular proliferation and
`amino acid metabolism . This review will focus
`mainly on those aspects of mammalian folate me(cid:173)
`tabolism relevant to cell proliferation since these
`are the most germane to the use of antifolares in
`cancer therapy . The textbook by R.L. Blakley- is a
`comprehensive work covering all aspects of folate
`metabolism.
`
`ASPECTS OF FOLATE METABOLISM
`
`Folace Pachways Associaced Wich Cell Proliferation
`
`Folic acid functions mainly in its fully reduced
`form, 5,6,7,8-tetrahydrofolate (FH 4; Fig I) . FH4
`serves as a carrier for one-carbon moieties within
`the cell. These are obrained from a variety of
`sources that include serine. In this reaction, serine
`hydroxvmerhyl
`transferase forms 5,1O-methylene
`converting
`(CH zFH4 ) while
`tetrahvdrotolare
`serine to glycine (Fig 2). CHzFH" may be con(cid:173)
`verted within the cell to one-carbon carrying fo(cid:173)
`late derivatives of various oxidation states. One of
`l Oeforrnvl retrahvdrofolare, is the substrate
`these,
`for two enzymes involved in the de novo synthesis
`of purines. These are glycinamide ribonucleotide
`formyl transferase (GARFT) and arninoimidaz ole
`carboxamidc ribonucleotide formyl
`tran sferase
`(AlCARFT). Thus , two of the carbon atoms in
`the purine skeleton are derived from folate . The
`folate-dependent reactions of purine synthesis use
`the carbon atom from the l Ovformyl group and
`release unsubsrituted tetrahydrofolate as the folate
`product. Thus,
`the folate molecule can then ac(cid:173)
`quire another carbon atom from serine and con(cid:173)
`tinue to cycle through GARFT and AICARFT,
`allowing continued purine synthesis without any
`overall consumption of folate . CHzFH4 is also the
`substrate for
`the enzyme thvmidyl ate synthase
`(TS) . Thymidyl arc synthase converts deoxyuri -
`
`Seminars in Oncology. Vol 26. No 2. Suppl 6 (April), 1999: pp 3·10
`
`dine rnonophosphate into thymidine monophos(cid:173)
`phate and is a key enzyme involved in cell prolif(cid:173)
`eration because it is the rate-lim iting step in the de
`novo synthesis of thym idvlare, which is required
`exclusively for DNA synthesis. The folate product
`of TS is not
`tctrahvdrofolare , but
`the oxidized
`form, dihydrofolate (FHz). This product cannot
`continue to functi on in folate metabolism until it
`is converted back to FH" by the enzyme dihydro(cid:173)
`folate reductase (DHFR).
`
`The Role of Folate and Antifolate Polyglutamates
`Folic acid possesses a glutamate residue shown
`at the right-hand side of the folate structures in Fig
`I. Naturally occurring folates within the cell are
`converted to polygluramare forms by the addition
`of glutamate residues via a l'-peptide linkage. An (cid:173)
`tifolares that possess a glutamate residue (known
`as classical antitolares) are also frequently con(cid:173)
`verted into their corresponding polyglutamate
`forms. The process of polvgluramation is accom(cid:173)
`plished by the enzyme folvlpolv-v-gluramate syn(cid:173)
`thetase. This reaction is illustrated in Fig 3 using
`the antifolate LY231514 (MTA) as an example.
`The process is analogous for natural folares and
`many other classical antifolates. In Fig 3, the car(cid:173)
`boxylate groups of the glutamic acid residue are
`shown in their ionized form, carrying a negat ive
`charge, showing that polyglutamation increases
`the overall negative charge on the folate molecule
`by one unit for each additional glutamate. The
`negatively charged polyglutarnates cannot cross
`the cell membrane and are therefore retained and
`concentrated with in the cell. This is probably the
`major physiologic role of polvgluramanon . Cells
`that are deficient
`in folylpolv-v-glutamate syn(cid:173)
`thetase are auxotrophic for the end products of
`
`From the Cancer Re.learch Unit , Department of Oncology,
`University of Newcastle upon Tyne.
`S/lORSored Iry Eli !..illy andComlXlny"
`DrCalvert is a consultant for and has received research funding
`from EliLilly and Company and Zeneca .
`Address reprint requesrs to Hilary Calvm , MD. Cancer Re·
`search Unit, Department of Oncology, FremlinglOn Place. Univer(cid:173)
`sity of Newcaslle u/xm Tyne, NE2 4HH.
`Col,yright © /999 /ry W.B. Saunders Company
`0093·7754/99/2602·0602$/0.00/0
`
`' J
`
`r-
`
`-
`
`ACCORD EX 1005
`
`

`

`
`
`HILARY CALVER‘
`
`FolicAcid
`
`COOH
`
`Dihydrofolic
`Acid (FHZ)
`
`Tetrahydrofolic
`Acid (FH4)
`
`tetrahydrofolic
`
`Acid (CHZFH4)
`
`10-formyl
`tetrahydrofolic
`
`ACld (CHOFH4)
`
`5-methyl-
`tetrahydrofolic
`acid
`
`Fig I. Forms of folic acid.
`
`m 5,1 O-Methylene
`
`4
`
`WXJIHDTOW
`
`“3:6;3'Otl'uj
`
`A" 'NH
`
`TEN—On!"
`will}:")2
`
`fin"
`
`coon
`
` )ENmH—ermgCOOH
`
`folate metabolism (thymidine, hypoxanthine, and
`glycine)‘ In addition to being retained within the
`cells, the polyglutamate forms of natural folates
`also may be better substrates for the various folate
`metabolizing enzymes.
`The formation of polyglutamates of those anti-
`folates that are subsn'ates for folylpoly-'y«glutainate
`synthetase also has profound effects on their 210
`tivity. The polyglutamates may be retained within
`the cell for very long periods,3 thus increasing the
`potency of the cytotoxic action of these com-
`pounds.
`in addition.
`the addition of glutamate
`residues frequently renders the compounds much
`more potent inhibitors of their target enzyme. For
`example,
`raltitrexed pentagluramate is
`roughly
`
`lOO-fold more potent as a T8 inhibitor than the
`parent molecule.4 The effects of polyglutamation
`on the potency of molecules such as these is so
`profound that they may be considered as prodtugs
`for their polyglutainate forms. Indeed, cellular re-
`sistance to antifolates can be caused by a reductioz‘.
`in the ability of the cell to form the polyglutamate
`derivatives.5 A more complete and in—depth re:
`view of polyglutamation and its relevance to can-
`cer therapy is given by Richard G. Moran else-
`where in this supplement.
`
`Cell Membrane Transport of Folaies and Antifolates
`Folates do not cross the cell membrane to an
`
`appreciable extent by passive diffusion but require
`
`

`

`
`
`OVERVIEW OF FOLATE METABOLISM
`
`PYRlMlDlNE SYNTHESIS
`\
`
`PURINE SYNTHESIS
`
`TMP
`
`DNA
`
`Fig 2. Metabolic pathways of folate metabolism.
`
`DNA
`
`RNA
`
`specific transport mechanisms. There are several
`mechanisms that have been characterized;
`these
`
`are reviewed in depth by Sierra and Goldman in
`this supplement. Of these,
`the most extensively
`characterized mechanism involves the reduced fo«
`
`late carrier (RFCI). This is an anion exchange
`concentrative process and is known to be capable
`of transporting methotrexate and a number of
`other antifolates as well as tetrahyclrofolate itself.
`
`Changes in this carrier that alter its relative affin-
`ity for antifolates have been shown to be a cause of
`drug resistance."’ The reduced folate carrier has a
`relatively low affinity for natural folates (1 to 5
`panel/L) compared with their physiologic extracel-
`lular concentrations (typically in the nanomolar
`region). A second mechanism of folate transport.
`the folate receptor, has a much higher affinity for
`folates which, after binding,
`are
`internalized
`
`SQWW: Pentaqlutamate
`
`H2N
`
`MTA
`
`0
`
`,NH 00.
`
`,,co
`
`,NH 00
`
`,,co
`
`C,NH 0°
`/,
`o
`
`0,,C-o-
`
`oo‘
`
`FPGS
`
`Fig 3. Formation of polyglutarnates.
`
`NH
`
`MTA
`
`

`

`6
`
`HILARY CA
`
`within a membrane vesicle and subsequently re,
`leased into the cytoplasm. Three genes for folate
`receptors have been cloned (see Sierra and Gold—
`man). Folate receptors may be responsible for the
`transport of some antifolates, for example, lomc'
`trexol. In addition to these two mechanisms, the
`
`level of folates and antifolates within the cell may
`be affected by an energycdependent efflux pump
`and by a low pH transporter.
`
`Actions of Various Antifolates
`
`Having been introduced nearly 50 years ago,
`methotrexate (Fig 4) is the antifolate with the
`
`longest history. It acts mainly by inhibitit
`DHFR. The result of this inhibition is tha
`tracellular folate accumulates in the forl
`
`dihydrofolate. There is a consequent inhib
`of the de novn synthesis both of purines
`thymidine. This may be due in part to a t
`notion in the intracellular pools of tetrflh\
`folates, but additionally, methotrexate pol
`tamates and the accumulated dihydrof-
`polyglutamates are capable of inhibiting
`TS and AICARFT directly.7'9 Characte
`cally,
`the intracellular pools of dihydrof-
`and deoxyuridinc will increase following e
`
`NH:
`
`NH
`
`/
`
`fljchbr“I
`
`CH
`3
`
`0
`
`cNHijj’fl‘mN
`
`CH
`3
`
`CW3
`
`NH:
`
`J\
`%
`
`0
`
`NHJICj/NtOYN
`Nib/KN
`NH
`
`NH
`
`NH,
`
`OH
`
`:J/NCHFQTNH
`
`0
`
`COO"
`
`COOH
`
`OOH
`
`COOH
`
`00H
`
`cm“
`
`OOH
`
`COOH
`
`con
`
`coon
`
`Methotrexate
`
`Raltitrexed
`
`(TomudexTM,
`ZD 1694)
`
`CB37TZ
`
`PDDF
`
`Lometrexol
`
`D DATH F
`
`LY 309887
`
`~~
`
`"H2
`
`cXCDHTW mm
`a?
`
`COOH
`
`s”
`
`"H
`
`LY 231514
`
`MTA
`
`Fig 4. Structures of \
`antifolates
`
`
`
`

`

`OVERVIEW OF FOLATE METABOLISM
`
`these effects are illus'
`
`sure to methotrexatelo;
`trated in Fig 5.
`It has been argued that the effects of methotrex-
`ate on reduced folate pools, and consequently, the
`indirect inhibition of purine de novo synthesis and
`amino acid interconversions, may be detrimental
`to its main antiproliferative action, namely, the
`inhibition of the synthesis of thymidylate that is
`required exclusively for DNA synthesis.“1 For this
`reason, many researchers have developed antifov
`lates designed to inhibit TS directly while not
`affecting other folate enzymes. The first of these to
`be used clinically was CB 3717,12 but this has been
`superseded by raltitrexed (Tomudex, ZD 1694;
`Zeneca Pharmaceuticals, Cheshire, England), which
`is licensed for the treatment of colon cancer in some
`
`countries. These specific TS inhibitors produce the
`elevation of the deoxyuridine pool in a manner sim—
`ilar to that observed following methotrexate but,
`importantly, dihydrofolate pools do not increase and
`purine synthesis is unaffected (Fig 6).
`Both direct TS inhibitors (such as raltitrexed
`and CB 3717) and drugs that inhibit TS indirectly
`(such as methOtrexate) lead to a marked increase
`in the intracellular pool of deoxyuridine mono«
`phosphate. The reduction in thymidine nucleo’
`tides caused by these drugs leads to activation of
`the pyrimidine synthetic pathways producing de-
`
`to a disproportionate in-
`thus,
`oxyuridine and,
`crease in the concentration of deoxyuridine. It has
`been shown that this increase in the intracellular
`
`pool of deoxyuridine monophosphate is mirrored
`by a corresponding increase in the extracellular
`pool of deoxyuridine,” presumably due to intra-
`cellular phosphatases allowing the release of de-
`oxyuridine from the cells (Fig 7). This provides a
`useful surrogate for in vivo TS inhibition. The
`plasma deoxyuridine levels can be monitored and
`an elevation compared with baseline indicates the
`inhibition, in vivo, of T5.14
`In addition, selective inhibitors of GARFT, the
`first
`folate-dependent enzyme involved in the
`pathway of de novo purine synthesis, have been
`developed. Examples of these are lometrexol and
`LY309887 (Fig 4). These compounds have good
`antitumor activity in preclinical systems with the
`suggestion that their activity may be preserved in
`tumor cells that have a nonfunctional p53 path—
`way. The clinical toxicity of many antifolates is,
`nor surprisingly, affected by the pretreatment fo—
`late status of the patient.
`In the case of the
`GARFT inhibitors, the effect of the folate status is
`particularly marked, with the maximum tolerated
`dose being at least 10«fold higher in patients who
`have received folate supplementation compared
`with those who have riot.‘5
`
`PURINE SYNTHESIS
`
`GARFT
`
`AICARFT
`
`-._
`
`‘
`
`V Y
`
`.\'
`
`Vl
`
`'
`
`Ilav-
`
`<1
`
`FH4TTN”
`
`I
`
`pHER- s u
`fl TFHZ "
`i
`
`DNA
`
`Methotrexate
`
`Fig 5. Effects of DHFR inhibition.
`
`
`
`PYRIMIDINE SYNTHESIS
`\
`1
`ldUMP CHFH4 ‘*
`¢
`CHOFH4-~ v
`,' CHZFH4
`\
`
`" - " Reduced flux
`
`\l
`
`+
`
`Increased 000! size
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`PYRiMIDINE SYNTHESIS
`
`PURINE SYNTH E5
`
`HILARY CAl
`
`Raiiitrexed
`
`CB 3717
`
`
`
`
`
`
`
`' ' ' ' Reduced flux
`
`+ Increased 000] size
`
`Kev
`
`Fig 6. Effects of TS inhibition.
`
`Clinical Measurement of Functional Foiate Status
`
`Although the effect of folic acid supplementa-
`tion on reducing the toxicity of antifolate drugs
`(particularly the GARFT inhibitors) is clear.
`it
`always has been difficult
`to correlate antifolatec
`induced toxicity with pretreatment folate levels.
`One possible explanation for this is that the folate
`levels do not adequately reflect the functioning of
`folic acid within proliferating cells at the time of
`measurement.
`in addition to the pathways dis«
`cussed so far, folic acid is also involved in cellular
`methylation reactions by virtue of its role in me—
`
`thionine synthesis. CHlFl-i4 can be reducer
`Scmethyltetrahydrofolate (Fig 1). This is a
`strate for the enzyme methionine synthase, wl
`uses the methyl group to convert homocystein
`methionine, Methionine in turn takes part in
`lular methylation reactions regenerating hornc
`teine. Methionine synthase is Blz’dependent
`also uses 5-methyltetrahydrofolate as the CO'
`srrate. Thus. any functional deficiency eithe
`Bl; or foiate will result in reduction in the
`through methionine synthase and a conseq:
`increase in the plasma level of homocystei
`
`Plasma
`
`Intracellular fluid
`
`UdR<—'* dUMP
`
`g3" eli Membrane
`
`TS
`
`UdR I
`*‘I‘HI'll:’5‘}lh-'i'l'.'-||'I"'-I‘:3-*:uin.lI.
`
`
`
`
` -
`
`TMP
`
`Fig 1. Plasma denxyuridi
`surrogate for T5 inhibition.
`
`

`

`Folate or 312
`Deficiencv
`
`CH3FH4 <———~—- CHzFl-l4
`
`.....n.
`
`
`
`
`Methionine Svnthetase
`
`(BR: dependent)
`
`
`
`
`
`FH4
`
`OVERVIEW OF FOLATE METABOLISM
`
`Homocvsteine
`/
`Cellular Meth vlation
`reactions
`
`S-adenosvl-
`Fig 8. Role of 5-methy1 tetra- methionine
`hydrofoiic acid: a reduction in \
`functional
`folate
`increases
`plasma homocysteine levels.
`
`methionine
`
`(Fig 8). The measurement of pretreatment plasma
`homocysteine has proved to be a sensitive way of
`predicting the toxicity of MTA.”
`
`LY231514 (MTA)
`
`MTA was developed by Eli Lilly and Company
`(Indianapolis, 1N).
`initially as a T8 inhibitor.
`However, it rapidly became clear that, unlike any
`of the other antifolates discussed, MTA is capable
`of inhibiting two other enzymes involved in folate
`metabolism, GARFT and DHFR (see Mendelsohn
`et al,
`this supplement). MTA also has a broad
`spectrum of preclinical activity, displays different
`patterns of cross-resistance to other antifolates,
`and has an encouraging level of activity docu-
`mented in early phase 11 clinical
`trials.IS It
`is
`possible that its capability of inhibiting more than
`one locus contributes to these results by increasing
`the spectrum of biochemical profiles of tumors
`potentially sensitive to the drug and discouraging
`the development of drug resistance. Reports that
`follow in this supplement address these issues in
`detail.
`
`CONCLUSIONS
`
`Naturally occurring folates have complex met:
`abolic pathways and are involved in a number of
`biochemical processes essential
`to life,
`including
`cell proliferation. In addition to their direct role in
`various metabolic pathways, 3 number of other
`phenomena will significantly affect
`the actions
`both of natural folates and their analogues acting
`as antifolates. These include cell membrane trans«
`
`the formation of polyglutamates. and the
`port,
`pretreatment folate status of the patient con-
`cerned. The very complexity of the processes in—
`volved suggests ways in which the action of anti;
`
`folates
`
`could be
`
`tuned to have a selective
`
`advantage against tumors compared with normal
`tissues. Several clinically active drugs have already
`been developed. LY231514 (MTA) may establish
`itself as an important addition and advance those
`currently available.
`
`REFERENCES
`
`l. Farber 5. Diamond LK. Mercer RD. et al: Temporary
`remissions in acute leukaemia in children produced by folic
`acid antagonist 4ramino pteroyl‘glutamic acid (aminopterin).
`N Eng!) Med 238:787'793. 1948
`2. Blakeley RL: The Biochemistry of Folic Acid and Related
`Pteridines. Amsterdam. The Netherlands, North Holland Pub-
`lishing Co, 1969
`3. Sikora E. Jackinan AL, Newcll DR, et al: Formation and
`retention and biological activity of Nm-propargyl'Sfi-dide
`azafolic acid (C3371?) polyglutamates in 1.1210 cells in vitro.
`Biochem Pharmacol 37:4047-4054. 1983
`4. Bisset GMF, Pawelczak K. Jackinan AL, et al: The
`synthesis and thymiclylatc synthase inhibitory activity of the
`poly—g-glutainyl conjugates of N-[S-[NrUA-dihydro‘Z-methyl-
`4-oxoquinazlolin-6«ylinethyl)—N-methy|aminol-thhenoyllL—
`glutamic acid (1C1 D1694) and other quinazoline antifolates.
`J Med Chem 35:859-866. 1992
`5. Takemura Y, Kobayashi H. Miyachi H, et al: Biological
`activity and intracellular metabolism of ZD1694 in human
`leukemia cell
`lines with different resistance mechanisms to
`antifolate drugs. Jpn J Cancer Res 87:773-780. 1996
`6. Schuetz JD, Matherly LH, Westin EH. et al: Evidence for
`a functional defect in the translocation of the methotrcxate
`transport carrier in a methotrexate-resistant murine L1210
`leukemia cell line. J Biol Chem 263:9840'9847. 1988
`7. Allegra C], Chabner BA. Drake JC. et al: Enhanced
`inhibition of thyinidylate synthase by methotrexate polygluta-
`mates] Biol Chem 2602972043726, 1985
`8. Allegra C], Fine RL. Drake JC. et. al: The effect of
`methotrcxate on intracellular folate pools in human MCF'7
`breast cancer cells. Evidence for direct
`inhibition of purine
`synthesis J Biol Chem 261:6478-6485. 1986
`9. Allegra C}. Drake JC. Jolivet J. et al: Inhibition of phos'
`phoribosyIaminoimidazolecarboxamide
`transformylase
`by
`
`
`
`

`

`l0
`
`methotrexate and dihvdrofolic acid polyglutamates. Proc Natl
`Acad Sci U S A 82:4881»4885, 1985
`10. Jackson RC, Jackman AL, Calvert AH: Biochemical
`effects of the quinazoline inhibitor of thymidylate synthetase,
`CB3717, on human lymphoblastoid cells. Biochem Pharmacol
`3237833790, 1983
`
`11. Jones TR, Calvert AH, Jackman AL, et al: A potent
`antitumout quinazoline inhibitor of thymidylate synthetase:
`Synthesis, biological properties and therapeutic results in mice.
`Eur] Cancer 17:11'19, 1981
`[2. Calvert AH, Alison DL. Harland S]. et al: A phase I
`evaluation of the quinazoline antifolate thymidylate synthetase
`inhibitor N'"Apropargvl-5,81dideazafolic acid.
`] Clin Oncol
`4:1245-1252, 1986
`13. Taylor GA, Jackman AL. Calvert AH, et al: Plasma
`nucleoside and base levels following treatment with the new
`thymidylate synthetase inhibitor, C337”,
`in De Bruyn C,
`Simmons HA, Muller M (eds): Purine Metabolism in Man IV,
`Part B: Biochemical,
`lrnmunological and Cancer Research.
`New York, NY, Plenum. 1983, pp 379-382
`
`HILARY CALVERT
`
`
`
`
`14. Rafi I, Taylor GA, Calvete JA, et al: Clinical pharma.
`cokineric and pharmacodynatnic studies with the non-classical
`antifolate thymidvlate synthase inhibitor 3:4'dthdFO'Z-aming.
`6rmethyl—4—0xo-5—(pyridylthio)vquinazoline
`dihydrochloride
`(A0337) given by 24 hour continuous intravenous infusion,
`Clin Cancer Res 1:1275-1284, 1995
`[5. Laohavinij S, Wedge SR, Lind M], et al: A phase I
`clinical
`study of
`the
`antipurine
`antifolate
`lometrexol
`(DDATHF) given with oral folic acid. Invest New Drugs 14:
`325—335, 1996
`
`16. Savage DG, Lindenbaum], Stabler SP, et al: Sensitivity
`of serum methylmalonic acid and total homocysteine determi.
`nations for diagnosing cobalamin and folate deficiencies. Am]
`Med 96:239—246. 1994
`
`17. Nivikiza C, Walling l, Thornton D. et al: LY2315I4
`(MTA): Relationship of vitamin metabolite profile to toxicity.
`Proc Am Assoc Clin Oncol 34:2139, 1998 (abstr)
`18. Calvert AH, Walling JM: Clinical Studies with MTA.
`Br] Cancer 78:35-40, 1993 (Suppl 3)
`
`
`
`
`
`l
`
`.r.I
`
`..'_-.—.-_-:.'-.d'-
`
`