028
`
`An Overview of Folate Metabolism: Features Relevant to the
`Action and Toxicities of Antifolate Anticancer Agents
`
`Hilary Calvert
`
`SINCE the observation of reduced folate levels
`
`in children with leukemia made by Farber et
`al 1 in the 1940s, the study of folic acid metabolism
`and the action of antifolate drugs has been inti(cid:173)
`mately linked to the development of cancer 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 antifolates in
`cancer therapy. The textbook by R.L. Blakley2 is a
`comprehensive work covering all aspects of folate
`metabolism.
`
`ASPECTS OF FOLATE METABOLISM
`
`Folate Pathways Associated With Cell Proliferation
`
`Folic acid functions mainly in its fully reduced
`form, 5,6, 7 ,8-tetrahydrofolate (FH4; Fig 1 ). FH4
`serves as a carrier for one-carbon moieties within
`the cell. These are obtained from a variety of
`sources that include serine. In this reaction, serine
`hydroxymethyl transferase forms 5, IO-methylene
`(CH2FH4 ) while converting
`tetrahydrofolate
`serine to glycine (Fig 2). CH2FH4 may be con(cid:173)
`verted within the cell to one-carbon carrying fo(cid:173)
`late derivatives of various oxidation states. One of
`these, 10-formyl tetrahydrofolate, is the substrate
`for two enzymes involved in the de novo synthesis
`of purines. These are glycinamide ribonucleotide
`formyl transferase (GARFT) and aminoimidazole
`carboxamide ribonucleotide formyl
`transferase
`(AICARFT). 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 10-formyl group and
`release unsubstituted 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. CH2FH4 is also the
`substrate for the enzyme thymidylate synthase
`(TS). Thymidylate synthase converts deoxyuri-
`
`Seminars in Oncology. Vol 26. No 2. Suppl 6 (April). 1999: pp 3- I 0
`
`dine monophosphate into thymidine monophos(cid:173)
`phate and is a key enzyme involved in cell prolif(cid:173)
`eration because it is the rate-limiting step in the de
`novo synthesis of thymidylate, which is required
`exclusively for DNA synthesis. The folate product
`of TS is not tetrahydrofolate, but the oxidized
`form, dihydrofolate (FH2). This product cannot
`continue to function in folate metabolism until it
`is converted back to FH4 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
`1. Naturally occurring folates within the cell are
`converted to polyglutamate forms by the addition
`of glutamate residues via a y-peptide linkage. An(cid:173)
`tifolates that possess a glutamate residue (known
`as classical antifolates) are also frequently con(cid:173)
`verted into their corresponding polyglutamate
`forms. The process of polyglutamation is accom(cid:173)
`plished by the enzyme folylpoly-y-glutamate 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 folates 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 negative
`charge, showing that polyglutamation increases
`the overall negative charge on the folate molecule
`by one unit for each additional glutamate. The
`negatively charged polyglutamates cannot cross
`the cell membrane and are therefore retained and
`concentrated within the cell. This is probably the
`major physiologic role of polyglutamation. Cells
`that are deficient in folylpoly-y-glutamate syn(cid:173)
`thetase are auxotrophic for the end products of
`
`From the Cancer Research Unit, Department of Oncology,
`University of Newcastle upon Tyne.
`Sponsored by Eli Lilly and Company.
`Dr Calvert is a consultant for and has received research funding
`from Eli Lilly and Company and Zeneca.
`Address reprint requests to Hilary Calvert. MD, Cancer Re(cid:173)
`search Unit, Department of Oncology, Fremlington Place. Univer(cid:173)
`sity of Newcastle upon Tyne, NE2 4HH.
`Copyright© 1999 by W.B. Saunders Company
`009 3-7754/99/2602-0602$10 .00/0
`
`Teva – Fresenius
`Exhibit 1014-00001
`
`

`
`4
`
`HILARY CALVERT
`
`1._N
`
`., ~OOH
`/j\..
`NH,_.)l ,--NH~.H
`NH2~ N
`
`COOH
`
`Folic Acid
`
`JL _N
`•• ~OOH
`/j\..
`NH,_.J. JNH~.H
`NH2~ NH
`
`Dihydrofolic
`Acid (FH 2)
`
`COOH
`
`0
`
`HN~ NH~COOH
`Jl_NHJ "=fir
`NH: .. )L )
`NH,AN I NH
`
`0
`
`COOH
`
`Tetrahydrofolic
`Acid (FH4 )
`
`5, 10-Methylene
`tetrahydrofolic
`Acid (CH2FH4)
`
`10-formyl
`tetrahydrofolic
`Acid (CHOFH4 )
`
`5-methyl(cid:173)
`tetrahydrofolic
`acid
`
`Fig I. Forms of folic acid.
`
`ff
`JL .NHJ···'==fH
`ff-\~ N~OOH
`N~.,,l J
`O
`NH,A I NH
`
`COOH
`
`folate metabolism (thymidine, hypuxanrhin.:. ;111,l
`glycine). In addition to being retained \\·ithin rhc
`cells, the polyglutamate forms of natural f,ilate,
`also may be better substrates for the various folate
`metabolizing enzymes.
`The formation of polyglutamates of those anti(cid:173)
`folates that are substrates for folylpoly-y-glutamate
`synthetase also has profound effects on their ac(cid:173)
`tivity. The rolygluramates may be retained within
`the cell for very long periods,' thus increasing the
`potency of the cytotoxic action of these com(cid:173)
`pounds. In addition, the addition of glutamate
`residues frequently renders the compounds much
`more potent inhibitors of their target enzyme. For
`example, raltitrexed pentaglutamate is roughly
`
`I 00-fold more potent as a TS inhibitor than the
`parent molecule.4 The effects of polyglutamation
`, in the plltency of molecules such as these is so
`pnlfound that they may be considered as prodrugs
`for their pulyglutamate forms. Indeed, cellular re(cid:173)
`sistance tu antifolates can be caused by a reduction
`in the ahilitY ,if the cell to form the polyglutamate
`derivati\·es.' A more complete and in-depth re(cid:173)
`view of polyglutamation and its relevance to can(cid:173)
`cer therapy is gi\·en by Richard G. Moran else(cid:173)
`where in this supplement.
`
`Cell Membrane Transport of Folates and Antifolates
`folates do not cross the cell membrane to an
`appreciable extent by passive diffusion but require
`
`Teva – Fresenius
`Exhibit 1014-00002
`
`

`
`OVERVIEW OF FOLATE METABOLISM
`
`PYRIMIDINE SYNTHESIS
`~
`
`TMP I FH2 - - - - - - ' ....
`
`DHFR
`
`DNA
`
`PURINE SYNTHESIS
`
`GARFT
`
`AICARFT
`
`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(cid:173)
`late carrier (RFCl). 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 tetrahydrofolate itself.
`
`Changes in this carrier that alter its relative affin(cid:173)
`ity for antifolates have been shown to be a cause of
`drug resistance. 6 The reduced folate carrier has a
`relatively low affinity for natural folates ( 1 to S
`µmol/L) compared with their physinlogic extracel(cid:173)
`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
`
`,.\J-0-f ·•-{°0
`MTA
`- PentaQlutamate
`< 100
`,NH1oo·
`,NH1oo·
`
`,f'NH
`0
`
`~c
`0
`
`~c
`0
`
`,....,..,..·
`~.,".H....._ ........ ._
`
`<
`
`Hz~N NH
`
`FPGS
`
`MTA
`
`Fig 3. Formation of polyglutamates.
`
`Teva – Fresenius
`Exhibit 1014-00003
`
`

`
`6
`
`HILARY CALVERT
`
`within a membrane vesicle and subsequently re(cid:173)
`leased into the cytoplasm. Three genes for folate
`receptors have been cloned (see Sierra and Gold(cid:173)
`man). Folate receptors may be responsible for the
`transport of some antifolates, for example, lome(cid:173)
`trexol. In addition to these two mechanisms, the
`level of folates and antifolates within the cell may
`be affected by an energy-dependent 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 inhibition of
`DHFR. The result of this inhibition is that in(cid:173)
`tracellular folate accumulates in the form of
`dihydrofolate. There is a consequent inhibition
`of the de novo synthesis both of purines and
`thymidine. This may be due in part to a dimi(cid:173)
`nution in the intracellular pools of tetrahydro(cid:173)
`folates, but additionally, methorrexate polyglu(cid:173)
`tamates and
`the accumulated dihydrofolate
`polyglutamates are capable of inhibiting both
`TS and AICARFT directly. 7- 9 Characteristi(cid:173)
`cally, the intracellular pools of dihydrofolate
`and deoxyuridine will increase following expo-
`
`~h:r-Orf COOH
`
`Methotrexate
`
`Raltitrexed
`(Tomudex™,
`ZD 1694)
`
`CB 3717,
`PDDF
`
`Lometrexol
`DDATHF
`
`LY 309887
`
`LY 231514
`MTA
`
`Fig 4. Structures of various
`antifolates.
`
`Teva – Fresenius
`Exhibit 1014-00004
`
`

`
`OVERVIEW OF FOLATE METABOLISM
`
`sure to methotrexate 10; these effects are illus(cid:173)
`trated in Fig 5.
`It has been argued that the effects of methotrex(cid:173)
`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. 11 For this
`reason, many researchers have developed antifo(cid:173)
`lates designed to inhibit TS directly while not
`affecting other folate enzymes. The first of these to
`be used clinically was CB 3 717, 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(cid:173)
`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 3 717) and drugs that inhibit TS indirectly
`(such as methotrexate) lead to a marked increase
`in the intracellular pool of deoxyuridine mono(cid:173)
`phosphate. The reduction in thymidine nucleo(cid:173)
`tides caused by these drugs leads to activation of
`the pyrimidine synthetic pathways producing de-
`
`oxyuridine and, thus, to a disproportionate in(cid:173)
`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, n presumably due to intra(cid:173)
`cellular phosphatases allowing the release of de(cid:173)
`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 TS.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(cid:173)
`way. The clinical toxicity of many antifolates is,
`not surprisingly, affected by the pretreatment fo(cid:173)
`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 not.' 5
`
`PURINE SYNTHESIS
`
`GARFT
`
`AICARFT
`
`PYRIMIDINE SYNT~IS
`tdUMP CHFH4 ~.._.
`, * CHOFH4 - ... t
`
`Kev:
`
`t
`
`Increased oool size
`
`Reduced flux
`
`I
`
`- '
`
`~ I
`
`TMP,
`
`I
`
`I
`
`I
`
`I
`
`1
`DNA
`
`F H2--:t.,,._• ·:-. --~ FH4 .. ·~: :.· ._. :· - - - -
`
`I Methotrexatel
`
`i
`DNA
`Fig 5. Effects of DHFR inhibition.
`
`t
`
`I
`
`RNA
`
`Teva – Fresenius
`Exhibit 1014-00005
`
`

`
`- - - -----------------------
`
`8
`
`HILARY CALVERT
`
`PYRIMIDINE SYNTHESIS
`
`PURINE SYNTHESIS
`
`Raltitrexed
`
`CB3717
`
`I
`
`~ t dUMP CHFH4 ~ I
`t CHOFH4 ~ f
`,
`GARFT
`\~I
`TMP ' ~ CH,FH,
`,.:\ t
`TS t
`.·
`I
`i AICARFT
`/DHFR ~t
`
`.. - - - .....
`
`/
`1'
`
`FH2
`
`FH4
`
`DNA
`
`Kev: t Increased oool size
`
`Reduced flux
`
`I
`
`DNA
`
`RNA
`
`Fig 6. Effects of TS inhibition.
`
`Clinical Measurement of Functional Folate Status
`Although the effect of folic acid supplementa(cid:173)
`tion on reducing the toxicity of antifolate drugs
`(particularly the GARFT inhibitors) is clear, it
`always has been difficult to correlate antifolate(cid:173)
`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(cid:173)
`cussed so far, folic acid is also involved in cellular
`methylation reactions by virtue of its role in me-
`
`thionine synthesis. CH2FH4 can be reduced to
`5-methyltetrahydrofolate (Fig 1). This is a sub(cid:173)
`strate for the enzyme methionine synthase, which
`uses the methyl group to convert homocysteine to
`methionine. Methionine in turn takes part in cel(cid:173)
`lular methylation reactions regenerating homocys(cid:173)
`teine. Methionine synthase is B12-dependent but
`also uses 5-methyltetrahydrofolate as the co-sub(cid:173)
`strate. Thus, any functional deficiency either in
`B12 or folate will result in reduction in the flux
`through methionine synthase and a consequent
`increase in the plasma level of homocysteine 16
`
`Plasma
`
`UdR
`
`Intracellular fluid
`
`UdR~dUMP
`
`ell Membrane
`
`TS
`
`TMP
`
`Fig 7. Plasma deoxyuridine: a
`surrogate for TS inhibition.
`
`Teva – Fresenius
`Exhibit 1014-00006
`
`

`
`OVERVIEW OF FOLATE METABOLISM
`
`9
`
`Folate or Bi2
`Deficiency
`
`Homocvsteine
`~
`Cellular Methvlation
`
`reactions •
`
`S-adenosvl-
`methionine
`...___methionine
`
`Fig 8. Role of 5-methyl tetra(cid:173)
`hydrofolic acid: a reduction in
`functional
`folate
`increases
`plasma homocysteine levels.
`
`(Fig 8). The measurement of pretreatment plasma
`homocysteine has proved to be a sensitive way of
`predicting the toxicity of MTA.17
`
`LY231514 (MTA)
`
`MT A was developed by Eli Lilly and Company
`(Indianapolis, IN), initially as a TS inhibitor.
`However, it rapidly became clear that, unlike any
`of the other antifolates discussed, MT A is capable
`of inhibiting two other enzymes involved in folate
`metabolism, GARFT and DHFR (see Mendelsohn
`et al, this supplement). MT A 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(cid:173)
`mented in early phase II 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 profi le.s 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(cid:173)
`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, a number of other
`phenomena will significantly affect the actions
`both of natural folates and their analogues acting
`as antifolates. These include cell membrane trans(cid:173)
`port, the formation of polyglutamates, and the
`pretreatment folate status of the patient con(cid:173)
`cerned. The very complexity of the processes in(cid:173)
`volved suggests ways in which the action of anti-
`
`folates could be
`to have a selective
`tuned
`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
`
`1. Farber S, Diamond LK, Mercer RD, et al: Temporary
`remissions in acute leukaemia in children produced by fol ic
`acid antagonist 4-amino pteroyl-glutamic acid (aminoptcrin).
`N Engl J Med 238:787-793, 1948
`2. Blakeley RL: The Biochemistry of Folic Acid and Related
`Pteridines. Amsterdam, The Netherlands, North Holland Pub(cid:173)
`lishing Co, 1969
`3. Sikora E, Jackman AL, Newell DR, et al: Formation and
`retention and biological activity of N 10-propargyl-5,8-dide(cid:173)
`azafolic acid (CB3717) polyglutamates in LIZ!O cells in vitro.
`Biochem Pharmacol 37:4047-4054, 1988
`4. Bisset GMF, Pawelczak K, Jackman AL, et al: The
`synthesis and thymidylate synthase inhibitory activity of the
`poly-g-glutamyl conjugates of N-15-[N-(3,4-dihydro-2-methyl-
`4-oxoquinazlolin-6-ylmethyl)-N-methylaminol-2-thenoyl]-L(cid:173)
`glutamic acid (IC! Dl694) and other quinazoline antifolates.
`J Med Chem .15:859-866, 1992
`5. Takemura Y, Kobayashi H, Miyachi H, et al: Biological
`activity and intracellular metabolism of Z01694 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 methotrexate
`transport carrier in a methotrexate-resistant murine LI 210
`leukemia cell line. J Biol Chem 263:9840-9847, 1988
`7. Allegra CJ, Chabner BA, Drake JC, et al: Enhanced
`inhibition of thymidylate synthase by mcthotrexate polygluta(cid:173)
`mates. J Biol Chem 260:9720-9726, 1985
`8. Allegra CJ, Fine RL, Drake JC, et al: The effect of
`methotrexate 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 CJ, Drake JC, Jolivet J, et al: Inhibition of phos-
`by
`phoribosylaminoimidazolecarboxamide
`transformylase
`
`Teva – Fresenius
`Exhibit 1014-00007
`
`

`
`10
`
`HILARY CALVERT
`
`methotrexate and dihydrofolic acid polyglutamatcs. Proc Natl
`Acad Sci US A 82:4881-4885, 1985
`10. Jackson RC, Jackman AL, Calvert AH: Biochemical
`effects of the quinazoline inhibitor of thymidylate synthctase,
`CB3 717, on human lymphoblastoid cells. Biochem Pharmacol
`32:.3783-3790, 1983
`11. Jones TR, Calvert AH, Jackman AL, et al: A potent
`antitumour quinazoline inhibitor of thymidylate synthetase:
`Synthesis, biological properties and therapeutic results in mice.
`Eur J Cancer 17:11-19, 1981
`12. Calvert AH, Alison DL, Harland SJ, ct al: A phase l
`evaluation of the quinazoline antifolate thymidylate synthetase
`inhibitor N 10-propargyl-5,8-dideazafolic acid. J 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, CB3 717, in De Bruyn C,
`Simmons HA, Muller M (eds}: Purine Metabolism in Man JV,
`Part B: Biochemical, Immunological and Cancer Research.
`New York, NY, Plenum, 1983, pp 379-382
`
`14. Rafi I, Taylor GA, Calvete JA, et al: Clinical pharma(cid:173)
`cokinetic and pharmacodynamic studies with the non-classical
`antifolate thymi<lylate synthase inhibitor 3,4-dihydro-2-amino-
`6-methyl-4-oxo-5-(pyridylthio)-quinazoline <lihydrochlnride
`(AG337) given by 24 hour continuous intravenous infusion.
`Clin Cancer Res 1:1275-1284, 1995
`15. Laohavinij S, Wedge SR, Lind MJ, et al: A phase I
`clinical study of
`the antipurinc antifolate
`lometrexol
`(DDATHF) given with oral folic acid. Invest New Drugs 14:
`325-335, 1996
`16. Savage DG, Lindenbaum J, Stabler SP, ct al: Sensitivity
`of serum methylmalonic acid and total homocysteine determi(cid:173)
`nations for diagnosing cobalamin and folate deficiencies. Am J
`Med 96:239-246, 1994
`17. Niyikiza C, Walling J, Thornton D, et al: LY231514
`(MTA): Relationship of vitamin metabolite profile to toxicity.
`Proc Am Assoc Clin Oncol 34:2139, 1998 (ahstr)
`18. Calvert AH, Walling JM: Clinical Studies with MT A.
`Br J Cancer 78:35-40, 1998 (suppl 3)
`
`Teva – Fresenius
`Exhibit 1014-00008