2 Combinations of Antimetabolites and Ionizing Radiation
Hiroshi Harada, Keiko Shibuya, and Masahiro Hiraoka
H. Harada, MD K. Shibuya, MD M. Hiraoka, MD PhD
Department of Radiation Oncology and Image-Applied Therapy, Graduate School of Medicine, Kyoto University, Sakyo, Kyoto, 606-8507, Japan
2.1
Antimetabolites: Classes, Intracellular Metabolism, and Mechanisms of Action
Antimetabolites have antineoplastic activity, which is attributed to the fact that their structure is very similar to the normal metabolites required for cell function and replication. After intracellular modi- fication, the antimetabolites interact with intracel-
lular enzymes and show cytotoxic effects by (a) substituting for a normal metabolite incorporated into key molecules, such as DNA and RNA, and (b) occupying the catalytic site of a key enzyme and competing with a normal metabolite. Consequently, they interfere with DNA synthesis and proliferation of the cancer cell. Until recently, several kinds of antimetabolites have been developed and are cat- egorized into three major groups, pyrimidine ana- logs (see Fig. 2.1a), folic acid analogs (see Fig. 2.1b) and purine analogs (see Fig. 2.1c). The mechanisms of action of representative agents in each class are briefly described herein.
2.1.1
Pyrimidine Analogs
2.1.1.1 5-Fluorouracil
5-Fluorouracil (5-FU) is a nucleoside analog of uracil in which a fluorine atom is inserted into the C-5 posi- tion in place of hydrogen (see Fig. 2.1a; Longley et al. 2003). It has been widely applied for the treatment of various kinds of cancers, particularly for colorec- tal and breast cancers. 5-FU requires metabolic acti- vation to form its cytotoxic metabolites (see Fig. 2.2;
Malet-Martino and Martino 2002; Longley et al. 2003). 5-FU is converted to 5-fluorouridine-5’- monophosphate (5-FUMP) directly by orotate phos- phoribosyltransferase (OPRT) or indirectly via an intermediate metabolite, 5-fluorouridine (5-FUrd), by uridine phosphorylase and uridine kinase. 5- FUMP is subsequently phosphorylated to 5-fluo- rouridine-5’-diphosphate (5-FUDP) by pyrimidine monophosphate kinase and further to a cytotoxic metabolite, 5-fluorouridine-5’-triphosphate (5- FUTP) by pyrimidine diphosphate kinase. The 5- FUTP can mimic uridine-5’-triphosphate (UTP), and be recognized by RNA polymerases as the sub- strate. This leads to the incorporation of 5-FU in all classes of RNA, disrupting normal RNA func-
CONTENTS
2.1 Antimetabolites: Classes, Intracellular Metabolism, and Mechanisms of Action 19
2.1.1 Pyrimidine Analogs 19 2.1.1.1 5-Fluorouracil 19 2.1.1.2 Gemcitabine 21 2.1.2 Folic Acid Analogs 22 2.1.2.1 Methotrexate 22 2.1.2.2 Pemetrexed 23
2.1.3 6-Mercaptopurine as an Example for Purine Analogs 23
2.2 Mechanisms of Radiosensitization with Antimetabolites 24
2.2.1 dNTP Depletion 24 2.2.2 Cell Cycle Distribution 25 2.2.3 Checkpoint and p53 25 2.2.4 Apoptosis 26
2.3 Interactions of Antimetabolites with Radiation:
Preclinical to Clinical 26
2.3.1 5-Fluorouracil and its Prodrugs: Capecitabine, UFT, and S-1 26
2.3.1.1 5-Fluorouracil 26
2.3.1.2 Prodrugs of 5-FU: UFT; S-1; and Capecitabine 28 2.3.2 Gemcitabine 29
2.3.3 Pemetrexed 30 2.4 Conclusion 30 References 31
a
c
b
SH
N
N N
N H SH
N
N N
N H SH
N N N
N NN
N H N H NH H
N O
O F
NH2
N O N
O
FH OH H HOH2C
F
H HCl
NH H
N O
O F NHNH
H N H
N O
O F
NH2
N O N
O
FH OH H HOH2C
F
H HCl
NH2
N O N
O
FH OH H HOH2C
NH2
N N O O NN
O O
FH OH H HOH2C
F
H HCl
CH2CH2COOH NH2
H2N N
CH2N CH3
CONH COOH H C N
N N
CH2CH2COOH NH2
H2N N
CH2N CH3
CONH COOH H C N
N N
CH2CH2COOH NH2
H2N NN
CH2N CH3
CONH COOH H C N
N
N N N N
ONa O
N NH H2N
N
O
HN O
O NaO
ONa O
N NH H2N
N
O
HN O
O NaO
ONa O
O N
N NH NH H2N H2N
N N
O O
HN O O
O O NaO Pyrimidine analog
5-Fluorouracil Gemcitabine hydrochloride Methotrexate Folic acid analog
Purine analog
Pemetrexed
6-mercaptopurine
Fig. 2.1a-c. Structures of antimetabolites. a Pyrimidine analogs, gemcitabine hydrochloride, and 5-fl uorouracil. b Folic acid analogs, methotrexate and pemetrexed. c Purine analog, 6-mercaptopurine
inhibition facilitation
5-FUrd
UK UP
Incorporation into RNA
5 5--FUFU
5-FUMP
5-FUDP
5-FUTP
RNA damage
RNAP OPRT
PMK
PDK
Urd
UMP
UDP
UTP
RNAP OPRT
PMK
PDK
Incorporation into RNA
RNA function
Incorporation into DNA 5-FdUrd
5-FdUMP
5-FdUDP
5-FdUTP
DNA damage
DNAP TK
PMK
PDK
dUMP
dTMP
TS
Incorporation into DNA
DNA replication DNA repair TS inhibition
dUTP
DNAP TP
inhibition facilitation
5-FUrd
UK UP
Incorporation into RNA Incorporation
into RNA
5 5--FUFU
5-FUMP
5-FUDP
5-FUTP
RNA damage RNA damage
RNAP OPRT
PMK
PDK
Urd
UMP
UDP
UTP
RNAP OPRT
PMK
PDK
Incorporation into RNA Incorporation
into RNA
RNA function RNA function
Incorporation into DNA Incorporation
into DNA 5-FdUrd
5-FdUMP
5-FdUDP
5-FdUTP
DNA damage DNA damage
DNAP TK
PMK
PDK
dUMP
dTMP
TS
Incorporation into DNA Incorporation
into DNA
DNA replication DNA repair DNA replication
DNA repair DNAP TS inhibition
dUTP
DNAP TP
Fig. 2.2. Metabolism and mechanism of action of 5-fl uorouracil. 5-fl uorouracil (5-FU), 5-fl uorouridine-5’-monophosphate (5-FUMP), orotate phosphoribosyltransferase (OPRT), 5-fl uorouridine (5-FUrd), uridine phosphorylase (UP), uridine kinase (UK), 5-fl uorouridine-5’-diphosphate (5-FUDP), pyrimidine monophosphate kinase (PMK), 5-fl uorouridine-5’-triphosphate (5-FUTP), pyrimidine diphosphate kinase (PDK), RNA polymerase (RNAP), uridine (Urd), Uridine-5’-monophosphate (UMP), uridine-5’-diphosphate (UDP), uridine-5’-triphosphate (UTP), 5-fl uoro-2’-deoxyuridine (5-FdUrd), thymidine kinase (TK), 5-fl uoro-2’-deoxyuridine-5’-monophosphate (5-FdUMP), 5-fl uoro-2’-deoxyuridine-5’-diphosphate (5-FdUDP), 5-fl uoro-2’- deoxyuridine-5’-triphosphate (5-FdUTP), thymidylate synthase (TS), thymidine-5’-monophosphate (dTMP), 2’-deoxyuridine- 5’-monophosphate (dUMP), thymidine-5’-triphosphate (dTTP), 2’-deoxyuridine-5’-triphosphate (dUTP), DNA polymerase (DNAP)
tion. The misincorporation inhibits the processing of pre-ribosomal RNA to mature ribosomal RNA (Kanamaru et al. 1986), the modification of tRNAs (Santi and Hardy 1987) and the splicing of mRNAs (Doong and Dolnick 1988), leading to profound effects on cellular metabolism and viability.
After the conversion of 5-FU to 5-fluoro-2’- deoxyuridine (5-FdUrd), the 5-FdUrd is sequentially phosphorylated to cytotoxic 5-fluoro-2’-deoxyuri- dine-5’-monophosphate (5-FdUMP), to 5-fluoro- 2’-deoxyuridine-5’-diphosphate (5-FdUDP), and finally to cytotoxic 5-fluoro-2’-deoxyuridine-5’- triphosphate (5-FdUTP). The 5-FdUMP and the 5-FdUTP exert their antitumor effects through the inhibition of thymidylate synthase (TS) (Santi et al.
1974; Sommer and Santi 1974) and incorporation into DNA, respectively. TS is responsible for the pro- duction of thymidine-5’-monophosphate (dTMP) from 2’-deoxyuridine-5’-monophosphate (dUMP) through the transfer of a methyl group from 5,10- methylenetetrahydrofolate (CH2THF). This reaction is fully responsible for the production of de novo source of thymidylate, which is necessary for DNA replication and repair. The 5-FdUMP binds to the nucleotide-binding site of TS, forms a stable ternary complex with TS and CH2THF, and interferes with the access of normal substrate dUMP to the nucleotide- binding site, resulting in the inhibition of dTMP syn- thesis (Santi et al. 1974; Sommer and Santi 1974).
Depletion of dTMP directly results in the subsequent depletion of thymidine-5’-triphosphate (dTTP) and induces an imbalance of the deoxynucleotide pool through various feedback mechanisms. As a result of these actions, 5-FU inhibits DNA synthesis and repair, leading to lethal DNA damage (Yoshioka et al. 1987;
Houghton et al. 1995). In addition, TS inhibition is accompanied by the accumulation of its substrate, dUMP, which might be subsequently metabolized to 2’-deoxyuridine-5’-triphosphate (dUTP; Mitrovski et al. 1994; Aherne et al. 1996). Both the dUTP and a 5-FU metabolite, 5-FdUTP, can be misincorporated into DNA. UracilDNAglycosylase (UDG) hydro- lyzes the (F)uracil-deoxyribose glycosyl bond of the dUTP and the 5-FdUTP residues in DNA, generating a single strand break; however, since the depletion of dTTP, which is caused by the TS inhibition, as mentioned above, reduces the efficacy of the repair of such strand breaks, the misincorporated dUTP and 5-FdUTP are hard to be removed, leading to cell death. The misincorporation of 5-FU-metabolites into DNA and the imbalance of intracellular nucleo- tides results in various detrimental effects on DNA synthesis.
2.1.1.2 Gemcitabine
Gemcitabine (dFdCyd), 2’-deoxy-2’,2’-difluoro- cytidine, is a nucleoside analog of deoxycytidine in which two fluorine atoms are inserted into the deoxyribofuranosyl ring (see Fig. 2.1a). It shows broad-spectrum activity in the treatment of solid malignancies, particularly for pancreatic (Kaye 1994; Rothenberg et al. 1996) and non-small cell lung cancer (Gatzemeier et al. 1996; Crino et al.
1999). In order for dFdCyd to produce its cytotoxic effects, dFdCyd requires the following intracellular modifications (see Fig. 2.3): The dFdCyd is firstly phosphorylated by deoxycytidine (dCyd) kinase to the 5’-monophosphate of dFdCyd (dFdCMP;
Heinemann et al. 1988). Subsequent phosphory- lations yield the active metabolites diphosphate (dFdCDP) and triphosphate (dFdCTP) nucleotides (Heinemann et al. 1988). These metabolites have the potential to interfere with multiple steps of DNA synthesis and are directly and indirectly responsible for the cytotoxic properties of dFdCyd.
The dFdCTP form competes with dCTP for incor- poration into DNA, and the incorporation of dFdCTP into DNA is strongly correlated with the inhibition of further DNA synthesis (Huang et al. 1991). Once the dFdCTP is incorporated into the end of the elon- gating DNA strand, only one more deoxynucleotide is added, and thereafter the DNA polymerases are unable to proceed, resulting in the termination of the DNA synthesis. Moreover, proof-reading exonu- cleases, such as DNA polymerase epsilon, are unable to remove the incorporated dFdCTP from the penul- timate position and are unable to repair the growing DNA strands. This action is termed “masked chain termination” (Plunkett et al. 1995).
The dFdCDP form, on the other hand, can inhibit ribonucleotide reductase, which is responsible for the reactions that synthesize the deoxynucleotides required for DNA synthesis and repair (Baker et al. 1991). The inhibition of this enzyme causes a reduction in the concentrations of the DNA precur- sor pool (Heinemann et al. 1990). The reduction in the dNTP (particularly dCTP) leads to the following
“self-potentiation.” Firstly, the reduction enhances the incorporation of dFdCyd-derived nucleo- tides into DNA, because dFdCTP competes with dCTP for incorporation into DNA, as mentioned above (Huang et al. 1991). Secondly, the reduction enhances the phosphorylation of the dFdCyd and increases the intracellular dFdCDP and dFdCTP because the dCyd kinase is negatively regulated
by the dCTP (Shewach et al. 1992a). Finally, the reduction suppresses the deamination (inactiva- tion) of dFdCyd and its derivatives because dCTP is essential as a co-factor for the activity of dCMP- deaminase (Hertel et al. 1990). As a result of these mechanisms, dFdCyd exhibits cell phase specific- ity, primarily killing cells undergoing DNA synthe- sis (S-phase) and also blocking the progression of cells through the G1/S-phase boundary (Hertel et al. 1990). Yet, additional observations suggest that other effects are implicated in the interaction with ionizing radiation also (Rosier et al. 2004).
2.1.2
Folic Acid Analogs
2.1.2.1 Methotrexate
Methotrexate (MTX), 4-amino 10-methyl folic acid (see Fig. 2.1b), is an analog of folic acid that has been widely used for the treatment of childhood acute lymphoblastic leukemia (ALL) and a number of other malignant diseases, such as lymphoma,
osteosarcoma, breast cancer, and head and neck cancer (Chu et al. 1996). The MTX is taken up by cells via the reduced folate carrier (RFC), such as RFC1, and is then converted within the cells by folyl- polyglutamate synthase to methotrexate polygluta- mates (MTXPGs; see Fig. 2.4; Chabner et al. 1985).
The MTXPGs are retained longer in cells compared with MTX (Jolivet et al. 1983) and compete with some cellular folate cofactors (e.g., 10-formyl-tet- rahydrofolate, 5,10-methylene-tetrahydrofolate and 5,10-methenyl-tetrahydrofolate) for the interaction with dihydrofolate reductase (DHFR). This results in the inhibition of the activity of DHFR, thereby decreasing the amount of reduced folate, which is the carbon donor for the purine ring formation in the critical pathways for DNA synthesis, DNA repair and cell replication, such as de novo purine synthe- sis (DNPS) and thymidine synthesis (Allegra et al. 1985a,b). In addition, MTXPGs directly inhibit phosphoribosyl pyrophosphate amidotransferase (PRPPAT), glycinamide ribonucleotide formyltrans- ferase (GARFT) and 5-aminoimidazole-4-carbox- amide ribonucleotide formyltransferase (AICARFT), which are also key enzymes in the DNPS pathway (Segal et al. 1990; Kremer 1994). The DNPS inhi-
Incorporation into DNA
dCyd
dCMP
dCDP
dCTP
dFdCyd
dFdCMP
dFdCDP
dFdCTP CDP
dCyd kinase
DNA Synthesis
DNAP
Incorporation into DNA
Masked chain termination
DNAP
Termination of DNA synthesis depletion
of dCTP
dFdUMP dFdU
dCMP deaminase
Incorporation into DNA Incorporation
into DNA dCyd
dCMP
dCDP
dCTP
dFdCyd
dFdCMP
dFdCDP
dFdCTP CDP
Ribonuc leotide reduc
tase
dCyd kinase
DNA Synthesis DNA Synthesis
DNAP
Incorporation into DNA Incorporation
into DNA
Masked chain termination Masked chain
termination
DNAP
Termination of DNA synthesis depletion
of dCTP depletion of dCTP
Cyd / dCyd dFdU
deaminase
dCMP deaminase
Fig. 2.3. Metabolism and mechanism of action of gemcitabine. Gemcitabine (dFdCyd), gemcitabine-5’-monophosphate (dFdCMP), gemcitabine-5’-diphosphate (dFdCDP), gemcitabine-5’-triphosphate (dFdCTP), 2’deoxycytidine (dCyd), 2’-deox- ycytidine-5’-monophosphate (dCMP), 2’-deoxycytidine-5’-diphosphate (dCDP), 2’-deoxycytidine-5’-triphosphate (dCTP), cytidine-5’-diphosphate (CDP), DNA polymerase (DNAP), 2’-deoxy-2’,2’-difl uorouridine (dFdU), dFdU-5’-monophosphate (dFdUMP)
inhibition facilitation inhibition facilitation inhibition facilitation
bition by both MTX and its metabolite results in purine depletion, leading to the inhibition of DNA synthesis, decreased cell proliferation and signifi- cant cytotoxicity.
2.1.2.2 Pemetrexed
Pemetrexed, L-glutamic acid, N-[4-[2-(2-amino- 4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin- 5- yl)ethyl]benzoyl]-, disodium salt, heptahydrate (see Fig. 2.1b), is a multi-targeted folate analog that exerts its antineoplastic activity in many kinds of human malignancies, e.g., breast, pancreatic, colorectal, head and neck, gynecological, and non- small cell lung cancers (Hanauske et al. 2001), by disrupting folate-dependent metabolic processes. It gains entry to cells via the reduced folate carrier, and once inside a cell, it is intracellularly converted to polyglutamate forms, tri- and pentaglutamate, by folylpolyglutamate synthetase. This modification prolongs its intracellular retention (Mendelsohn et al. 1999). The polyglutamate forms inhibit DHFR, TS, and GARFT, all of which are folate-dependent enzymes responsible for the de novo biosynthesis of thymidine and purine nucleotides (Shih et al. 1997).
As a result of these processes, pemetrexed inhibits DNA synthesis by almost the same mechanism as
MTX (see Fig. 2.4), leading to a subsequent decline in cell proliferation, which is particularly significant in rapidly proliferating tumor cells.
2.1.3
6-Mercaptopurine as an Example for Purine Analogs
6-Mercaptopurine (6-MP) is a 6-thiopurine analog of purine bases (see Fig. 2.1c), such as hypoxanthine and guanine, and has been in clinical use for over 30 years as an antileukemic agent. 6-MP is a prodrug that requires activation to exert its cytotoxic effect (see Fig. 2.5). It is first converted by hypoxanthine- guanine phosphoribosyl transferase (HGPRT) into 6-thioinosine monophosphate (6-TIMP; Lennard 1992). It is subsequently metabolized by a two-step process involving inosine monophosphate dehydro- genase (IMPDH) into 6-thioxanthine monophos- phate (6-TXMP), and by guanosine monophosphate synthetase into 6-thioguanosine 5’-monophosphate (6-TGMP). 6-TGMP is further metabolized by a series of kinases and reductases to deoxy-6-thio- guanosine 5’-triphosphate (dGS). The resultant dGS can be incorporated into DNA and trigger cell cycle arrest and apoptosis (Swann et al. 1996). In addi- tion, thiopurine methyltransferase (TPMT) also can MTXMTX
MTXPGs
Intracellular Extracellular
DHFR
PRPPAT GARFT AICARFT
DNPS
TS DNA synthesis
DNA repair cell replication
RFC1
MTX
Folylpolyglutamate synthase
DNPS
inhibition facilitation MTXMTX
MTXPGs
Intracellular Extracellular
DHFR
PRPPAT GARFT AICARFT
DNPS
TS DNA synthesis
DNA repair cell replication
RFC1
MTX
Folylpolyglutamate synthase
DNPS
inhibition facilitation inhibition facilitation
Fig. 2.4. Metabolism and mechanism of action of methotrexate. Methotrexate (MTX), MTX polyglutamates (MTXPGs), dihydrofolate reductase (DHFR), phosphoribosyl pyrophos- phate amidotransferase (PRPPAT), glycinamide ribonucleotide formyltransferase (GARFT), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), de novo purine synthesis (DNPS), thymidine synthase (TS). Pemetrexed has been reported to show antineoplastic activity through almost the same mechanism as MTX
convert the 6-TIMP into S-methyl-thioinosine 5’- monophosphate (MeTIMP). The resultant MeTIMP acts as a strong inhibitor of PRPPAT, which is respon- sible for de novo purine synthesis (Tay et al. 1969;
Allan and Bennett 1971); thus, 6-MP treatment results in purine depletion, leading to the inhibition of DNA synthesis and proliferation, and antileuke- mic effect.
2.2
Mechanisms of Radiosensitization with Antimetabolites
In addition to the cytotoxic effect of these antime- tabolites themselves, they have been reported to enhance the effect of radiation as potent radiosensi- tizers in vitro. Recent in vivo studies have confirmed
these activities and have reported significant tumor growth delay with the combination of antimetabo- lites and ionizing radiation in animal models; how- ever, the mechanism of radiosensitization has yet to be fully understood. Initial studies have focused, as the mechanism, on the modulation of the metabo- lism of deoxynucleotides, the changes in cell cycle distribution, the role of the tumor suppressor pro- tein p53, and the induction of apoptosis.
2.2.1
dNTP Depletion
Antimetabolite-induced perturbation of the intra- cellular dNTP pool and its radiosensitizing effect are summarized in Table 2.1. In the case of dFdCyd, it increases the radiosensitivity of cells even under conditions in which the drug alone shows no cytotoxicity (Shewach et al. 1994). The dFdCyd- mediated sensitization has been reported not to be dependent either on the intracellular concen- tration of dFdCTP or on the dFdCTP/dCTP ratio, but correlates to the dFdCDP-mediated decrease in intracellular dATP pools (Shewach et al. 1994);
therefore, it has been hypothesized that the deple- tion of the dATP pool level by the dFdCyd treat- ment is a key factor in enhancing radiosensitivity (Shewach et al. 1994; Shewach and Lawrence 1995; Lawrence et al. 1996, 1997). In addition, it has been reported that a 5-FU-derivative, 5- FdUrd, which depletes the intracellular TTP level but not dATP pools under the radiosensitizing condition, decreases the efficiency of repair of radiation-induced DNA damage (Bruso et al. 1990;
Heimburger et al. 1991). Likewise, a thymidine analog, 5-bromo-2’-deoxyuridine (BrdU), which is known to be incorporated into DNA and decrease the intracellular dCTP and TTP pools (Shewach et al. 1992b), increases the radiation-induced DNA damage, and moreover, decreases damage repair (Iliakis et al. 1989; Ling and Ward 1990). Further- more, hydroxyurea, whose primary activity is the depletion of intracellular dNTP levels, can enhance the sensitivity of the cell to radiation (Sinclair 1968a). Taken together, perturbation of the bal- ance of intracellular nucleosides seems to play an important role in causing radiosensitization, at least in part. This hypothesis is consistent with the reports showing that imbalances of the intracellu- lar dNTP pool produce errors in DNA replication (Kunz 1982; Bebenek et al. 1992; Martomo and Mathews 2002).
Fig. 2.5. Metabolism and mechanism of action of 6-Mercap- topurine. 6-Mercaptopurine (6-MP), hypoxanthine-guanine phosphoribosyl transferase (HGPRT), 6-thioinosine mono- phosphate (6-TIMP), inosine monophosphate dehydroge- nase (IMPDH), 6-thioxanthine monophosphate (6-TXMP), guanosine monophosphate synthetase (GMS), 6-thioguano- sine 5’-monophosphate (6-TGMP), deoxy-6-thioguanosine 5’-triphosphate (dGS), DNA polumerase (DNAP), thiopurine methyltransferase (TPMT), S-methyl-thioinosine 5’-mono- phosphate (MeTIMP), phosphoribosyl pyrophosphate amido- transferase (PRPPAT)
2.2.2
Cell Cycle Distribution
The cell cycle for mammalian cells is composed of four sequential phases: mitosis (M-phase); gap 1 (G1); DNA synthetic phase (S-phase); and gap 2 (G2), followed again by M-phase (see Fig. 2.6). It is known that cells are the most radioresistant in the S-phase, while they are radiosensitive in the M- and G2- phases (Sinclair 1968b; Pawlik and Keyomarsi 2004). Moreover, cells at the G1/S-boundary and in early S-phase are more radiosensitive than those in late S-phase (Latz et al. 1998); therefore, cell cycle modulation with an antimetabolite also seems to be very important for the radiation-enhancing effect.
The cell cycle effect of dFdCyd seems to be concen- tration dependent (Tolis et al. 1999; Cappella et al.
2001). It was reported that low concentrations (IC50 values) of dFdCyd cause cell cycle arrest in early S- phase (Merlin et al. 1998), and increasing concen- trations of the drug results in a shift of this arrest to the early S-phase and moreover to the G1/S-bound- ary (Pauwels et al. 2003). The radiation-enhancing effect of dFdCyd, therefore, could be explained by the accumulation of dFdCyd-treated cells in these radiosensitive phases. Indeed, the percentage of early S-phase cells was reported to correlate with the radiosensitizing effect (Pauwels et al. 2003); how- ever, it is still unclear both why radiosensitization occurs in cells at early S-phase and whether such a mechanism is responsible for the radiosensitizing property of the other antimetabolites, which exhibit the same kind of cell phase specificity.
In addition, it has also been reported that both dFdCyd and 5-FU selectively radiosensitize cells in S-phase, which are relatively radioresistant, whereby the fluctuation of radiosensitivity within the cell cycle is eliminated or at least reduced (Latz et al.
1998). Although the mechanism causing the above
phenomenon still remains unclear, the treatment may reduce the repair of radiation-induced cellular damage and increase the probability of the fixation of damage being lethal in S-phase cells.
2.2.3
Checkpoint and p53
In response to a stress signal, p53 protein is activated by post-translational modifications and leads to the transcription of various genes, which determine whether the cell will continue to progress, arrest in checkpoints and repair the DNA damage, induce cell senescence, or trigger apoptosis (Levine 1997;
Jin and Levine 2001). Cells expressing wild-type p53 were reported to accumulate in the G1/S cell-
Fig. 2.6. Cell cycle phase-dependent radiosensitivity of cells.
The cell cycle for mammalian cells is composed of four sequential phases: mitosis (M-phase); gap 1 (G1), DNA syn- thetic phase (S-phase) and gap 2 (G2), followed by M-phase again. The DNA content in each cell phase (dashed curve) and the relative radioresistance (solid curve) of the cell are shown Table 2.1. Antimetabolite-induced imbalance of intracellular dNTP pool and its radiosensitizing effect
Antimetabolite Imbalance of dNTP Effect Reference
dFdCyd (gemcitabine)
dATP depletion Radiosensitization in vitro and in vivo Shewach et al. (1994),
Shewach and Lawrence (1995), Lawrence et al. (1996), Lawrence et al. (1997) 5-FU
(5-FdUrd)
TTP depletion Decrease in repair of DNA damage induced by radiation
Bruso et al. (1990), Heimberger et al. (1991) BrdU dCTP and TTP depletion Increase in radiation-induced DNA
damage and decrease in damage repair
Iliakis et al. (1989), Ling et al. (1990) Hydroxyurea dNTPs depletion Radiosensitization in vitro Sinclair et al. (1968a,b)
Cell Cycle
Relative Radioresistance
G
M S G2 M
DNA content (per cell)
1 2 3 4 Radioresistance
DNA content per cell
Cell Cycle
Relative Radioresistance
G1
M S G M
DNA content (per cell)
1 2 3 4 Radioresistance
DNA content per cell Radioresistance DNA content per cell
cycle checkpoint in response to many DNA-damag- ing agents including irradiation (Linke et al. 1997;
Ostruszka and Shewach 2000). This arrest allows the cells not only to repair the DNA damage, but also to inhibit the replication of the cells with dam- aged genomic DNA (Sak et al. 2000). On the other hand, cells expressing mutant p53 were reported to continue to progress into S-phase and G2-M after the treatment and irradiation (Ostruszka and Shewach 2000). This may affect the radiosensitiv- ity of cells. Indeed, the p53 status and the inap- propriate progression of cells into S-phase in the presence of drugs, such as 5-FU and 5-FdUrd as well as dFdCyd, has been reported to affect radiation sensitivity. This conclusion is firstly derived from the result that 5-FdUrd enhances the radiosensitiv- ity of cells expressing G1/S cyclins in the presence of the drug, but not of cells expressing no activated cyclins (Lawrence et al. 1996). It is further con- firmed by the report that 5-FdUrd greatly increases the radiosensitivity of a cell, which can progress into S-phase after irradiation because of the expres- sion of mutant-type p53, but hardly shows a radio- sensitizing effect on a cell expressing wild-type p53 (Naida et al. 1998). Likewise, a previous study sug- gested that a cell line that has no ability to progress through S-phase after dFdCyd and radiation is not radiosensitized because of the expression of wild- type p53 (Ostruszka and Shewach 2000).
Yet, other reports have suggested that, regardless of the p53 status, dFdCyd can enhance the radiosen- sitivity of cells, as shown by Robinson and Shewach (2001), in whose study both wild-type p53-express- ing cells and mutant p53-expressing cells displayed high dATP depletion and S-phase accumulation after dFdCyd treatment, both of which we recog- nize as important factors in radiosensitization. That article suggests that p53 function alone might not determine the radiosensitivity in the presence of antimetabolites.
2.2.4 Apoptosis
Although antimetabolites are known to induce the expression of proapoptotic effector proteins, the level is not sufficient to induce cellular apoptosis.
In the case that the intracellular checkpoint system is working, the damage of genomic DNA is repaired and the intracellular level of proapoptotic factors decreases during the cell cycle arrest; however, during the above period, if the cells are exposed
to irradiation, the expression of proapoptotic fac- tors is further induced, leading to apoptosis. This mechanism may be a reason why the maximum radiosensitization with dFdCyd and with 5-FU occurs when cells are incubated with the drug before radiation.
2.3
Interactions of Antimetabolites with Radiation: Preclinical to Clinical
2.3.1
5-Fluorouracil and its Prodrugs: Capecitabine, UFT, and S-1
2.3.1.1 5-Fluorouracil
Since 5-FU was first introduced in 1957, it has been playing an essential part in the treatment of a wide range of solid tumors, such as epithelial malignan- cies in the gastrointestinal tract, pancreatic cancer, breast cancer, or head and neck cancer. Although the single-agent response rates are not so high, 5-FU-containing treatment regimens produced a highly significant survival improvement for sev- eral malignancies. Heidelberger et al. (1958) demonstrated very early that doses of radiation, which were inhibitory but not curative for rodent tumors, were made curative by combination with 5-FU. Subsequently, results in experimental ani- mals (Vermund et al. 1961) and quantitative stud- ies showed that 5-FU could enhance cell killing by radiation ( Bagshaw 1961; Berry 1966). These early observations led to a series of clinical investi- gations from the 1960s to 1970s. Firstly, controlled studies in gastric adenocarcinoma (Moertel et al. 1969) and head and neck squamous carcinoma (Gollin et al. 1972) showed improved response and survival rates by combined therapy with radiation and 5-FU; however, other studies could not achieve any remarkable results (Helsper and Sharp 1962;
Hall et al. 1967; Stein and Kaufman 1968; Sato et al. 1970). These negative, or less impressive results, were considered to be caused not only by the organ or tumor-specific characteristics, but also by some factors which depend on the administration sched- ule. During those periods, bolus 5-FU had been usually given with fractionated radiotherapy. In 1982, Byfield et al. (1982) demonstrated that cell killing was maximized if the cells were continu-
ously exposed to 5-FU following irradiation. In 1994, a well-controlled study in patients with rectal cancer [Mayo/North Center Cancer Treatment Group (NCCTG) 86-47-51’s protocol], comparing protracted venous infusion (PVI) with bolus injec- tion during radiotherapy, showed that PVI resulted in a significant improvement in both the time to relapse and survival (O’connell et al. 1994). As 5-FU has cell-cycle specificity and a short plasma half-life, the prolonged exposure of cells to the agent would theoretically result in enhanced cell killing. Presently, as a standard method, 5-FU is administered by PVI or by bolus infusion for con- secutive days during radiation.
Table 2.2 shows the representative studies con- ducted to test the efficacy of chemoradiotherapy with 5-FU for several cancers.
As for the patients with colorectal lesions, we need to be very careful about gastrointestinal toxicities.
Miller et al. (2002) analyzed the NCCTG trial and showed that patients who received 5-FU by PVI had a higher risk of severe or life-threatening diarrhea compared with those with bolus infusion during pelvic radiotherapy. Patients with locally advanced (T3 or T4) rectal cancer had been recommended to receive radiotherapy and chemotherapy either pre- operatively or postoperatively. Although the results
of the clinical trials of the National Surgical Adju- vant Breast and Bowel Project (NSABP) showed that chemoradiation altered neither the incidence of dis- tant metastases nor survival, there was a reduction in the locoregional relapse (see Table 2.2; Wolmark et al. 2000). To reduce the toxicities and to enhance the survival for colorectal cancer, optimizing the regimen of chemoradiation is still under active investigation.
For the treatment of esophageal cancer, the ran- domized study, EST-1282, was undertaken by the Eastern Cooperative Oncology Group (ECOG) to determine whether the combined use of 5-FU, mito- mycin C (MMC) and radiation therapy improved the survival of patients compared with radiation therapy alone. In that study, 5-FU was delivered by continuous infusion for 96 h, initiated on day 2 and day 28 during radiation therapy (Smith et al.
1998). The result was that patients treated with chemoradiation had a longer median survival than patients receiving radiation therapy alone.
The Radiation Therapy Oncology Group (RTOG) compared radiotherapy of 64 Gy in 32 fractions alone with chemoradiotherapy of 50 Gy in 25 frac- tions with cisplatin/5-FU, showing that combined therapy significantly increased the overall survival (Cooper et al. 1999).
Table 2.2. Randomized trials of chemoradiation with 5-fl uorouracil (5-FU)
Study Treatment No. of patients Median survival
(months)
Survival (%)
Follow-up (years) Rectal cancer
Krook et al. (1991) S+RT 100 35 7
(median)
Mayo/NCCTG79-47-51 S+RT+5-FU 104 55 7
(median)
O’Connell (1994) S+RT+bolus 5-FU 332 60 4
Mayo/NCCTG86-47-51 S+RT+infusional 5-FU 328 70 4
Wolmark et al. (2000) S+5-FU/LV 348 6265 5
NSABP R-02 S+5-FU or MOF/LV+RT 346 6265 5
Pancreatic cancer
Moertel et al. (1981) RT 60 Gy 5.2
Gastrointestinal Tumor RT 40 Gy+5-FU 9.6
Study Group (GISTG) RT 60 Gy+5-FU 9.2
GISTG (1988) RT 54 Gy+5-FU 10.5
GISTG (1988) SMF 8
Esophageal cancer
Cooper et al. (1985) RT64 Gy 62 9.3 0 (5 years) 8
RTOG 85-01 RT50 Gy+CDDP/5-FU 61 14.1 26 (5 years) 8
S surgery, RT radiotherapy, SMF STZ, mitomycin C and 5-FU, MOF 5-FU, semustine, and vincristine, LV leucovorin, CDDP cisplatin.
2.3.1.2
Prodrugs of 5-FU: UFT; S-1; and Capecitabine
A prodrug is defined as a pharmacologically inac- tive compound that is converted into an active agent by a metabolic biotransformation. The prodrugs of 5-FU are characterized by a pyrimidine ring with a fluorine atom in position 5. The first generations of prodrugs of 5-FU were represented by 5-fluoro- 2’-deoxyuridine (5-FdUrd), which was more effi- ciently metabolized by the liver than 5-FU. As for the second generation, ftorafur (FTO, 1-(2-tetrahy- drofuryl)-5-fluorouracil, Tegaful or Futraful) and 5’-deoxy-5-fluorouridine (5’d5-FUrd, doxifluridine or Furtulon) were developed with the intention of possible oral administration. They are designed to be well absorbed from the gastrointestinal tract and enzymatically converted into 5-FU by hepatic microsomal cytochrome P-450.
The third-generation compounds include those preferentially activated in the tumor: capecitabine, and the dehydropyrimidine dehydrogenase (DPD) inhibitory compounds; UFT (FTO+uracil); and S-1 [FTO+5-chloro-2, 4-dihydroxypyrimidine (CDHP;
gimestat)+potassium oxonate (OXO; otastat)].
2.3.1.2.1 UFT
Although prolonged continuous exposure has been demonstrated to have advantages over bolus admin- istration in rectal, head and neck, and esophageal cancers, another dimension for the timing of FU in the clinic is illustrated by the animal models, which demonstrate daily cyclic patterns of mitosis and enzyme activity. The activities of DPD, which inacti- vates FU, have been shown to vary significantly over 24 h. Because the pharmacokinetics and sensitivity of 5-FU are determined by DPD, stabilization of the pharmacokinetics of 5-FU and the enhancement of its efficacy have been attempted by means of DPD inhibition. The UFT is a small molecule, a combina- tion agent utilizing FTO and uracil in molar propor- tions of 1:4. Uracil is a competitive and irrevers- ible inhibitor of DPD. This combination ratio was chosen based on preclinical models that suggested tumor selectivity, which produces a constant reserve of 5-FU and its active metabolites and minimizes production of inactive and potentially toxic metabo- lites (Fujii et al. 1979). Later, some other preclinical studies demonstrated that this combination resulted in significant improvements in the tumor to normal tissue and tissue serum ratios of 5-FU (Taguchi
1997). By the stabilization and prolongation of the effective half-life of FU, the opportunity for synergy with radiation had been considered to be greater. To date, there has been a limited number of published studies combining UFT with radiation therapy, but recently a phase-I trial examined the use of the oral delivery of UFT/leucovorin with conventional RT of 45 Gy for pancreatic cancer. Results compared favorably with continuous-infusion regimens show- ing the potential benefit (Childs et al. 2000). In preoperative and postoperative rectal cancer ther- apy, several studies are ongoing (Rosenthal et al.
2000). For unresectable non-small cell lung cancer, Ichinose et al. (2005) conducted a multi-institu- tional phase-II study in which the combination of chemotherapy of UFT plus cisplatin was given with concurrent radiotherapy, showing a satisfactory response (overall response rate 81%) with no severe toxicities.
2.3.1.2.2 S-1
An oral formulation of 5-FU with DPD inhibitors makes oral absorption very reliable and allows protracted exposure without the need for a venous catheter infusion pump; however, the results of sev- eral phase-I trials showed that the dose-limiting toxicity (DLT) was gastrointestinal reactions (diar- rhea, nausea, vomiting; Gonzalez Baron et al.
1993; Muggia et al. 1996; Pazdur et al. 1998). The pharmacology study showed that the occurrence of these toxic effects correlated significantly with the maximum plasma concentration and area under the concentration-vs-time curve (AUC 0–6 h) of 5-FU (Ho et al. 2000). The next strategy in the 1990s was to develop new kinds of drugs to alleviate the gastrointestinal toxicities, without reduction of the high plasma concentration of 5-FU. S-1 (or TS-1) is a combination of FTO and two compounds, CDHP and OXO, which was developed in 1996 by Japanese groups. The CDHP and OXO were designed to act as modulators of 5-FU. The CDHP is a reversible and strong inhibitor of DPD. In vitro, CDHP is 180 times more potent than uracil (Tatsumi et al.
1987). The OXO accumulates in the gastrointesti- nal tissues and competitively inhibits the enzyme orotate phosphoribosyl transferase (OPRT), which converts 5-FU to 5-FUMP. The phosphorylation of 5-FU within the digestive tract by OPRT has been considered the cause of gastrointestinal toxicities.
In a preclinical study of Yoshida sarcoma-bearing rats, the administration of OXO with UFT mark-
edly reduced the injury of gastrointestinal tissues and/or severe diarrhea without influencing the antitumor effect of UFT (Shirasaka et al. 1993).
The optimal molar ratio of the three constituents (FTO/CDHP/OXO) in S-1 is 1:0.4:1. Several pre- clinical studies in experimental models of rodent tumors or human xenografts demonstrated that S- 1 significantly inhibited tumor growth with lower gastrointestinal toxicities (Takechi et al. 1997;
Fukushima et al. 1998; Cao et al. 1999). Clinically, S-1 has been approved for use in Japan for gastric cancer, head and neck cancer, colorectal cancer, and non-small cell lung cancer, and in Korea for advanced or recurrent gastric cancer, head and neck cancer, and unresectable metastatic or recur- rent colorectal cancer. Presently, several trials of newly combined chemotherapy of S-1 plus cispla- tin or CPT-11 with radiotherapy for head and neck cancer, colorectal cancer, and non-small cell lung cancer are ongoing.
2.3.1.2.3 Capecitabine
Capecitabine (N4-pentyloxycarbonyl-5’-deoxy-5- fluorovytidine) is an oral fluoropyrimidine car- bonate that is converted to 5-FU through a cascade of three enzymes: carboxylesterase; cytidine (Cyd) deaminase; and thymidine phosphorylase (TP).
These enzymes have been known to have unique tissue localization patterns: carboxylesterase is almost exclusively located in high concentrations in the liver and hepatoma, but not other tumors and normal tissues, Cyd deaminase is located in high concentration in the liver and various types of solid tumors, and TP is more concentrated in vari- ous types of tumor tissues than in normal tissues (Miwa et al. 1998). Oral capecitabine passes intact through the intestinal tract almost completely to be first converted in the liver; thus, theoretically, gastrointestinal injury should not occur with this compound. Schuller et al. (2000) demonstrated that in the resected samples of patients with colon cancer, the concentration of 5-FU was on average 3.2 times higher than in adjacent healthy tissues. In 1995, TP was reported to be identical with platelet- derived endothelial cell growth factor (PD-ECGF;
Haraguchi et al. 1994; Moghaddam et al. 1995), which is expressed at higher levels in a wide variety of solid tumors compared with adjacent normal tis- sues. PD-ECGF/TP was suggested to be able to confer resistance to apoptosis induced by hypoxia, and deg- radation products of thymidine are involved in this
resistance (Kitazono et al. 1998). Takebayashi et al. (1996) reported that higher levels of PD-ECGF/
TP expression in colorectal carcinomas were asso- ciated with more aggressive malignant growth and unfavorable clinical outcome; thus, a tumor-specific enzyme is responsible for the local production of the cytotoxic agent, 5-FU, from capecitabine, i.e., capecitabine should increase the concentration of active FU in tumor site and decrease the concen- tration in healthy normal tissues with a reduction of toxicity. Moreover, in human cancer xenografts, a number of chemotherapeutic agents, such as paclitaxel and docetaxel (Sawada et al. 1998), and cyclophosphamide (Endo et al. 1999), and radia- tion (Sawada et al. 1999), upregulate TP. Sawada et al. (1999) demonstrated that a single-dose local irradiation of 5 Gy increased the TP levels by up to 13-fold 9 days after irradiation. They also observed that whole-body irradiation upregulated TP in a tumor, but it did not increase the enzyme level in the liver. Capecitabine has been approved in more than 50 countries, e.g., in metastatic breast cancer (Blum 2001). On the basis of two large phase-III studies, capecitabine has been approved as a first- line alternative to IV FU/LV in advanced metastatic colorectal cancer (Hoff et al. 2001; Van Cutsem et al. 2001) and recently, capecitabine has been used in combination therapy with CPT-11 or oxaliplatin with promising results in phase-II studies. Phase- III studies comparing capecitabine plus oxaliplatin with standard FU/LV/oxaliplatin (FOLFOX) are in progress. The clinical benefits of combined therapy with capecitabine and radiation are under investi- gation. The efficacy and safety of combined chemo- therapy with capecitabine lends support for its use in chemoradiotherapy.
2.3.2 Gemcitabine
Gemcitabine exhibits cell phase specificity, primar- ily killing cells undergoing DNA synthesis (S-phase) and also blocking the progression of cells through the G1/S-phase boundary. Gemcitabine has recently been shown to be a potent radiosensitizer in preclin- ical studies using human tumor cell lines, caused by perturbation of nucleotide metabolism, cell cycle modulation, or caspase activation and apoptosis (see section 2.1.1.2); however, the mechanisms have not been fully elucidated. Even at very low concentra- tions, gemcitabine has been shown to be a power- ful radiation sensitizer (Mason 1999). Lawrence
et al. (1996, 1997) showed that 100-nm gemcitabine, which was noncytotoxic, radiosensitized HT29 cells up to 48 h after drug removal. During this period, there was an increase in the S-phase population and dNTP pools remained depleted throughout the 72-h period after drug treatment. Actually, the most common method of clinical administration is by short-term infusion (30–90 min), which is relevant to the minimalization of normal tissue toxicity, while maintaining synergistic effects during radio- therapy. Gemcitabine has shown promising clini- cal effectiveness against a range of solid tumors, most importantly non-small cell lung cancer, breast cancer, and pancreatic cancer; however, because of the radiosensitization of both tumor and normal tissues, the therapeutic window is extremely narrow (Rube et al. 2004). Some clinical trials of concur- rent radiotherapy with gemcitabine for non-small cell lung cancer or head and neck cancer revealed severe pulmonary or esophageal toxicities. Many investigators are now attempting to define the opti- mal dosing and schedule for concurrent gemcitabine and radiation therapy.
2.3.3 Pemetrexed
Pemetrexed is a novel agent that inhibits several enzymes of thymidylate and purine synthesis, dis- rupting metabolic processes essential for cell rep- lication. A large, randomized phase-III trial was conducted to compare pemetrexed/cisplatin with cisplatin in the treatment of malignant mesothe- lioma, and the combination of pemetrexed/cispla- tin demonstrated superiority in the response rate, survival, and quality of life (Hazarika et al. 2005).
Now several clinical trials are ongoing for malignant mesothelioma, non-small cell lung cancer, pancre- atic cancer, head and neck cancer, breast cancer, and colorectal cancer. Among new combination thera- pies, those in progress include pemetrexed with carboplatin, oxaliplatin (Scagliotti et al. 2005), or gemcitabine (Monnerat et al. 2004) for lung cancer, with gemcitabine for pancreatic cancer (Oettle et al. 2005), and with irinotecan for colorectal cancer (Hochster et al. 2005); however, a randomized phase-III study of pemetrexed plus gemcitabine vs gemcitabine in patients with advanced pancreatic cancer showed that pemetrexed plus gemcitabine regimen was not superior in overall survival or toxicities, but other studies demonstrated that the combinations had good tolerance and were at least
as active as previous, more toxic regimens. As for the combination with radiation, there are some preclini- cal studies to assess the radiosensitizing potential of pemetrexed. Bischof et al. (2002) demonstrated that pemetrexed enhanced the radiation-induced cell inactivation at moderately toxic exposures and over many hours after drug removal by in vitro survival assays. They also demonstrated the high antiendothelial/antitumoral efficacy of the concur- rent administration of irradiation, pemetrexed and VEGFR inhibitor (SU5416) in vitro (Bischof et al.
2004). Clinical trials of combinations of pemetrexed and radiation with or without other chemothera- peutic agents for malignant mesothelioma and non- small cell lung cancer are now ongoing.
2.4
Conclusion
Antimetabolite agents have remarkable antineo- plastic activities, and their radiosensitizing effects have been reported for several decades both preclin- ically and clinically. Although the mechanisms of radiosensitization have not been fully understood, several preclinical studies have focused on the modulation of the metabolism, the changes in cell cycle distribution, the role of p53, and the induction of apoptosis. Chemoradiation with antimetabolites has become the standard therapy for the treatment of a wide range of solid tumors. Especially 5-FU has been one of the most commonly used anticancer drugs in combination with radiotherapy. Recently, oral 5-FU prodrugs are emerging in the clinical area, such as UFT, S-1, and capecitabine. They have new characteristics related to unique metabolic pat- terns or enzymatic inhibitors of degradation. These pharmacological features and oral formulation allow protracted exposure of FU to the tumor tissues without the need for a central venous catheter or infusion pump. The clinical trials have just started, but the preliminary results of the combination of oral 5-FU prodrugs and radiation are very encour- aging. Other newer antimetabolite agents, such as gemcitabine or pemetrexed, have been developed and clinically investigated for several years. As for the combination with radiation, gemcitabine is being established in the key role of systemic therapy in locally advanced pancreatic cancer, and chemo- radiation with gemcitabine is becoming one of the standard therapies, as an alternative to 5-FU and radiation.
Currently, several combined chemotherapies of these antimetabolites with other active agents, such as cisplatin, CPT-11, or taxanes, are under investiga- tion. We need to determine the efficacy of chemora- diation using these newly combined chemotherapy regimens, as compared with previous standard chemoradiotherapies for each tumor. In the future, the assessment of combinations with novel molecu- lar targeted therapies, such as, for example, those studied in tumor xenografts treated with C225, gem- citabine, and radiation (Buchsbaum et al. 2002), is also warranted.
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