Annals of Oncology 13: 1841–1851, 2002

Review DOI: 10.1093/annonc/mdf337

Irinotecan: mechanisms of tumor resistance and novel strategies

for modulating its activity
Y. Xu & M. A. Villalona-Calero*
Department of Medicine and the Experimental Therapeutics Program, Comprehensive Cancer Center, Arthur G. James Cancer Hospital and
Richard J. Solove Research Institute, Ohio State University, Columbus, OH, USA

Received 27 February 2002; revised 1 July 2002; accepted 17 July 2002

Camptothecins are broad-spectrum anticancer drugs that specifically target DNA topoisomerase I
(Topo I). The formation of a cleavable drug–Topo I–DNA complex results in lethal double-strand DNA
breakage and cell death. However, de novo or acquired clinical resistance to camptothecins is common.
Studies of the camptothecin analog irinotecan suggest the following general mechanisms of resistance:
(i) variable levels of the enzymes involved in the conversion of irinotecan; (ii) reduced cellular accu-
mulation from active drug efflux; (iii) reduced levels of Topo I expression; (iv) alterations in the
structure of Topo I from different mutations; (v) alterations in the cellular response to camptothecin–
Topo I–DNA complex formation, which involves proteasome degradation of Topo I and/or enhanced
DNA repair; and (vi) activation of the transcription factor nuclear factor kappa B by DNA damage and
subsequent suppression of apoptosis. Multiple approaches using pharmacological and biological
modulation to circumvent the above mechanisms of resistance have been incorporated into ongoing
clinical trials and are expected to enhance the antitumor activity of irinotecan and reduce its systemic
toxicity.
Key words: apoptosis, irinotecan, modulation, nuclear factor kappa B, proteasome, resistance

Introduction failure of 5-fluorouracil (5-FU) and leucovorin [2], and more

Irinotecan (CPT-11) is a semisynthetic analog of campto-
thecin, originally isolated from the Chinese/Tibetan ornamen-
tal tree Camptotheca acuminata. It is a chemotherapy agent
that causes S-phase-specific cell killing by poisoning topo-
isomerase I (Topo I) in the cell. It was first discovered and
synthesized in Japan in 1983, and it has now demonstrated
potent antitumor activity against a wide range of tumors
[1]. Numerous studies have been done to uncover possible
mechanisms for the cellular resistance to this agent, and mul-
tiple experimental approaches are being tested in clinical trials
for their potential ability to circumvent individual mechan-
isms for this resistance. This paper summarizes the recent
advances in this field.
Clinical significance and toxicity
Irinotecan has shown activity against colorectal, esophageal,
gastric, non-small-cell and small-cell lung cancers, leukemia
and lymphomas, as well as central nervous system malignant
gliomas [1]. Initial approval in the United States was as a
second-line treatment for metastatic colorectal cancer, after
*Correspondence to: Dr M. A. Villalona-Calero, B406 Starling-Loving
Hall, 320 West 10th Avenue, Columbus, OH 43210, USA.
Tel: +1-614-293-7511; Fax: +1-614-293-7529;
E-mail: Villalona-1@medctr.osu.edu
recently it has been approved for use in combination with
5-FU/leucovorin as a first-line treatment for this disease [3, 4].
Studies evaluating irinotecan as adjuvant chemotherapy in
node-positive colorectal cancer after resection are underway.
Likewise, phase II studies on advanced esophageal and gastric
cancer also showed encouragingly high response rates [5, 6].
In addition, the combination of irinotecan and cisplatin is cur-
rently being compared with cisplatin and etoposide in patients
with extensive stage small-cell lung cancer in randomized
trials in the United States, based on exciting results from a
Japanese randomized trial. This combination showed marked
superior response rate (84.4% versus 67.5%, P = 0.02) and
prolonged survival (median survival 12.8 versus 9.4 months,
P = 0.002) [7]. In non-small-cell lung cancer, the combination
of cisplatin and irinotecan is at least comparable with other
active combination regimens containing cisplatin [8].
The major toxicities of irinotecan in clinical use are
myelosuppression and diarrhea. It can cause either acute
diarrhea related to a cholinergic surge from inhibition of
acetylcholinesterase, or a delayed diarrhea syndrome, which
is possibly related to the accumulation of the active metabolite
of irinotecan in the bowel [9]. On occasion, these side-effects
can be life-threatening, calling for pharmacological studies to
optimize dosing and schedules or for biological modulation of
undesirable effects.

© 2002 European Society for Medical Oncology

1842

Pharmacology by uridine diphosphate glucoronosyltransferase isoform 1A1

Understanding the pharmacology of irinotecan provides in-
sights into the basis of irinotecan toxicity and tumor resistance
[10]. Irinotecan is a unique semisynthetic camptothecin,
characterized by the presence of a bulky piperidino side-chain
at the C-10 position [11]. This side-chain can be cleaved
enzymatically by carboxylesterase to 7-ethyl-10-hydroxy-
camptothecin (SN-38), which is 1000-fold more potent than
irinotecan [12]. Both irinotecan and SN-38 are in equilibrium
with their active lactone and inactive carboxylate forms, an
equilibrium that is pH- and protein-dependent [13, 14]. The
metabolism and mechanism of action of irinotecan are sum-
marized in Figure 1.
Carboxylesterase activity is found in serum, liver, intestine
and other sites [15]. Genetic variability of carboxylesterase
expression and/or activity is suspected, as variations of SN-38
levels are observed from individual to individual for a given
dose of irinotecan [16]. After conversion from irinotecan by
carboxylesterase, SN-38 is deactivated through conjugation
(UGT 1A1) in the liver to an inactive form, SN-38 glucuron-
ide (SN-38G). This isoenzyme, which is also responsible for
the glucuronidation of bilirubin, is mutated in Gilbert’s syn-
drome [17] and deficient in Crigler–Najjar syndrome type I
[18]. As a result, patients with these disorders are at increased
risk for severe irinotecan-induced toxicity. The deficiency in
Gilbert’s syndrome is due to a homozygous TA insertion in
the TATAA promoter of UGT 1A1, leading to a mutated allele
[19]. Pretreatment of rats with valproic acid caused 99%
inhibition of the formation of SN-38G by inhibiting UGT 1A1
enzyme activity, leading to a 270% increase in the area under
the plasma concentration–time curve (AUC) [20]. In contrast,
pretreatment with phenobarbital, an inducer of UGT 1A1,
caused a 1.7-fold increase in the AUC of SN-38G, and 31 and
59% reduction in the AUC of SN-38 and irinotecan, respect-
ively [20].
The elimination of irinotecan and SN-38 is through biliary
excretion, which is dependent on the canalicular multispecific

Figure 1. Depicted are the steps involved in the activation and metabolism of irinotecan, as well as its mechanism of action. Numerals reflect areas in
which enhancement (+) or interference (–) with irinotecan action and toxicity have been reported: 1, phenytoin and carbamazepine (–); 2,
carboxylesterase (CE) gene transfer (+); 3, genetic profiling (+), phenobarbital and dexamethasone (–), valproic acid (+); 4, antisense canalicular
multispecific organic anion transporter (cMOAT) (+), cyclosporin (+); 5, antibiotics (–); 6, intestinal alkalinization (–); 7, multidrug resistance-
associated protein, P-glycoprotein and breast cancer resistance protein inhibitors (+); 8, topoisomerase I (Topo I) increase (+), Topo I mutation (–);
9, proteasome inhibition (+); 10, gene transfer (IκBα) (+), proteasome inhibition, thalidomide and cyclooxygenase inhibition (+). APC,
aminopentanecarboxylic acid; B Gluc, β-glucuronidase; CYP 3A4, cytochrome P-450; ds, double strand; NF-κB, nuclear factor kappa B;
NPC, 7-ethyl-10[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin; SN-38, 7-ethyl-10-hydroxycamptothecin; SN-38G, SN-38 glucuronide;
ss, single strand; SUMO, small ubiquitin-like modifier; UGT, uridine diphosphate glucoronosyltransferase.

organic anion transporter (cMOAT), a member of the trans-
porters with an ATP cassette [21, 22]. Treatment with cyclo-
sporin A, which decreases biliary flow and inhibits cMOAT,
increases the AUC of irinotecan and SN-38 [23]. Subse-
quently, SN-38G is deconjugated to SN-38 by β-glucuro-
nidase produced by the intestinal bacterial flora, which may
account for the late SN-38 double peaks in the plasma, and
may be responsible for the delayed intestinal toxicity of irino-
tecan [24]. Indeed, β-glucuronidase activity is correlated with
irinotecan-induced cecal damage in the rat [25]. The toxicity
in this model was attenuated by antibiotic administration,
which presumably inhibits the intestinal microflora prolifera-
tion [25].
A second major metabolite of irinotecan is aminopentane-
carboxylic acid (APC), produced by oxidation of the terminal
piperidine ring by the cytochrome P-450 CYP3A4 enzyme
[26]. APC does not hydrolyze to SN-38 and is a poor inhibitor
of the Topo I–DNA cleavable complex.
Mechanism of action
Irinotecan interacts with cellular Topo I–DNA complexes and
has S-phase-specific cytotoxicity [27]. Topoisomerases reduce
DNA twisting and supercoiling that occur in selected regions
of DNA as a result of essential cellular processes such as trans-
cription, replication and repair recombination. They cleave
and reseal the phosphodiester backbone of DNA, and form a
covalent enzyme–DNA linkage, which allows the passage of
another single- or double-stranded DNA through the nicked
DNA. Topo I binds to single-strand DNA breaks, and the
reversible Topo I–irinotecan–DNA cleavable complex is not
lethal to the cells by itself. However, upon their collisions with
the advancing replication forks, the formation of a double-
strand DNA break occurs, leading to irreversible arrest of the
replication fork and cell death [27]. The collision of the irino-
tecan–Topo I complex with the replication fork also results in
G2 arrest/delay by signaling the presence of DNA damage to
an S-phase checkpoint mechanism [28]. At higher concentra-
tions of irinotecan, non-S-phase cells can also be killed. The
mechanism of non-S-phase cell killing appears to be related to
transcriptionally mediated DNA damage, and through the
mechanism of apoptosis [29].
Mechanisms of irinotecan resistance
Pharmacokinetics and metabolism of irinotecan
Irinotecan is subject to extensive metabolic conversion by
various enzymatic systems in the body. The conversion of
irinotecan to the more active form SN-38 requires carboxy-
lesterase. A clear relationship between carboxylesterase level
and the chemosensitivity of human small-cell and non-small-
cell lung cancer cell lines has been demonstrated in vitro,
where irinotecan resistance was encountered in cell lines with
low carboxylesterase expression [30]. Furthermore, as SN-38
1843
is inactivated to SN-38G by glucuronidation by UGT 1A1,
overexpression of UGT at the protein and mRNA levels was
found to account for SN-38 resistance in a human lung cancer
cell line [31].
Irinotecan’s complex metabolism makes this agent a target
of potential interactions with other medications. The most com-
pelling example of interference with irinotecan pharmaco-
kinetics comes from studies in patients with central nervous
system tumors [32]. Irinotecan’s clearance in these patients
was approximately two-fold higher than reported in previous
trials. The observation that the overwhelming majority of
these patients were receiving enzyme-inducing antiepileptic
drugs (phenytoin, carbamazepine and phenobarbital) led to
the suspicion that induction of hepatic cytochrome P-450
enzymes, including CYP3A4, resulted in increased conver-
sion of irinotecan to its APC inactive metabolite. In addition,
phenobarbital and dexamethasone are known to induce
glucoronyl transferase, enhancing the conversion of SN-38 to
SN-38G [20]. Conversely, another anticonvulsant, valproic
acid, inhibits SN-38 conjugation, decreasing therefore irinote-
can clearance [20]. A phase I study in patients with malignant
glioma receiving irinotecan and concurrent enzyme-inducing
antiepileptic drugs demonstrated that doses of irinotecan that
are two-fold higher than the usually recommended doses can
be administered safely to these patients [33].
Decreasing the intracellular level of irinotecan by
active efflux
Multidrug resistance, a major obstacle encountered in cancer
chemotherapy, is characterized by overexpression of ATP-
binding cassette (ABC) transmembrane transporters such as
P-glycoprotein (P-gp) and multidrug resistance-associated
protein (MRP), which actively transport chemotherapy agents
out of the cell [34]. P-gp accepts amphipathic cationic or
neutral compounds as substrate, whereas MRP acts as a
glutathione S-conjugate (GS-X) export pump. Several ABC
proteins have been shown to be able to efflux camptothecins.
Using membrane vesicles from human cell lines overexpress-
ing P-gp and MRP, it was found that both P-gp and MRP are
involved in the active efflux of SN-38 and irinotecan [35]. The
involvement of MRP was further shown in MRP-overexpress-
ing transfectants, where the ATP-dependent efflux of irino-
tecan and SN-38 was inhibited by its specific inhibitor
PAK-104P [36]. Another ATP-dependent transporter, cMOAT
(also known as MRP2), which shares 49% amino acid identity
with MRP1, also exhibits GS-X pump activity [37]. cMOAT
is predominantly located in hepatic cells, where it is involved
in the excretion of irinotecan to the bile. Transfection of
cMOAT antisense cDNA into a human hepatic cancer cell line
results in a reduction in cMOAT protein and increased sensit-
ivity to camptothecin derivatives [38].
In addition, the breast cancer resistance protein (BCRP),
another member of the ABC family of drug transporters, also
mediates resistance to camptothecins [39]. The affinities to
1844
BCRP are as follows in descending order: SN-38, topotecan,
9-aminocamptothecin (9-AC), irinotecan. Administration of a
BCRP inhibitor, GF120918, reverses the resistance almost
completely at 100 nM, further confirming the involvement of
BCRP in irinotecan efflux [40]. Overexpression of BRCP has
been described in ovarian, breast, colon and gastric cancer cell
lines [40], implying their chemoresistance. In summary, the
above results indicate that the P-gp and MRP family of
transporters play important roles in the efflux and active
excretion of irinotecan and can be targets for pharmacological
modulation.
Quantitative Topo I level
As Topo I is the cellular target of irinotecan, it is conceivable
that the cellular level of Topo I would be proportional to
irinotecan cytotoxic effects. This notion is supported by
experimental evidence from yeast systems and mammalian
cell lines [41, 42]. In irinotecan-resistant cell lines rendered
resistant by stepwise, continuous treatment with irinotecan,
the total activity of Topo I was shown to be reduced compared
with the irinotecan-sensitive parental cell line [43]. Theoret-
ically, the expression of Topo I in tumor specimens may serve
as a predictor for sensitivity to irinotecan chemotherapy.
Increased levels of Topo I are demonstrated in colon cancer
and prostate cancer, but not in renal cancer [43, 44]. Topo I
levels are higher in colorectal tumors compared with normal
colon mucosa, suggesting a favorable therapeutic index [43].
Pathological specimens from breast cancer and seminoma
examined for Topo I expression using immunohistochemical
staining showed great variability, with increased expression
observed in only 41% of the breast and 30% of the seminoma
specimens tested, respectively [45, 46]. Although no consist-
ent association has been described between pretreatment
tumoral Topo I expression and antitumor response to irino-
tecan, this question has not been properly addressed in pro-
spectively designed clinical trials. In addition, tumor cells
may escape irinotecan cytotoxic effects by reducing the level
of Topo I expression. Therefore, dynamic studies, evaluating
the behavior of Topo I in cancer patients during a treatment
period are warranted.
Multiple mechanisms contribute to the regulation of Topo I
level in the cell. In tumors demonstrating increased levels of
enzyme expression, increased levels of mRNA were also
observed [44]. This would indicate either increased trans-
cription or increased mRNA stability. On the other hand,
specimens with reduced expression were found to have a non-
productive rearrangement of the Topo I genome, leading to
decreased transcription and thus reduced enzymatic produc-
tion of Topo I [47]. The rearranged allele in these specimens is
hypermethylated, resulting in transcriptional silencing.
Topo I mutations
Certain cell lines resistant to camptothecin have been found to
harbor mutations in Topo I [48, 49]. Mutations have been
found in the area that may alter DNA cleavage or Topo I–
DNA–irinotecan interactions, so that camptothecin can no
longer enter the complex [50]. Mutations immediately flank-
ing the catalytic tyrosine may affect the catalytic activity of
the enzyme, altering its ability to relax supercoiled DNA [51].
Recently, the crystal structure of Topo I–DNA covalent com-
plexes became available, which will allow structural mapping
of these mutations and may shed light on the functional roles
of the mutations [52, 53].
DNA repair: ubiqitin/26S proteasome-mediated
degradation of Topo I
Another cellular mechanism of resistance to irinotecan is
repair of irinotecan-induced DNA damage; a mechanism which
is coupled with RNA transcription [27]. According to this
model, the collision between the elongation RNA polymerase
complex and the Topo-I cleavable complex (on the template
strand) results in transcription arrest and the formation of a
Topo I linked single-strand break. This collision triggers
degradation of Topo I through an ubiquitin/26S proteasome-
dependent system [54–56]. Subsequent to Topo I destruction,
repair of the single-strand break can presumably occur. The
model is illustrated by studies in breast cell lines, demonstrat-
ing that the most sensitive cell line was completely defective
in irinotecan-induced Topo I reduction, whereas the least-
sensitive line exhibited effective Topo I reduction. Tumor
sensitivity to proteasome degradation can therefore serve as
an important parameter for determining irinotecan sensitivity/
resistance [55]. However, studies on a panel of breast, colon
and leukemia cell lines have demonstrated that irinotecan-
induced Topo I reduction is defective in most tumor cell lines
[27], whereas Topo I reduction in peripheral blood mono-
nuclear cells has been observed in patients treated with
irinotecan [57]. It remains to be established whether irino-
tecan-induced Topo I reduction occurs preferentially in normal
cells to protect them from irinotecan toxicity, while tumor
cells remain sensitive.
In addition to the ubiquitin degradation pathway, a small
ubiquitin-like modifier (SUMO-1) can also conjugate Topo I
[58]. The role of SUMO–protein conjugation is still unclear,
and diverse functions have been proposed. One speculation is
that SUMO may target Topo I for relocation to a different
cellular compartment, so it cannot participate in the formation
of Topo I cleavable complexes [58]. Likewise, a gene that
encodes an enzyme that can hydrolyze the bond between Topo
I–DNA has been isolated in yeast systems [59]. Enzyme-
defective mutants of this gene are hypersensitive to treatments
that increase the amount of covalent complexes. Identification
of this gene in humans may have implications for the effect-
iveness of Topo I interactive agents in the clinic.
Nuclear factor kappa B activation
As a result of irinotecan–Topo I–DNA complex formation and
its collision with the replication fork, a double-stranded DNA

break ensues. The double-stranded DNA damage is lethal and
causes cell-cycle arrest and apoptosis. Recently, multiple
experiments also showed that treatment of irinotecan activates
nuclear factor kappa B (NF-κB), an ubiquitous transcription
factor which controls the transcription of a wild variety of
genes involved in inflammation and immunity [60–64]. The
activation was dependent on initial nuclear DNA damage,
followed by cytoplasmic signaling events [62]. This very
important discovery has raised awareness of another mechan-
ism of chemoresistance, as many experiments on the diverse
roles of activated NF-κB indicated that NF-κB acts as an
antiapoptotic factor, especially in early transforming tumor
cells. Activated NF-κB can suppress the apoptotic cascade
induced by tumor necrosis factor-alpha (TNF-α), oncogenic
Ras, and chemotherapy agents, particularly irinotecan [61–65].
Inhibition of NF-κB activation augments irinotecan-induced
apoptosis [63, 64]. In addition, NF-κB knockout mice die
embryonically from extensive apoptosis within the liver [66],
indicating again that NF-κB may normally be antiapoptotic.
How does NF-κB protect cells from apoptosis? It has been
suspected that the antiapoptotic effect of NF-κB involves the
regulation and activation of pro-survival genes; however, only
a few of them have been identified. Major recent discoveries
have revealed that the NF-κB cell-survival pathway cross-
talks with the c-Jun N-terminal kinase (JNK) pathway, sub-
stantially blunting this pathway [67, 68]. JNKs are part of the
evolutionarily conserved mitogen-activated protein kinase
family, and are implicated in cell-death pathways stimulated
by environmental stresses and TNF-α. Two JNK inhibitory
proteins have been discovered. In one model, activation of
NF-κB mediates transcriptional increase of a protein called
gadd45b/myd118, which then lowers JNK signaling induced
by the TNF-α receptor [67]. The other protein (NF-κB-
induced X-chromosome-linked inhibitor of apoptosis) is
switched on by TNF-α in an NF-κB-dependent manner and
also blunts JNK activation [68]. These discoveries undoubtedly
shed light on elucidating the downstream events of NF-κB
activation, and its role of antiapoptosis and cell survival
following TNF-α stimulation and chemotherapy.
Strategies to overcome resistance to
irinotecan
In vivo transfer of carboxylesterase cDNA to solid
tumors
Irinotecan is activated by carboxylesterase to SN-38 in vivo.
Although plasma and liver carboxylesterase presumably pro-
duce sufficient SN-38 for cytotoxicity, a lack of such conver-
sion at the tumor site may result in decreased sensitivity to
irinotecan. Conversely, selective increase of carboxylesterase
levels in tumors may produce tumor-specific activity. Carb-
oxylesterase expression has been detected in some, but not all,
squamous cell lung carcinomas and adenocarcinomas at vari-
ous levels [69, 70]. Thus, conceivably it would be desirable to
1845
identify tumors deficient in carboxylesterase and stimulate its
expression. A gene-therapy-based approach of transferring the
carboxylesterase gene directly to the tumor followed by sys-
temically administered irinotecan has been attempted in cell
lines and a nude mouse tumor model with success [71, 72].
This strategy may have potential as a local therapy for solid
tumors, such as the treatment of intrabronchial tumors, lung
tumors invading into the chest wall and inoperable intra-
pulmonary tumors. Another very interesting application of
this gene therapy-based approach is to purge contaminating
tumor cells from their mixture with CD34+ cells while prepar-
ing for autologous stem cell transfer. Adenovirus-mediated
transfer of carboxylesterase followed by exposure to irino-
tecan successfully killed neuroblastoma cells mixed with
CD34+ cells in vitro, while the cytotoxic effect did not affect
the colony formation by CD34 cells [73]. This approach
shows potential clinical utility to reduce the tumor burden of
peripheral stem cells in autologous transplantation.
Increased cellular level of Topo I
In preclinical systems, coadministration of irinotecan and mito-
mycin C (MMC) had a synergistic effect [74]. It was further
demonstrated that MMC increases Topo I catalytic activity as
measured by relaxation of supercoiled DNA, although Topo I
protein level was not increased [75]. Based on these pre-
clinical data, our group has evaluated the combination of
MMC and irinotecan (given 24 h after MMC) in patients with
refractory solid malignancies [76]. Topo I gene expression
was measured in pre-MMC and post-MMC peripheral mono-
nuclear specimens by RT-PCR studies and quantified with a
competitive, reaction-specific internal standard and analysis
by capillary electrophoresis with laser-induced fluorescence
[77]. This combination chemotherapy showed promising
activity in heavily pretreated gastric, esophageal, breast and
non-small-cell lung cancers. Interestingly, patients experi-
encing a major response to treatment (complete and partial
responses, n = 5) had a 46-fold induction of Topo I at 3 h after
MMC compared with baseline, and a 312-fold induction at the
end of the irinotecan infusion (P = 0.00021, ANOVA) [77]. In
contrast, non-responding patients (stable disease or tumor
progression, n = 29) did not experience significant Topo I
induction (P = 0.64). Although these results are of interest, it
is not certain if the dynamics of Topo I following MMC in
peripheral mononuclear cells adequately reflect intratumoral
events. Ongoing phase II tumor-specific studies with the
combination, in which tumor tissues are sampled pre- and
post-treatment for Topo I and carboxylesterase gene expres-
sion, will help clarify this issue.
Studies on another Topo I-interactive agent, topotecan,
noted the occurrence of depleted cellular levels of Topo I in
relation to prolonged infusion of topotecan (for 21 days). Topo
I levels decreased progressively in a weekly fashion and
returned to baseline levels 1 week after stopping the infusion
[78, 79]. This association indicates that prolonged infusions
1846
may have advantages because of their ability to target
maximum amounts of free Topo I to form DNA–drug–Topo I
complexes. This regimen was used in a phase II trial as
second-line therapy in patients with previously treated ovarian
cancers [79]. The regimen was well tolerated, and produced a
response rate of 35%, which is at the upper level for topotecan
therapy in this group of patients. Laboratory data suggested
that the depletion of free Topo I was partly due to the forma-
tion of cleavable complexes [78]. However, this experiment
cannot distinguish from two other reported possibilities of free
Topo I depletion: (i) translocation of Topo I from the nucleus
to cytoplasm in the presence of low doses of topotecan [80],
and (ii) the transcription-triggered proteasome degradation of
Topo I from the Topo I–DNA complex as described above
[54–56]. Randomized studies are required to elucidate the
therapeutic advantage and true mechanism in this administra-
tion schedule.
Sequential treatment with Topo I and Topo II
inhibitors
Cell lines selected for resistance to camptothecin often demon-
strate collateral sensitivity to Topo II poisons and increases in
Topo II enzyme activity or content [48]. Reciprocal changes in
Topo I activity have been observed in cell lines resistant to
Topo II poisons [81]. This phenomenon was also observed in
cancer patients. Several patients demonstrated decreased
levels of Topo I accompanied by increased levels of Topo II
when treated with the Topo I inhibitor 9-AC [82]. In colon
cancer cell lines, sequential exposure to camptothecin and
etoposide leads to additive cytotoxicity [83]. Sequential
treatment of athymic mouse bearing human colon tumor with
topotecan followed by etoposide also demonstrated increased
tumor sensitivity to etoposide [84]. However, results from a
recent phase I translational study of sequential administration
of the Topo I and II inhibitors, topotecan and etoposide, did
not fully support the above proposed mechanism. By assaying
Topo I and II levels in the tumor specimens after treatment, the
increase in Topo II was only clearly demonstrated in one out
of six patients tested [85]. Thus, the rationale of sequential
administration of Topo II and Topo I requires further invest-
igation.
Inhibition of the activation of NF-κB to enhance
irinotecan-induced apoptosis
Irinotecan exposure frequently results in increased production
of TNF-α, interleukin-1 (IL-1), phorbol esters and lipopoly-
saccharides, as well as in activation of the transcription factor
NF-κB [86]. Activation of NF-κB leads to inhibition of apop-
tosis. NF-κB is a heterodimer consisting of two proteins, p65
and p50. In unstimulated cells, NF-κB is located in the
cytoplasm and is bound to IκBα and IκBβ, which prevents it
from entering the nuclei. The external stimuli modulate signal
transduction pathways leading to IκB phosphorylation,
causing its rapid degradation by proteasomes. The release of
NF-κB from IκB results in the passage of NF-κB into the
nucleus, where it binds to specific sequences in the promoter
regions of target genes [64]. As has recently been shown, the
NF-κB complexes negatively cross-talk to the JNK cascade,
reducing the TNF-α-mediated JNK activation [67, 68].
A number of strategies have been employed to inhibit the
activation of NF-κB. The first approach is the molecular gene-
therapy approach of introducing a dominant negative variant
of IκBα via adenovirus vector to tumor cells [63]. This form
of IκBα is a ‘super-repressor’, which is resistant to phos-
phorylation by IκB kinase, and presumably remains bound to
NF-κB, thus sequestering NF-κB in the cytoplasm. This
approach has been tested in multiple tumor cell lines and
mouse tumor models (fibrosarcoma, colorectal cancer) by
different groups. It consistently demonstrated increased cell
death or tumor regression in the presence of irinotecan and
increased induction of apoptosis [63, 87]. Inhibition of NF-κB
using the ‘super-repressor IκBα’ was found to sensitize non-
small-cell lung cancer cells to gemcitabine-induced apoptosis
[88]. However, using the same approach, enhanced chemo-
sensitivity was not confirmed in similar studies done by two
other groups [89, 90]. This discrepancy may represent the
intricate cellular protein interaction and control of apoptosis.
NF-κB has been connected with multiple aspects of oncogen-
esis, including promotion of cell proliferation, cell migration,
angiogenesis, metastasis and blockage of cell differentiation
and apoptosis [64]. In many late-stage tumor cell lines and
other experimental tumor systems, NF-κB appears to be
proapoptotic [64].
As the use of gene therapy to deliver NF-κB inhibitors is
relevant to certain cancers, but is limited when considering
widely disseminated metastases, an alternative approach is to
use pharmacological inhibitors of proteasome function. The
inhibition of proteolytic function effectively blocks degrada-
tion of cellular proteins that have undergone ubiquitination,
such as IκB [91]. MG-132, an inhibitor of proteasomes, has
been shown to significantly inhibit NF-κB activation induced
by irinotecan [90]. However, in that experimental system,
apoptosis was decreased, again raising the question of the role
of NF-κB to be pro- or antiapoptotic in that tumor line.
PS-341, a potent boronic acid dipeptide that is highly selective
for proteasome inhibition, was also tried, in order to inhibit
proteasome function in human colon cancer cell lines [92].
Pretreatment of cancer cells with PS-341 can effectively block
the activation of NF-κB that is induced by exposure to SN-38/
irinotecan. NF-κB inhibition using PS-341 also augmented
the sensitivity of tumor cells to chemotherapy in tumor xeno-
graft models. The increased antitumor effect was attributable
in part to regulation of apoptosis, with markedly increased
levels of apoptosis compared with single-agent treatment
alone [92]. In pancreatic xenografts, the combination of
PS-341 and irinotecan results in 89% inhibition of tumor
growth and increased cellular apoptosis [93]. While using
PS-341 appears to be very promising, it is important to keep in
mind that the increased antitumor effect may in part be attrib-

uted to the prolonged binding of irinotecan to Topo I, rendered
by the inhibition of degradation of bound Topo I–irinotecan
complex, in the presence of proteasome inhibition [54, 55]. In
addition, proteasome inhibition may also stabilize a wide
range of key cell-cycle regulatory proteins, although it has
been shown to be at least independent of p53 [93].
Thalidomide and cyclooxygenase-2 inhibitors
The teratogenic sedative and antinausea drug thalidomide has
come back to the clinic based on its anti-inflammatory and
anti-oncogenic properties. It is an inhibitor of TNF-α by
enhancing the degradation of TNF-α mRNA [94]. Thalido-
mide has been recently demonstrated to block NF-κB activa-
tion through a mechanism that involves the inhibition of
activity of the IκB kinase [95]. Thalidomide could inhibit the
activation of NF-κB induced by stimulation with TNF-α and
IL-1β, and could inhibit the transcription of many endogenous
downstream genes controlled by NF-κB [95]. This study
suggests the inhibition of NF-κB as the mediator for the anti-
inflammatory and anti-oncogenic properties of thalidomide;
in addition, it also provides a rationale for the use of thalido-
mide in combination with irinotecan to modulate irinotecan-
induced chemoresistance. A clinical trial with the combination
of irinotecan and thalidomide in 5-FU-refractory metastatic
colorectal cancer is ongoing at the University of Arkansas.
Patients receive 350 mg/m2 irinotecan every 3 weeks and
400 mg thalidomide daily. Preliminary results are encouraging,
including one complete response and three partial responses
among 14 evaluable patients. Interestingly, grade 3–4 diarrhea,
a toxicity frequently associated with single-agent irinotecan,
has rarely been observed [96].
Aspirin and non-steroidal anti-inflammatory drugs
(NSAIDs) inhibit cyclooxygenases (COXs) to prevent prosta-
glandin synthesis. They also play important roles in the
chemoprevention of colon cancer. COX-1 is constitutively
expressed in many types of tissue, while COX-2 expression is
induced by cytokines and growth factors, and is increased in
90% of sporadic colon cancers and 40% of colonic adenomas,
but is not elevated in the normal colonic epithelium [95].
Both the non-specific COX inhibitor sulindac and the COX-2
inhibitor celecoxib have been shown to cause regression of
polyps in patients with familial adenomatous polyposis [97,
98]. The mechanism of chemoprevention by COX-2 inhibitors
may involve an increase in apoptosis [97]. In addition,
NSAIDs also act through COX-independent mechanisms by
inhibiting the NF-κB pathway [99]. The NSAIDs specifically
bind to IκB kinase b, inhibiting its kinase activity. This inhibi-
tion results in prevention of IκB degradation and nuclear
translocation of NF-κB. The above studies provide evidence
that NSAIDS, or more specifically COX-2 inhibitors, are
potential agents that may inhibit the activation of NF-κB.
In order to evaluate if the target of thalidomide and COX-2
inhibitors mediating favorable interactions with irinotecan is
NF-κB, we are conducting a phase I clinical trial in patients
1847
with solid malignancies in which the combination of irino-
tecan with thalidomide and subsequently the triple drug com-
bination irinotecan/thalidomide/celecoxib are evaluated. The
principal end point is demonstration of inhibition of NF-κB
nuclear translocation in peripheral mononuclear cells. Pre-
liminary results in 10 patients receiving irinotecan 125 mg/m2
on days 1 and 8 every 3 weeks in combination with thalido-
mide 400 mg daily confirms the attenuated toxicity profile
previously reported with the irinotecan/thalidomide combina-
tion and no effect of thalidomide on the pharmacokinetics of
irinotecan.
Pharmacological modulations to reduce
irinotecan toxicity
Pharmacological modulation to reduce irinotecan toxicity has
attracted many investigations. Myelosuppression and diarrhea
are the two major toxicities associated with irinotecan treat-
ment. It is believed that toxicity correlates with levels of
SN-38 in the plasma and bowel. Systemic SN-38 levels
depend on the expression and activity of carboxylesterases in
serum and liver, which convert irinotecan into SN-38 [15],
and on hepatic glucoronosyltransferase (UGT 1A1) activity,
which converts SN-38 into its glucoronidated and inactive
form SN-38G [18]. Although genetic variability in carboxyl-
esterase expression is suspected, and wide variability on the
ability of carboxylesterases to metabolize irinotecan has been
documented among its different isoenzymes [100, 101], no
genetic disorders of deficiency of this enzyme have been
characterized. UGT 1A1, however, is mutated in Gilbert’s
syndrome and deficient in Crigler–Najjar type 1 syndrome,
resulting in increased risk for irinotecan-induced severe toxic-
ity [17, 18]. A clinical trial has been planned at the University
of Chicago to test the predictive value of hepatic UGT 1A1
genotyping for irinotecan pharmacokinetics and toxicity [19].
In addition, pharmacological modulation with cyclosporin A,
an inhibitor of the active irinotecan/SN-38 transporter
cMOAT on hepatic cells [23], and phenobarbital, an inducer
of UGT 1A1 [20] has been tested in cancer patients in a phase
I clinical trial [102]. Cyclosporin A significantly inhibited
the clearance of irinotecan while phenobarbital induced
glucuronidation of SN-38, both consistent with preclinical
findings.
SN-38G is largely eliminated by biliary excretion [103]. In
the bowel, fecal bacteria-derived β-glucuronidase converts
the non-active SN-38G to active SN-38, which can produce
direct injury to the mucosa. Since the conversion to SN-38 is
believed to be a major factor in the diarrhea produced by
irinotecan, inhibition of bowel β-glucoronidase is a potential
strategy to reduce irinotecan toxicity. In a small controlled
trial, the broad-spectrum antibiotic neomycin was given to
patients receiving irinotecan in order to inhibit microflora in
the intestines. Amelioration of diarrhea was found in six out of
seven patients treated [104]. However, a recent study has
1848
detected up to 30% of irinotecan excreted unchanged in the
bile [105]. Thus, the potential exists for direct bowel con-
version of irinotecan into SN-38 by the recently identified
intestinal carboxylesterases [101]. In the absence of a direct
inhibitor of intestinal carboxylesterases, strategies aimed at
ameliorating irinotecan-induced diarrhea must consider the
contribution of intestinal irinotecan conversion.
The pH dependency of the equilibrium between the active
lactone forms and less active carboxylate forms of both SN-38
and irinotecan [13] provides an additional opportunity to use
the understanding of irinotecan pharmacology to ameliorate
toxicity. Under acidic conditions, the lactone form is favored,
but at physiological or higher pH, the lactone form is unstable,
and hydrolysis to open the lactone ring into its carboxylate
form is favored. In addition, the lactone forms are passively
transported in intestinal cells, resulting in an intestinal uptake
10 times greater than the carboxylate forms [106]. Therefore,
intestinal alkalinization has been proposed as a potential strat-
egy to decrease irinotecan gastrointestinal toxicity [107, 108].
In a phase II trial, oral alkalinization utilizing sodium bicarbon-
ate, magnesium oxide, base water and ursodeoxycolic acid
combined with ‘controlled’ defecation resulted not only in
a decreased incidence of diarrhea compared with a non-
randomized control group, but also in decreased myelo-
suppression, while maintaining anticancer activity [108].
Finally, oral administration of immunomodulators, which
induce endogenous IL-15 for protection of intestinal epithel-
ium, and Chinese/Japanese herbal concoctions with an unclear
mechanism of action have been used [109, 110]. A need for
carefully designed randomized clinical trials will be needed to
assess the efficacy (decreased toxicity without affecting anti-
cancer activity) of all the strategies discussed above. In addi-
tion, feasibility aspects need to be considered, since efficacy
and simplicity will both be needed to guarantee the success of
these approaches in clinical practice.
Future directions
The intricate pathways of metabolic activation/deactivation of
irinotecan make this agent especially suitable for interactions
with commonly used medications. Therefore, it is extremely
important to be mindful of the potential for increased clear-
ance or increased toxicity that these medications may have
when used in combination with irinotecan. However, know-
ledge of these metabolic pathways can be used to identify
modulation strategies aimed at increasing the antitumor activ-
ity and reducing the toxicity of this unique agent. Similarly,
with the increasing understanding of irinotecan intracellular
interactions and the intracellular mechanisms mediating
resistance to this agent, the potential to prospectively identify
patients with tumors especially susceptible to irinotecan
therapy warrants careful attention. Clinical trials of irinotecan
should incorporate, whenever feasible, analyses of potential
markers of clinical activity and toxicity.
In addition, based on this knowledge, pharmacologically
based or biologically based novel therapeutic approaches to
modulate irinotecan activity and toxicity have been incorpor-
ated into clinical trials. Although none of the novel strategies
discussed in this manuscript has yet been validated, modula-
tion of NF-κB activity and Topo I expression and activity
appears to hold the most promise for improvement in clinical
activity in early clinical trials. Randomized phase II trials
evaluating several novel strategies may help to identify the
most promising regimens for further study. It is the bias of the
authors that these approaches should first be tested in irino-
tecan-naive patients and later in irinotecan-refractory patients,
in the event that encouraging activity is found on the former.
Given the broad spectrum of activity of irinotecan, validation
of these approaches in any of these settings might translate
into significant advances in clinical practice.
Acknowledgements
We would like to thank J. Kuhn, the University of Texas
Health Science Center at San Antonio, for critically reviewing
this manuscript.
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