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Introduction
Hepatocellular carcinoma (HCC) is one of the most
common cancers worldwide, causing nearly 600 000 deaths each
year. More than half of these deaths occur in
China. Residents of Qidong, Jiangsu Province, China are at particularly
high risk. In this area, HCC is the leading cause of cancer
death. Exposure to dietary aflatoxins and chronic infection
with hepatitis B virus (HBV) may contribute to this
extraordinarily increased risk[1].
Aflatoxins are mycotoxins produced by the fungus
Aspergillus flavus, which are found in
contaminated foods such as corn and nuts. Aflatoxin
exposure and infection with HBV may be responsible for 90% or
more of the HCC cases in this
area[2]. Prevention strategies targeting HBV and aflatoxin exposure could drastically
impact rates of HCC. For example, since a universal HBV
vaccination program was implemented in Taiwan in 1984, the
incidence of HCC in children has
declined[3]. Aflatoxin exposure can be reduced by improving methods used to grow,
harvest, store, and process susceptible foods.
This could include the control of the moisture content and relative
humidity for food storage, sorting strategies to remove
contaminated items, or dietary changes, such as shifting to foods
which are less susceptible to aflatoxin contamination (for
example rice instead of corn)[4].
However, the complete elimination of aflatoxin contamination might not be
possible. For this reason, chemoprevention is a practical strategy to
reduce the incidence of HCC in populations with high dietary
aflatoxin exposure.
Aflatoxin metabolism and mechanisms of
aflatoxin-induced hepatocarcinogenesis are well known (Figure 1). This
knowledge provides the basis for evaluation of exposure to
aflatoxin, as well as modulation of aflatoxin disposition by
chemopreventive agents. Aflatoxin B1 is metabolized by
cytochrome P450s[5,6] to a reactive epoxide
(aflatoxin-8,9-epoxide) and other oxidation products, including aflatoxin
M1. The epoxide can react further by interacting with DNA
to form a promutagenic aflatoxin-N7-guanine adduct. This is
an unstable adduct which rapidly undergoes depurination
and excretion in urine[7]. The epoxide can also
react with serum albumin to form long-lived lysine
adducts[8]. In addition, the epoxide can be conjugated by glutathione
S-transferases (GSTs), which are further metabolized to form
aflatoxin-mercapturic acid detoxification products that can be excreted in
urine[9]. Products of aflatoxin DNA damage and toxicity as
well as other metabolites can be used as biomarkers to
evaluate the modulation of aflatoxin activation and detoxication.
For example, levels of both lysine and
N7-guanine adducts, which are excreted in urine, have been shown to correlate
with aflatoxin exposure[10_13]. In animals, changes in hepatic
aflatoxin-N7-guanine adduct formation are associated with
the degree of chemoprotection as measured by reduction in
preneoplastic foci and cancers (Figure
2)[14,15]. In addition, the levels of aflatoxin
M1 excreted in urine have been shown to reflect human
exposure[16] and predict chemopreventive
efficacy and liver cancer risk[17_19]. Furthermore, increased
formation of aflatoxin-mercapturic acid metabolites can be
measured in the urine of laboratory animals as well as
humans and is inversely associated with levels of aflatoxin
DNA adducts formed in liver and excreted into urine. These
biomarkers are critical tools for evaluation of
chemopreven-tive agents in animal models and clinical interventions.
Mechanisms for prevention
Induction of cytoprotective enzymes Induction of
xeno-biotic metabolism enzymes has been shown to be an
effective means of protection against carcinogenesis, mutagenesis,
and other toxicities mediated by electrophiles.
These cyto-protective enzymes (often referred to as "phase 2 enzymes")
include conjugating and antioxidative enzymes.
Conjugating enzymes, such as GSTs and UDP-glucuronosyl
transferases facilitate the elimination of
carcinogens. Additional protection is achieved through induction of antioxidative
enzymes that enhance the resistance of cells to oxidative
stress. Induction of phase 2 enzymes has been shown to be
sufficient for chemoprevention. Known chemopreventive
agents, such as oltipraz and sulforaphane, enhance the
expression of many of these detoxication and cytoprotective
genes.
Nrf2 activation Nrf2 (NF_E2-related factor 2) is a
member of the basic leucine zipper NF_E2 family of transcription
factors. Studies using Nrf2-deficient mice have defined the
crucial role of Nrf2 in chemoprevention (Figure
3). Nrf2-deficient mice are more susceptible to toxicity, DNA adduct
formation, and cancer development in several models of
chemical-induced carcinogenesis. While the basal
expression of some cytoprotective genes is Nrf2
dependent[20_24], the increased sensitivity caused by loss of Nrf2 is likely due
to an impaired ability to mount an adaptive response in the
face of repetitive carcinogenic challenges through
induction of a broad array of cytoprotective
genes[25_28]. For example, DNA adduct formation is increased in
Nrf2-deficient mice compared to wild-type mice following exposure to
carcinogens, such as diesel exhaust[29], aflatoxin
B1[30], and
benzo[a]pyrene[31]. Nrf2-deficient mice develop a higher
burden of gastric neoplasia following treatment with
benzo[a]pyrene compared to wild-type
mice[20]. Compared to wild-type mice, Nrf2-null mice also have increased incidence of
skin tumors and tumor numbers per mouse in a
7,12-dime-thylbenz(a)anthracene-induced skin tumorigenesis
model[32]. Chemopreventive agents, such as oltipraz and sulforaphane,
do not induce cytoprotective genes in Nrf2-deficient
mice. Moreover, the antitumorigenic actions of these agents are
lost in Nrf2-disrupted mice[20,32].
Nrf2 regulates a larger cytoprotective response beyond the conjugating and
anti-oxidative classes. This response includes the
ubiquitin/proteasome system, the molecular chaperones/stress
response system, and anti-inflammatory
responses[27]. This broad protective response makes Nrf2 and its interacting
partners important targets for chemoprevention.
Antioxidant response element-mediated regulation of
Nrf2 The 5'-flanking region of many cytoprotective and
detoxifying enzymes contain a common regulatory element,
the antioxidant response element (ARE). Many
cytopro-tective genes, such as rat and mouse GST, rat and human
NAD(P)H:quinone oxidoreductase (NQO1), and human glutamate cysteine ligase subunits contain
AREs[33_37].
Activation of these AREs is induced by a structurally
diverse group of chemicals, including oxidizable diphenols,
dithiolethiones, isothiocyanates, and Michael acceptors
(olefins or acetylenes conjugated to electron-withdrawing
groups)[38,39]. This activation occurs through Kelch
ECH-associating protein 1 (Keap1)_Nrf2_ARE signaling (Figure
3). Keap1 is an actin binding protein which interacts with
the N-terminal Neh2 domain of Nrf2. Keap1 sequesters Nrf2
in the cytoplasm, causing transcriptional repression by
preventing the nuclear translocation of
Nrf2[40]. Under homeostatic conditions, Keap1 enhances the rate of the proteasomal
degradation of Nrf2. Keap1 also acts as an adaptor for the
Cullin 3-based E3 ubiquitin
ligase[41_43], which acts as a scaffold to form an E3 ligase complex and recruit the
ubiquitin-conjugation (E2) enzymes. Oxidative stress may disrupt the
Keap1_Nrf2 interaction resulting in the stabilization of Nrf2
and the accumulation of Nrf2 within the
cell[44]. Keap1 contains reactive cysteine residues which may act as sensors
for electrophilic and oxidative stresses, as well as the
chemical inducers described
earlier[45]. Cysteine modification may
induce a conformational change in Keap1 causing the
dissociation of Nrf2. Nrf2 can then translocate to the nucleus
where it heterodimerizes with small Maf proteins and binds
to the ARE to act as a transcriptional
activator[22]. Recent studies have suggested an alternative mechanism where
nuclear accumulation of Nrf2 caused by electrophilic stress
requires de novo protein
synthesis[46]. In addition, cysteine
residues in Keap1, which do not participate in the
Keap1_Nrf2 interaction, are critical to the degradation of Nrf2 by the
ubiquitin_proteasome system[46].
These studies suggest that activation of Nrf2 occurs by impairing the Keap1-mediated
degradation of Nrf2.
Other mechanisms of Nrf2 regulation
(kinases) Other signaling pathways influence Keap1_Nrf2_ARE signaling
through post-transcriptional modification. Several kinase
pathways, including protein kinase C (PKC),
mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K),
and PKR-like endoplasmic reticulum kinase (PERK) have been
shown to influence Keap1_Nrf2_ARE
signaling[47_49]. For example, phosphorylation of Nrf2 by PKC promotes release
from Keap1[50]. Inhibition of PI3K attenuates the nuclear
translocation of Nrf2 and transcription of ARE-regulated
genes in vitro[51]. PERK phosphorylates Nrf2 and triggers
dissociation from Keap1 resulting in increased nuclear
translocation[52]. These in
vitro studies require further study to determine the significance of these pathways
in vivo and their suitability as pharmacological targets for modulating
Nrf2 signaling.
Protection in animal models
Several classes of chemical inducers of Nrf2 signaling
have been evaluated in animal models of chemical
carcinogenesis. These agents include phenolic antioxidants
used as food preservatives; dithiolethiones; isothiocyanates,
such as sulforaphane; and highly potent synthetic analogs
of naturally occurring triterpenoids. These agents induce
Nrf2-regulated cytoprotective enzymes and prevent
chemically-induced carcinogenesis (Table 1).
Phenolic antioxidants Phenolic antioxidants, such as
butylated hydroxyanisole (BHA) and butylated
hydroxytoluene (BHT), are added to foods to prevent oxygen-induced
lipid peroxidation. Rats fed diets containing 0.45% BHA or
BHT showed increased hepatic GST and UDP-glucuronosyl
transferase enzyme activity. In addition, 0.45% BHA or BHT
in the diet also reduced aflatoxin-DNA adduct formation in
the liver by 85% and 65%,
respectively[53]. Dietary
administration of BHA or BHT also reduces neoplasia in
chemically-induced carcinogenesis models targeting the forestomach,
lung, small intestine, breast, and
skin[54]. Another antioxidant, ethoxyquin, is also used as a preservative in
food. Ethoxyquin given in the diet at 0.05% or 0.5% inhibited liver
carcinogenesis in rats exposed to aflatoxin
B1[55]. It was subsequently
shown that the induction of hepatic cytoprotective enzymes
by these antioxidants is mediated by Nrf2
signaling[56,57].
Dithiolethiones Some members of the dithiolethione
class of chemopreventive agents are more potent inducers
of conjugating and detoxication enzymes than the phenolic
antioxidants. The most studied member of this class is
5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione, or
oltipraz. During studies evaluating oltipraz for its antischistosomal
activity, it was noted that oltipraz caused an increase in
glutathione levels in the liver, kidney, and forestomach of
mice. Furthermore, dithiolethiones increased the activity of
conjugating and detoxication enzymes such as GST, NQO1, and
glutathione reductase[58]. It was predicted that oltipraz would
be an effective chemopreventive agent due to this
induction. Subsequently, oltipraz was shown to be an effective
chemopreventive agent in chemical-induced cancer
models. Several studies have shown that oltipraz protects against
aflatoxin-induced hepatic tumorigenesis. Rats were fed
0.01%_0.1% oltipraz for 4 weeks. On the second and third
week of oltipraz feeding, the rats were gavaged with
aflatoxin B1 5 times per week. Twelve weeks after oltipraz
feeding ended, the rats were killed and liver sections were
analyzed for preneoplastic foci. All of the doses of oltipraz
reduce the volume of the liver occupied by preneoplastic foci
by more than 90%. Hepatic aflatoxin-DNA adduct formation
is reduced by 40%_80% over the dose range of
0.01%_0.1%. Furthermore, oltipraz increases hepatic GST enzyme activity,
which acts to increase detoxication of the ultimate
carcinogenic form of aflatoxin,
aflatoxin-8,9-epoxide[59]. These
studies were extended to chemopre-vention of HCC induced by
aflatoxin in a 2 year study. While aflatoxin-exposed rats
receiving the control diet developed HCC or hepatocellular
adenomas, dietary oltipraz at 0.075% provided complete
protection against both aflatoxin-induced hepatocellular
neoplasms. The rats in the oltipraz group also had a
significantly longer life span and an increased survival free of liver
tumors[60].
The costly synthesis and purification of oltipraz as well
as toxicities noted in clinical trials for the chemotherapy of
schistosomiasis[61] have indicated that further development
of oltipraz could be problematic. A second generation of
dithiolethione analogs was evaluated to identify analogs
which would be more suitable than oltipraz for further
development. The chemopreventive potential of these
analogs was evaluated by measuring inhibition of formation of
DNA adducts and formation of preneoplastic lesions in the
livers of rats treated with aflatoxin
B1. 3H-1,2-dithiole-3-thione (D3T) is the most potent with a greater than 90% reduction
in DNA adduct formation at the highest dose (0.3 mmol/kg
body weight) compared to an approximately 60% reduction
by oltipraz at the same dose[62]. Further studies have shown
that D3T is also the most potent inhibitor of hepatic
preneoplastic lesions[62].
The chemoprotective activity of dithiolethiones is
Nrf2-mediated. Increased accumulation of Nrf2 has been shown
in extracts from hepatic nuclei of D3T-treated
mice[28]. While oltipraz induces the cytoprotective ferritin H and L genes in
wild-type mouse embryonic fibroblasts (MEFs), this
induction is eliminated in Nrf2-deficient
MEFs[63]. In addition, oltipraz and D3T do not induce GSTs and NQO1 in
Nrf2-deficient mice[20,28]. A microarray analysis using wild-type
and Nrf2-deficient mice showed that D3T induces a large
number of Nrf2-dependent genes. In addition to the
previously identified Nrf2-dependent detoxication and
antioxida-tive genes, new gene clusters were also identified as
D3T-inducible and Nrf2-dependent. These include other groups
of cytoprotec-tive genes involved in the
ubiquitin/protea-some system and molecular
chaperones[27]. Furthermore, in
chemical-induced cancer models targeting the bladder and
forestomach, the chemopreventive efficacy of oltipraz is lost
in Nrf2-deficient mice[20,64].
Sulforaphane Glucosinolates are found in high
concentrations in cruciferous vegetables. Glucosinolates can be
hydrolyzed by myrosinase (an enzyme which is released when
the plant is chewed or in the intestinal microflora) to produce
isothiocyanates. Isothiocyanates are potent inducers of
cytoprotective and detoxication enzymes. Sulforaphane is a
potent isothiocyanate whose precursor is abundant in
broccoli, particularly in 3 d-old broccoli
sprouts. Sulfora-phane induces detoxication and cytoprotective enzymes,
such as GSTs and NQO1, in a Nrf2-dependent
manner[65_67]. Sulforaphane has been shown to directly interact with
Keap1[68]. Furthermore, sulforaphane reduces mammary tumor
incidence and multiplicity in rats treated with
DMBA[69].
Triterpenoids Synthetic triterpenoids have recently been
developed at Dartmouth Medical School (Hanover, NH, USA)
as anti-inflammatory agents. These agents are synthetic
derivatives of oleanolic acid, a plant-derived compound used
in traditional Asian medicine for its weak anti-inflammatory
and antitumorigenic activity[70,71]. The synthetic
optimization of these triterpenoids has greatly improved upon the
weak activity of the naturally occurring compound. These
compounds are also being evaluated for both cancer
chemotherapy and chemoprevention. In cancer cell lines, these
triterpenoids induce differentiation, inhibit growth, induce
cell cycle arrest, and induce
apoptosis[72_76]. An imidazolide triterpenoid,
1-(2-cyano-3,12-dioxooleana-1,9[11]-dien-28-oyl)imidazole (CDDO-Im), is one of the most potent
triterpenoids. While optimizing the synthetic triterpenoids
to maximize anti-inflammatory
activity[77_79], the presence of Michael acceptor groups was determined to be essential for
anti-inflammatory activity as well as the induction of
apoptosis, inhibition of proliferation, and induction of
differentiation. Because Michael acceptor groups had
previously been identified as critical for the induction of
cytoprotective enzymes[80,81], triterpenoid activity in
suppressing inflammation was compared to their activity in
inducing cytoprotective enzymes[82]. These studies show that
triterpenoids are very potent inducers of cytoprotective
NQO1 enzyme activity in vitro. However, triterpenoid
analogs do not induce NQO1 or suppress nitric oxide
production in Nrf2-deficient or Keap1-deficient mouse embryonic
fibroblasts, indicating that this activity occurs through
Keap1_Nrf2 signaling[82]. CDDO-Im and the parent molecule
CDDO also induce cytoprotective heme oxygenase-1
in vitro and in vivo[83]. Low concentrations of CDDO-Im also reduce
reactive oxygen species formation in U937 cells challenged
with tert-butyl hydroperoxide. However, this
cytoprotective activity is lost in
Nrf2-deficient cells[83].
Due to the impressive potency of CDDO-Im noted in the
studies described earlier, further studies were conducted to
assess its chemopreventive potential in
vivo. Microarray studies in wild-type and Nrf2-deficient mice showed that
CDDO-Im induces many hepatic cytoprotective genes in a
Nrf2-dependent manner. Furthermore, CDDO-Im is an
extremely potent chemopreventive agent against
aflatoxin-induced hepatic tumorigenesis in
rats[14]. CDDO-Im reduces the hepatic burden of preneoplastic foci by 85% at the
lowest dose of 1 µmol/kg body weight and more than 99% at the
highest dose of 100 µmol/kg body weight. CDDO-Im
inhibits aflatoxin-DNA adduct formation by 40%_90% over the
range of 1_100 µmol/kg body weight. In addition, CDDO-Im
increases RNA transcripts of aflatoxin metabolism genes,
including Gsta2 and Gsta5, following an oral dose of 1
µmol/kg body weight[14]. CDDO-Im is approximately 100-fold more
potent than oltipraz in this rat antitumorigenesis model.
The pharmacodynamic action of synthetic triterpenoids
has been further evaluated in vivo. The activation of
Keap1_Nrf2_ARE signaling has been shown using 2 lines of
transgenic reporter mice. Transgenic mice with the ARE of
Nqo1 linked to a luciferase reporter can be used to identify
both the localization and extent of ARE activation using
in vivo bioluminescence. When Nqo1_ARE_luciferase mice
are treated with triterpenoids, luciferase reporter gene
expression is induced in the kidneys, salivary gland, liver,
and intestines (Figure 4)[84]. A more precise localization of
pharmacodynamic action can be obtained using
Nqo1_ARE_human placental alkaline phosphatase (hPlap) reporter mice.
These mice carry the ARE of Nqo1 linked to an hPlap
reporter gene which can be detected by
immunohistochemistry[85]. Nqo1_ARE_hPlap reporter gene expression is
increased in hepatocytes following treatment with
CDDO-Im[84]. Furthermore, several triterpenoid analogs may be
effective chemopreventive agents in multiple target tissues
as indicated by potent induction of Nrf2-regulated
cytopro-tective genes in the liver, lung, small intestine mucosa, and
brain[84].
While other analogs have not been studied against
aflatoxin-induced hepatic tumorigenesis, the methyl ester and
ethyl amide derivatives (called CDDO-ME and CDDO-EA,
respectively) have been evaluated in a post-initiation lung
cancer mouse model. As suggested by their ability to
induce Nrf2-regulated cytoprotective genes in the
lung[84], CDDO-ME and CDDO-EA are effective chemopreventive
agents in the lung. CDDO-ME and CDDO-EA reduce the
number, size, and severity of tumors on the surface of the
lungs[86]. This study also has important implications for
cancer chemoprevention in other organ sites.
Post-initiation chemoprevention had not previously been evaluated with
these compounds and is of critical importance because most
interventions for chemoprevention would occur over a long
period of time.
Human trials
The evaluation of potential chemopreventive agents in
rodents has provided a wealth of information on which
human clinical trials have been developed. The animal
studies described earlier have identified agents with the greatest
chemopreventive potential. Furthermore, rodent studies have
elucidated the mechanisms of aflatoxin carcinogenesis and
validated critical biomarkers for use in human studies.
Oltipraz Clinical trials have shown that oltipraz
modulates the activities of both conjugating/detoxication enzymes
as well as cytochrome P450s. A single 125 mg oral dose of
oltipraz reduced CYP1a2 activity by 75% in healthy
individuals[87]. Similar doses also increased GST activity in
peripheral lymphocytes[88]. A dose-finding study using 125,
250, 500, or 1000 mg/m2 oltipraz showed increased GST
activity in peripheral mononuclear cells and colon mucosa
biopsies only at the lower
doses[89]. Nqo1 RNA transcripts were
also increased at 250 mg/m2[89].
Together, these studies confirm that oltipraz increases cytoprotective enzymes in
humans.
Phase IIa intervention trials evaluated modulation of
carcinogen metabolism following treatment with
oltipraz. Participants for this randomized, placebo-controlled,
double-blind study were recruited from Daxin township, Qidong,
China. These residents have high dietary exposure to
aflatoxins as well as a high risk of HCC.
Two hundred forty adults with good general health and detectable serum
aflatoxin_albumin adduct levels were randomized to receive
placebo, 125 mg oltipraz administered daily or 500 mg oltipraz
administered weekly. Urine samples were collected at 2 week
intervals during the 8 week intervention period and during
an 8 week follow-up period. Urine samples collected after
the first month of intervention were assayed for aflatoxin
metabolites[17]. These samples were evaluated for alterations
in the activation product, aflatoxin M1, and the detoxication
product, aflatoxin_mercapturic acid. After 1 month of weekly
doses of 500 mg oltipraz, the level of aflatoxin
M1 excreted in the urine was decreased by
51%. However, the aflatoxin-mercapturic acid levels were not significantly
altered. Poten-tial modulation of detoxication enzymes may be masked by
inhibition of the activation of aflatoxin
B1. The daily administration of 125 mg oltipraz increased aflatoxin_mercapturic
acid excretion 2.6-fold, but with only a modest effect on
aflatoxin M1 excretion. This trial shows that induction of
cytoprotective genes can be translated into the modulation
of aflatoxin disposition in humans.
Broccoli sprouts Broccoli sprouts contain an abundance
of glucosinolates and isothiocyanates, making them an
attractive food-based candidate for
chemoprevention. Clinical studies have evaluated metabolism, safety, tolerance, and
biomarkers of carcinogenesis using broccoli
sprouts. Evaluation of broccoli sprout preparations has shown that
isothiocyanates are approximately 6 times more
bioavailable than the precursor
glucosinolates[90]. A placebo-controlled,
double-blind, randomized phase I clinical study evaluated
broccoli sprout preparations containing either glucosinolates
or isothiocyanates (principally
sulforaphane)[91]. The treatment groups received doses of 25 µmol glucosinolates, 100
µmol glucosinolates, or 25 µmol
isothiocyanate. No significant or consistent toxicities were observed with any of the
broccoli sprout preparations[91].
Interventions using hot water infusions of broccoli sprouts were evaluated in
residents of Qidong, China[92]. Modulation of the disposition of
aflatoxin was evaluated. Two hundred healthy adults drank
infusions of either 400 µmol glucoraphanin or a placebo
beverage nightly for 2 weeks. Again, no problems with safety or
tolerance were observed. Urinary aflatoxin-DNA adducts
were not different between the 2 interventions.
However, measurement of urinary dithiocarbamate levels (sulforaphane
metabolites) showed interindividual differences in
bioavail-ability. Further analysis to control for the bioavailability of
sulforaphane showed a highly significant inverse
association between the levels of dithiocarbamates excreted and
aflatoxin-DNA adducts[92]. The reduction of aflatoxin-DNA
adducts is most likely due to induction of GST activity by
sulforaphane. This study shows that aflatoxin disposition
can be altered by the administration of glucosinolate-rich
broccoli sprout preparations. A parallel inverse association
was observed with the elimination of phenanthrene tetraols,
demonstrating that the metabolism of polycyclic aromatic
hydrocarbons can also be
modulated[92]. Future studies with
broccoli sprouts will require preparations which produce a
higher yield and consistent level of sulforaphane
bioavail-ability.
Triterpenoids CDDO and CDDO-ME are currently in
clinical development by Reata Pharmaceuticals (Dallas,
TX,USA). CDDO is in phase 1 studies in patients with relapsed
and refractory leukemia. It is also being evaluated in
patients with solid tumors. CDDO-ME is in phase 1 clinical
development in patients with solid tumors and lymphoid
malignancies[93]. While this class of synthetic triterpenoids
is not yet in clinical trials for chemoprevention, the clinical
trials for chemotherapy could provide critical data in
humans to facilitate chemoprevention studies in the future.
Future directions
The clinical development of cancer prevention has been
hampered by concerns about safety and liability, as well as
few resources directed towards cancer
prevention. The
design of clinical cancer prevention trials must address these
challenges efficiently. Clinical trials which evaluate disease
as the end-point may not always be the most appropriate
means to translate promising preclinical chemoprevention
strategies into the clinic. These very costly trials require
large numbers of participants and long-term follow
up. "Phase 0" trials may be a more efficient strategy to initiate
the translation of chemopreventive agents into the
clinic. Phase 0 trials have been suggested as a way to improve the
speed and success of chemotherapeutic drug
development[94], but this approach is also relevant to
chemoprevention.
Exploratory phase 0 trials can provide proof of concept,
determine the feasibility of assays for target modulation,
evaluate biomarker modulation, and provide pharmacokinetic
data to provide information for the subsequent development
of investigational chemopreventive agents. As these
end-points require fewer participants, short-term follow up, and
require participants to receive limited doses of
chemopreven-tive agents, this minimizes the risk of toxicity, reduces cost,
and can provide earlier feedback on the pharmacodynamic
activity of test agents in humans. However, phase 0 trials
are only practicable for agents where the mechanism of
action is well understood. Furthermore, phase 0 trials for
cancer chemoprevention require that biomarkers for the
targeted carcinogenic pathway are well
validated. The utility of this approach has already been shown in trials using
broccoli sprouts[90_92]. These studies provided proof of concept
showing that aflatoxin disposition can be altered by the
administration of glucosinolate-rich broccoli sprout
prepara-tions. In addition, the pharmacokinetic data collected during
these studies identified the need for methods to achieve a
consistent level of sulforaphane bioavailability in the
broccoli sprout preparations. This information will be critical in
designing further broccoli sprout interventions.
While research and resources are currently focused on
cancer chemotherapy, chemoprevention is a practical
strategy to reduce the incidence of HCC as well as many other
types of cancer. Induction of Nrf2-regulated cytopro-tective
response has been proven to be a successful
chemopre-ventive strategy in many animal models and has shown
promise in clinical trials. There are many challenges for the
clinical development of cancer chemopreventive agents, but
success will have tremendous impact on the incidence of cancer
development.
Acknowledgements
We would like to thank our colleagues both past and
present for their contributions to our studies on
chemo-prevention of liver cancer.
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