Chronomodulated administration of
Capecitabine - Aspects for clinical outcome
Anna Tzani
Supervisors:
Prof. Dr. Jan Schellens
Bart Jacobs, PharmD
Institute:
Division of Experimental Therapy
The Netherlands Cancer Institute, Amsterdam
Contents
Abstract
3
1. Introduction
4
Capecitabine metabolism
4
2A. Predictive biomarkers based on enzymes expression levels
5
2A1. Predictive and prognostic value of thymidylate synthase
5
2A2. Predictive significance of thymidine phosphorylase and orotate phosphoribosyltransferase
6
2A3. Predictive importance of dihydropyrimidine dehydrogenase
6
2B. Predictive biomarkers based on enzymes genetic polymorphism
6
2C. Prominence of 5-FU/capecitabine predictive biomarkers
7
3. Circadian timing system and circadian biomarkers
8
4. The relevance of circadian timing system for anticancer treatments
10
5 The circadian variation in fluoropyrimidine enzymes
11
6 Clinical trials assessment
16
6A. Intravenous administration
16
6B. Oral administration
18
7. Comments and perspectives
21
References
25
2
Abstract
The activity of the most chemotherapeutic agents is defined by the damage they can cause in healthy
host tissues. This renders the establishment of the maximum tolerated dose a key step in anticancer
treatment. The majority of anticancer agents, including fluoropyrimidines, exert their cytotoxicity during
cell division of tumor or healthy cells. In particular, 5-fluorouracil (5-FU) and capecitabine are more toxic
against cells that are in S-phase of the cell cycle. Consequently, cells that undergo DNA synthesis are
more sensitive to 5-FU and capecitabine. Hence, 5-FU and capecitabine cytotoxicity depends on the cell
cycle phase as well as drug metabolism and elimination. Cell division and drug exposure present
circadian variation which is regulated by the circadian timing system (CTS)1. Studies in rodents have
shown that several enzymes, which are important for fluoropyrimidine metabolism, show circadian
variation. These enzymes include uridine phosphorylase (UP), orotate phosphoribosyl-transferase
(OPRT), thymidine kinase (TK), dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS)
that is the target enzyme of the drug2–4. Clearance of 5-FU is rate-limited by DPD. The circadian rhythm
of TS and DPD has been studied in humans. Results have shown that DPD activity peaks and TS activity
plunges around the middle of resting span1,5. Therefore, 5-FU and capecitabine are probably best
tolerated during rest. In chronomodulated treatment, drugs are administered near their respective
times of best tolerability. 5-FU and capecitabine chronotherapy is tested in several clinical trials. In some
clinical trials, 5-U chronotherapy showed an improvement in tolerability versus conventional therapy.
Chronomodulated treatment with capecitabine was not very advantageous in comparison to
conventional treatment, for all patients. Great response variability has been observed according to
gender. Men will potentially benefit from chronotherapy more than women. There is evidence that
sensitivity to 5-FU and capecitabine depends on different molecular markers6. In addition, several
factors like age, gender, different chronotypes and scheduling of chronotherapy can critically affect the
outcome of the treatment. Therefore these factors should be taken into consideration in order to
achieve more consistent results from clinical studies.
3
1. Introduction
Colorectal and breast cancer remain two of the major health problems in the western world.
Approximately 60% and 40% of colon and breast cancer patients respectively will develop metastatic
disease and will require systemic anti-cancer therapy7,8. Fluoropyrimidines are commonly prescribed for
the adjuvant treatment of epithelial cancers such as colorectal and breast tumors9. The first pyrimidine
analog, 5-fluorouracil (5-FU), was synthesized more than 50 years ago and since then it has been used
intravenously as chemotherapeutic regimen. Several oral prodrugs have been developed in order to
overcome disadvantages from intravenous 5-FU administration. Capecitabine (N4-pentyloxycarbonyl-5deoxy-5-fluorocytidine; Xeloda, F. Hoffmann La-Roche, Basel, Switzerland) is an orally administered
prodrug of 5-FU and remains the most used one among other established oral prodrugs.10 Capecitabine
has been designed in a way to be mainly converted into its active form in the tumor tissue, as an
attempt to avoid systemic toxicity and to increase selectivity of the drug in cancer cells 11. Tumor
selectivity of capecitabine has been confirmed in animal and human studies7. Nowadays, capecitabine
has replaced 5-FU in many tumor indications. It is registered for advanced colorectal cancer as
monotherapy or as adjuvant treatment and for advanced breast cancer together with docetaxel or as
monotherapy after failure of taxanes or anthracyclines12.
Capecitabine metabolism
Capecitabine is transformed into its active metabolite 5-FU by multiple sequential enzymatic reactions
(fig 1).
Figure 1 Simplified scheme of capecitabine metabolism and mechanism of action13. Capecitabine after absorption gets
converted in three metabolic steps into 5-FU preferably in tumor tissue where the concentration of thymidine phosphorylase
(TP) is higher. Further metabolism can result in two directions: elimination of the drug or activation into cytotoxic metabolites
responsible for cell death. The main mechanism of action of capecitabine is inhibition of thymidylate synthase (TS) and
misincorporation of cytotoxic metabolites into RNA and DNA. Abbreviations: 5′DFCR (5′-deoxy-5-fluorocytidine), 5′DFUR (5′deoxy-5-fluorouridine), 5-FU (5-fluorouracil), TP (thymidine phosphorylase), UP (uridine phosphorylase), PRPP (phosphoribosyl
PPI) is a co-factor of OPRT (orotate phosphoribosyl transferase) enzyme (not shown here), DPD (dihydropyrimidine
dehydrogenase), FUTP (fluorouridine triphosphate), FdUTP (fluorodeoxyuridine triphosphate) FBAL (α-fluoro-β-alanine).
4
Upon oral administration, capecitabine gets rapidly absorbed as intact molecule from the intestinal
mucosa. After absorption, the molecule is metabolized by subsequently carboxylesterase (CES), cytidine
deaminase (CDD) and thymidine phosphorylase (TP). These steps lead to the active form 5-FU. The third
step is very important for the selectivity of the drug as TP has been found to be upregulated three to
ten-fold in tumor tissues in comparison to healthy tissues10,11. Further 5-FU metabolism takes place via a
number of different biochemical pathways resulting in anabolite production mainly of fluorouridine
triphosphate (FUTP) and fluorodeoxyuridine triphosphate (FdUTP) which are responsible for tumor cell
death. The major fraction of 5-FU is catabolised by dihydropyrimidine dehydrogenase (DPD) into
biologically inactive metabolites that are excreted in the urine and the bile. This pathway represents the
80% of the drug metabolism14. The main action of the drug is the inhibition of thymidylate synthase (TS).
The enzyme regulates thymidine synthesis which is essential for DNA replication. Inhibition of TS leads
to apoptosis. As a consequence, the sensitivity of cancer cells to 5-FU and capecitabine depends on the
extent of TS inhibition15. A different way in which the damage can occur is by misincorporation into RNA
with consequent disruption of the transcription process or by misincorporation into DNA which results
in DNA damage14.
2A. Predictive biomarkers based on enzymes expression levels
The expression levels of TP, OPRT and DPD, which are key enzymes for capecitabine metabolism and the
expression of TS, which is the main target of the drug, have been examined as possible predictive
biomarkers for drug responsiveness and drug toxicity. A predictive biomarker allows the identification of
the group of patients who will most likely respond to a specific therapy. Patient-tailored capecitabine
therapy would potentially improve treatment efficacy and tolerability. In attempt to find acceptable
biomarkers, numerous studies have focused on the relationship between TS and tumor response to
Capecitabine or 5-FU treatment. Fewer studies have focused on TP, OPRT and DPD enzymes16. Most of
these clinical studies were prevalently based on the expression levels of the above mentioned enzymes
in tumor tissues.
2A1. Predictive and prognostic value of thymidylate synthase
Jensen et al. presented a number of studies suggesting that low TS levels in tumor tissue correlate with
increased progression free survival (PFS) and/or overall survival (OS) of patients treated with
capecitabine and could possibly predict a better response to the drug as well16. In addition, a recent
study on breast cancer patients suggested that high TS and low TP levels may be related to a reduced
PFS after capecitabine treatment17. TS expression could be a promising biomarker for 5-FU and
capecitabine treatment, however more analysis is required in order to reach a sufficient level of
evidence18 and be recommended for routine clinical use. An obstacle in evaluation of TS levels is that
they may vary between primary tumor tissue and metastasis. So, attention should be paid to the type of
the tissue chosen for analysis when evaluating TS levels as a predictive biomarker19. In addition to its
predictive value, TS could be also used as a prognostic marker. There are studies showing that low TS
5
levels are significantly correlated with higher overall survival20, in patients treated only with surgery
(without 5-FU adjuvant treatment).
2A2. Predictive significance of thymidine phosphorylase and orotate phosphoribosyltransferase
TP and OPRT enzymes are involved in capecitabine metabolism in order to form its active metabolites,
as it is already discussed. Given that, one can easily conclude that high levels of these enzymes could
possibly predict a better response to the drug and also higher toxicity. However, the results from the
few studies available do not reveal such consistent conclusions21. Despite the important role of these
two enzymes in capecitabine metabolism, they can be bypassed by other pathways involved as well in
the activation of the drug. Additionally, TP is identical to platelet-derived endothelial cell growth factor.
With this function the enzyme can promote angiogenesis, tumor growth and metastasis22. Therefore
high TP levels in tumor of an untreated patient can confer bad prognosis, while could be possibly
beneficiary in the treatment with capecitabine22. Since the role of TP and OPRT expression on 5-FU and
capecitabine efficacy and toxicity is still unclear, we cannot use these markers in clinical practice yet 16.
2A3. Predictive importance of dihydropyrimidine dehydrogenase
Results from studies performed to examine DPD levels in correlation to capecitabine response,
suggested improved PFS and OS with low DPD levels23–25. Theoretically this is a logical conclusion as DPD
activity is the rate-limiting step in catabolism and elimination of the drug. This means that low DPD
levels possibly correspond to high intracellular concentrations of 5-FU and cytotoxic metabolites. On the
other hand, no untreated groups were included and tumor samples were collected from primary and
metastatic lesions as well. These observations lead to the hypothesis that these studies were not
properly designed to completely validate DPD as predictive biomarker16.
2B. Predictive biomarkers based on enzymes genetic polymorphism
A biomarker is useful not only to predict the efficacy of a certain drug but also the grade of toxicity that
may be caused from this drug. In case of capecitabine, 5-FU plasma levels higher than therapeutic levels
can result in increased toxicity. DPD is the rate-limiting enzyme in 5-FU catabolism and at least 80% of
the drug follows this pathway. So, there is evidence that DPD deficiency will result in high 5-FU levels
and increased toxicity. Loganayagam et al. reported four gene sequence variants (c.1905+1G4A,
c.2846A4T, c.1601G4A and c.1679T4G) of the DPYD gene (encoding for DPD enzyme) that are associated
with DPD deficiency26. In their study, all patients who were found to be heterozygous or compound
heterozygous for these variants experienced severe (grade 3-4) fluoropyrimidine toxicity like diarrhea,
mucositis and neutropenia26. Other studies also showed that decreased DPD enzyme activity is a
significant cause for grade 3-4 toxicity27–29 providing decent evidence to sustain measurement of these
variants before starting therapy. Moreover, Loganayagam et al. also agreed with other studies30–32 that
there must be a 50-60% dose reduction for patients with the c.1905+1G4A variant, as this is the most
6
prominent loss-of-function allele of the DPYD gene and about 25% dose reduction for the other
variants26.
Among the most frequent toxicities upon capecitabine treatment are diarrhea and hand-foot syndrome.
Loganayagam et al. identified two variants of the promoter region of the CDA gene (c.−92 A>G and
c.−451C>T) which were related to grade 2-4 diarrhea. They have also described a homozygous MTHFR
1298CC variant genotype to be associated with hand-foot syndrome after treatment with capecitabine.
However, in these cases further studies are needed in order to decide if a dose reduction strategy would
be clinically advantageous26.
It is true that most patients with profound DPD deficiency experience high grade of the typical 5-FU toxic
effects like myelosuppression, diarrhea, mucositis and neurotoxicity33. However, high 5-FU plasma levels
and consequent severe toxicity has been also seen in patients with normal DPD activity. A recent study
showed that 3 out of 188 patients treated with 5-FU presented low clearance and high 5-FU plasma
levels but normal DPD activity. Looking only at DPD activity, these 3 patients would not have been
identified at risk to develop toxicity34. This suggests that 5-FU plasma levels resulting from capecitabine
metabolism are not depending only on DPD activity and we should be very critical when a patient
experiences severe toxicity.
2C. Prominence of 5-FU/capecitabine predictive biomarkers
Despite the efforts to identify predictive biomarkers for capecitabine and 5-FU efficacy and toxicity,
there are no consistent results suggesting one single molecule that can be extensively used as a
biomarker in clinical practice, regarding the protein levels. Low levels of TS, that has been studied the
most, seem to be a promising biomarker for better response to capecitabine treatment. However, there
is a need for more properly designed studies to further validate this hypothesis. Regarding the
association of TS levels with toxicity, results from several studies are contradictory26. It is more likely
that variations in the gene encoding for TS (TYMS) resulting in higher TS protein levels could possibly be
associated with adverse events, however, the effect is rather small and pre-treatment testing would not
be clinically significant26,35. There is evidence that high TP/DPD enzyme ratio is associated with higher
disease-free survival rate, compared to lower TP/DPD ratio in colorectal cancer patients who follow
adjuvant therapy with fluoropyrimidines36. Intratumoral TP/DPD enzyme ratio could be a significant
marker for adjuvant fluoropyrimidine therapy. The role of TP and OPRT levels in the evaluation of drug
efficacy and toxicity is uncertain, suggesting that looking for the expression levels of only one enzyme in
such a complicated metabolic pathway might be too simple16. In conclusion, many genetic
polymorphisms have been identified to be related to toxicity. Therefore, upfront genotyping for the
DPYD 1905+1G4A mutation can be suggested for patients treated with capecitabine. In the following
paragraphs of this review we focus on a different phenomenon related to capecitabine efficacy and
toxicity: the role of circadian rhythms within the capecitabine pathway.
7
3. Circadian timing system and circadian biomarkers
Chronopharmacology studies the interaction of biological rhythms with the different medications. The
tolerability and the antitumor effect of capecitabine highly depend on the activity of the enzymes
involved in its metabolic pathway. The enzymatic activity, in many cases, presents circadian variation.
Every cell has an endogenous 24h molecular clock regulated by 15 clock genes37. Cell molecular clocks
constitute the circadian timing system (CTS) which drives 24h changes in xenobiotic metabolism and
detoxification, cell cycle, DNA repair, apoptosis and angiogenesis38. Chronotherapy is aiming to adjust
the administration of the drugs to the CTS in order to achieve better tolerability and/or efficacy of the
medication.
The CTS is synchronized by environmental parameters such as day-night alternation, everyday life
routines and meal times. Examples of circadian rhythms in humans are the sleep-wake cycle, the core
body temperature, the heart rate and the metabolism that fluctuate during a 24h period. In humans,
motor activity is usually higher during day and lower during night. The maximum of body temperature is
near early evening, cortisol secretion reaches the maximum near 8:00h, while melatonin secretion peaks
around 2:00h. Human circadian rhythms are coordinated by the suprachiasmatic nucleus (SCN) which is
a pacemaker located in the hypothalamus (fig 2)37. The SCN is regulated by daily environmental changes
and produces rhythmic physiological output signals like rest-activity, hormonal secretions and body
temperature. The CTS comprises the central pacemaker (SCN) and the peripheral cellular clock regulated
from the 15 clock genes.
Figure 2 Schematic view of the circadian mammalian system and correlation with chronotherapy 37. Circadian timing system
(CTS) is composed of the suprachiasmatic nuclei (SCN) in the hypothalamus, a set of SCN-originated rhythmic physiological
outputs and the molecular clock of each cell in all peripheral tissues. The CTS is synchronized by light-dark alternation and other
daily behavioral activities and environmental factors. Subsequently SCN coordinates and synchronizes the peripheral molecular
clocks through output signals like hormonal secretion, body temperature and rest-activity rhythm. These circadian outputs can
be monitored and measured and serve as biomarkers of the CTS. Chronotherapy consist of the adaptation of drug
administration to CTS in order to increase tolerability and efficacy of the drug.
8
Healthy individuals can present variations in their CTS due to differences in living habits, physiology and
clock gene expression39. CTS can also be shifted, lengthened or shortened by deficiencies in recognition
of external synchronizers (e.g. through blindness). Moreover, circadian and cell cycle disruption is a
hallmark of cancer especially in rapidly growing tumors and advanced stage malignancies38. In turn,
changes in secretion of chronobiotic agents like melatonin and glucocorticoids are happening in order to
restore circadian system. Changes in rest-activity, body temperature and hormonal secretion can be
used as CTS biomarkers that help in defining the circadian pattern38.
When rest-activity, body temperature, plasma cortisol and melatonin concentrations reveal altered CTS
activity, a personalized anticancer chronotherapy ought to be determined. Chronopharmacology
investigates the dependency of the CTS on drug pharmacokinetics (PK), pharmacodynamics (PD) and the
PK-PD relationship. Optimal time for drug administration, when medication will be possibly less toxic
and more effective, can be predicted based on the CTS. Chronotherapy aims to adapt drug delivery
schemes to circadian rhythm in order to optimize treatment effects37.
9
4. The relevance of circadian timing system for anticancer treatments
The CTS rhythmically controls drug metabolism (phase I and II) and detoxification and also regulates the
cell cycle of healthy cells40. CTS can modify the interactions between drugs and their molecular targets,
DNA repair as well as apoptosis during the 24h period (fig 3). Clock-controlled cell proliferation
determines cytotoxicity of anticancer agents as most anticancer drugs target a certain stage of cell
division cycle41.
Figure 3 The CTS determines the most advantageous circadian timing of anticancer treatment38. The CTS regulates
transportation, bioactivation, metabolism, elimination and molecular targets that account for the chronopharmacology of
anticancer drugs. The CTS also controls cell cycle events as well as DNA repair and apoptosis which elucidate the
chronopharmacodynamics of anticancer agents. Therapy for which the drug administration schedule is adapted to
chronopharmacokinetics and chronopharmacodynamics is named chronotherapy.
It has been demonstrated in experimental systems that both dose and circadian timing play a crucial
role on tumor growth inhibition and patient survival for at least 28 anticancer drugs, among which is the
5-FU42. The chronoefficacy was determined based on the administration of the drug, both as single
agent and in combination with up to four other drugs at certain circadian times42. Chronoefficacy usually
coincides with chronotolerance for 28 drugs tested and in general for cytostatics, interferons,
antiangiogenic agents, and cell cycle inhibitors38. Hence, experimental chronotherapeutics sustains
circadian timing as a possible method to enhance anticancer treatment. Chronoefficacy can arise from
the circadian control of cell cycle phase related to protein targets, like TS for 5-FU. It can also derive
from the circadian regulation of other enzymes involved in drug metabolism. Both concepts are
illustrated in figure 3.
10
Both 5-FU and capecitabine target cells on S-phase. The main target of capecitabine and 5-FU is
thymidylate synthase (TS) which presents circadian rhythm. Studies in mice have shown that the peak of
bone marrow TS activity is near the second half of darkness and coincides with the highest hematologic
toxicity of the drug1. Transcription and activity of TS are highest in early S-phase in proliferating
tissues43. Meanwhile, bone marrow cells of mice that are in S phase increase by almost 50% in the mid
dark44. This means that the circadian control of cell cycle in correlation with TS activity can affect 5-FU
chronoefficacy. Other enzymes of the fluoropyrimidine metabolic pathway that present circadian
organization are DPD, UP, OPRT and TK3,4. However, for some of them, their potency in the metabolism
and their exact circadian pattern in humans remain to be clarified. It is also unclear whether CTS directly
regulates the activity of these enzymes and it will be discussed below.
5. The circadian variation in fluoropyrimidine enzymes
Cellular toxicity caused by 5-FU mainly derives from thymidylate synthase (TS) inhibition. TS activity is
higher in proliferating cells than in non-proliferating45. Generally, TS activity is related to the
proliferative activity of the tissue and it has been associated to higher S-phase fraction and the relative
sensitivity of the tissue to 5-FU2,46. Both animal and human studies have shown that toxicity and efficacy
of 5-FU depend on the time of the day at which the drug is administered47. The differences in toxicity
and efficacy of the drug are possibly related to circadian organization of TS throughout the day. A recent
study investigated TS activity in mice normal tissues like bone marrow, intestinal mucosa and oral
mucosa, which the 5-FU damages the most2. Normal tissue damage limits 5-FU dosage. Consequently, it
is possible to achieve a higher dosage when the drug is administered at the time of the highest
tolerability. In this study, TS activity was monitored throughout a full 24h circadian cycle (fig 4). TS
activity was found to vary almost 10-fold among different tissues and 2-fold throughout the day for the
same tissue. Even though the value of their TS activity varied a lot, bone marrow and oral mucosa
presented similar TS circadian rhythm with two peaks in late activity (23:00 and 21:00 hours after light
onset (HALO) respectively) and in late sleep (09:30 and 09:00 HALO respectively). TS circadian rhythm in
mice intestinal mucosa was slightly different with one main peak in mid-activity phase (18:45 HALO) and
a smaller peak in mid-sleep phase (07:00 HALO)2. Due to circadian variability of TS, the incidence of
gastrointestinal toxicity in mice can be up to 2-fold higher when capecitabine is administered at the time
of lowest TS activity in comparison to administration at the time of highest TS activity. To extrapolate
these results in humans we must keep in mind that humans are more active during the day while mice
are more active during the night. Most physiological rhythms present a 12h phase shift between humans
and rodents48. DNA synthesis in human bone marrow, oral and intestinal mucosa is almost 50% lower in
the night (from 00:00 to 04:00h) in comparison to daytime49. This circadian organization of the dominant
molecular target of 5-FU can partly explain why the toxic-therapeutic ratio of the drug differs within the
day50. There are studies in humans that further sustain this conclusion, suggesting that CTS directly
regulates the time of the day with the highest and lowest TS activity and subsequently 5-FU toxictherapeutic index1. It has been found that the protein formed from the clock gene BMAL-1 coordinates
TS activity and DNA replication. High tumor nuclear BMAL-1 protein coincides with low tumor TS activity
11
and it was found to be early in the morning. Inversely, low nuclear BMAL-1 protein coincides with high
tumor TS activity (during late activity)1. Low TS activity in a tissue is associated with higher toxicity in
that tissue and higher tumor shrinkage upon 5-FU administration, while high TS activity is related with
poor tumor response1.
Figure 4 Representation of TS circadian rhythm in three different normal tissues of mice. In all three tissues we observe two
peaks of TS activity during the 24h cycle and almost 2-fold differences from the highest to the lowest value within the same
tissue2.
Besides the importance of the circadian organization of TS, circadian activity of other enzymes within
the 5-FU pathway may contribute to the toxic-therapeutic ratio of the drug. Important anabolic enzymes
for fluoropyrimidine drugs like uridine phosphorylase (UP), orotate phosphoribosyl transferase (OPRT) 3
and thymidine kinase (TK)4 present circadian rhythm. Moreover, the activity of the dominant
fluoropyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase (DPD), is rhythmically organized
throughout the day4,51.
Naguib et al. (1993), described the 24h activity of hepatic OPRT and UP in female mice kept in
standardized conditions of 12h light alternating with 12h darkness3. They observed a 24h cycle change in
12
the activity of the enzymes with peaks at 18 and 15 HALO and troughs at 6 and 3 HALO for OPRT and UP
respectively (fig 5).
Figure 5 Pattern of circadian rhythm of hepatic orotate phosphoribosyl transferase (OPRT) and uridine phosphorylase (UP) in
female mice maintained under a cycle of 12h light (06:00-18:00h) and 12h darkness (18:00-06:00h)3.
Rats treated with fluoropyrimidines usually experience toxicity in bone marrow, intestinal mucosa, liver
and spleen4. Zhang et al. demonstrated the circadian pattern of the anabolic enzyme TK in these tissues
of rats after treatment with fluoropyrimides4. The same study also showed a circadian rhythm of 5-FU
catabolizing enzyme DPD activity. Moreover, they presented the relationship between the circadian
organization of anabolic and catabolic enzymes activity. The animals maintained under standard light
conditions from 06:00 to 18:00 and darkness from 18:00 to 06:00. The peak of TK activity in bone
marrow and intestinal mucosa was at 22 HALO (04:00) and the trough at 10 HALO (16:00). TK activity,
observed in the liver and spleen of the rats, peaked at 20 HALO (02:00) and showed lowest activity at 8
HALO (14:00) (fig 6). Circadian alterations of TK activity can have a great influence on bone marrow and
intestinal toxicity as these are the major toxicity sites of fluoropyrimidine treatment.
13
Figure 6 Circadian pattern of TK activity in intestinal mucosa (A), bone marrow (B), liver (C) and spleen (D) of rats kept under a
cycle of 12h light (06:00-18:00h) and 12h darkness (18:00-06:00h)4. The four different types of tissue present similar TK
circadian rhythm.
Using the same animals under the same conditions, DPD activity was measured in liver and bone
marrow as well. The peak DPD activity was found to be at approximately 7 HALO (13:00) and 5 HALO
(11:00) and the trough at approximately 19 HALO (01:00) and 17 HALO (23:00) for liver and bone
marrow respectively (fig 7). The results revealed comparable circadian organization for TK and DPD in
different tissues examined. However, a comparison of TK with DPD activity demonstrated an inverse
correlation between them (when TK is at its maximum activity, DPD is reaching its minimum activity).
Figure 7 Circadian pattern of DPD activity in liver (A) and bone marrow (B) of rats kept under a cycle of 12h light (06:00-18:00h)
and 12h darkness (18:00-06:00h)4. The enzyme activity in the liver was shown to follow similar circadian pattern as bone
marrow activity.
14
Extrapolation of circadian organization from rodents to human beings is usually difficult due to different
life cycles. Rodents are more active in darkness (nocturnal) while humans are more active in the light
(diurnal). Moreover, life style among humans is different because of different work schedules and other
daily habits, while experimental animals under the strictly light-dark control tend to present similar
circadian pattern between them. However, results from a study performed to determine DPD activity in
human peripheral blood mononuclear cells5 agreed with the circadian pattern of DPD activity in rats
meaning that the peak DPD activity in rodents and humans occurred at the midle stage of resting span.
In this study, there was an obvious interpatient variability and the data was normalized in order to
conclude to an overall pattern for all patients. The peak of DPD activity was estimated at around 01:00h
and the trough at around 13:00h (fig 8).
Figure 8 Circadian pattern of DPD activity in peripheral blood mononuclear cells (PBMCs) of a patient receiving continuous
infusion of 5-fluorouracil5.
A direct connection between DPD activity and the CTS in humans has been proved by W. Krugluger et al.
They suggested that DPD transcription is controlled by the clock gene Per 1 as they noticed high
correlation between DPD mRNA and Per 1 mRNA levels in colon cancer tissue52.
Summarizing, the 5-FU anabolic enzymes UP, OPRT and TK, as well as the main drug-target TS and the 5FU catabolizing enzyme DPD, present circadian variations. In rodents, all these enzymes are more active
during activity phase except DPD which is more active during resting phase. Obviously, since when DPD
activity is low the anabolic enzymes activity is high, there may be a higher fraction of the drug entering
the anabolic pathway at that time. Therefore, the exposure of cellular targets to the toxic 5-FU
metabolites is lower during night, when 5-FU catabolism is accelerated. This is in agreement with the
observation that 5-FU is more toxic to healthy tissues during activity span53 when DNA synthesis is
higher, in both animal and humans38,54,55. In humans, it has been shown that DPD enzyme activity peaks
in the middle of resting phase4,5, while the circadian organization of the anabolic enzymes UP, OPRT and
TK has not been studied. So, for the human situation, data about circadian organization of 5-FU anabolic
15
enzymes is still lacking. However, regardless the circadian pattern, these anabolic pathways can be
bypassed by another pathway driven by TP enzyme. TP is not circadian but it possibly drives a more
dominant pathway as TP possesses high enzymatic activity, much higher than UP56. Moreover, a recent
study in xenografts revealed that among fluoropyrimidine metabolic enzymes, the efficacy of
capecitabine mainly depends on TP and DPD tumor levels6. Additionally, as it has been discussed, there
is a directed TS regulation from the CTS through BMAL-1 protein1. In this case, DPD and TS circadian
organization seem to be more important in the determination of the clinical outcome upon
fluoropyrimidines administration. The abovementioned conclusions led to the hypothesis that
administration of 5-FU or capecitabine at night would possibly be better tolerable from healthy tissues.
Moreover, it has been noticed a correlation between 5-FU plasma levels and the efficacy of
fluoropyrimidines treatment33. So, hypothetically, a higher dose administration at the time at which the
drug is less toxic could achieve higher plasma levels. Several clinical trials, with 5-FU or capecitabine,
have been performed in order to validate this hypothesis.
6. Clinical trials assessment
The feasibility of chronotherapy with fluoropyrimidines was investigated in several clinical trials of phase
I to determine safety of chronomodulation. Phase II studies were performed to determine toxicity and
response of chronomodulated fluoropyrimidines. In addition, in phase III trials the chronomodulated 5FU was compared to conventional therapy.
Regarding intravenous administration, different schedules were examined, where constant infusion of 5FU was mainly compared to chronomodulated infusion of 5-FU. For oral administration, different
schedules were used where the daily dose was subdivided in two or three doses. A part of the dose was
given during day and another part of it during evening.
When assessing the applicability of chronotherapy we must keep in mind that intravenous
administration has the drawback of requiring multichannel programmable pumps38, while variability in
absorbance and compliance between different individuals contribute in the result of per os
administration57. Chronopharmacokinetic studies are needed when drug absorbance presents high
interindividual variability58. Usually, subjects participating in clinical trials tend to be compliant.
However, it is useful to include assessments of compliance in the trial in order to avoid this kind of
error57,59.
6A. Intravenous administration
5-FU is usually given in combination with oxaliplatin because of their synergistic effects60. However, the
time of best tolerability for 5-FU is at night probably because of high DPD activity, as discussed in the
previous chapter, while oxaliplatin seems best tolerable during midday61. A phase I study revealed a
good safety profile for oxaliplatin with a peak delivery at 16:00h62. In a phase II trial, where patients with
metastatic colorectal cancer were included, the safety and efficacy of a chronomodulated schedule were
determined. The schedule consisted of oxaliplatin administrated from 10:15h till 21:45h, with a peak at
16
16:00h, combined with folinic acid and 5-FU administrated from 22:15h till 09:45h, with a peak at
04:00h. The dose of oxaliplatin, folinic acid and 5-FU were 20 mg/m²/day, 300 mg/m²/day and 600
mg/m²/day, respectively. This schedule was maintained on day 1-5 of each three-week course63. This
chronomodulated combination was compared to continuous administration of the same combination of
drugs in the same dosage given again for 5 consecutive days every third week as it has been investigated
in two independent randomised phase III trials50,64 (table 1). In the first trial, 92 patients were included
who have not previously received chemotherapy64. This study demonstrated that the chronomodulated
schedule was less toxic and more effective. Patients, upon chronomodulated treatment, presented
higher response rate and longer progression free survival and overall survival compared with the
constant rate infusion. However, this trial was terminated prematurely because of possible chemical
interactions between 5-FU and oxaliplatin when delivered simultaneously from the same catheter64. The
second trial involved 186 patients with previously untreated metastases50. Half of them received
chronotherapy and the other half constant rate infusion. The main clinical endpoint was the objective
response rate and it was found to be higher by 21.5% in the chronotherapy group than in the constant
infusion group (p = 0.003). Chronotherapy was also found to be less toxic. The incidence of severe
mucosal toxicity, peripheral sensitive neuropathy, as well as other common toxic effects like diarrhea,
hand-foot syndrome and neutropenia was 14% for chronotherapy vs 76% for constant infusion group (p
< 0.0001). In general, lower incidence of severe toxicities in the chronotherapy arm led to less
hospitalization for toxicity and lower number of patients who had to be withdrawn because of toxicity.
In addition, the reduction of severe toxicities in the chronotherapy arm allowed the increase of the drug
dose. This resulted in higher plasma levels and greater efficacy of the drug. In this trial, the median dose
of 5-FU per course was 40% higher for chronomodulated delivery (3500 mg/m²) compared to constantrate infusion (2500 mg/m²). Another important observation was that after treatment, more patients
that received chronotherapy were eligible for surgery of some metastases that were initially
unresectable. Finally, the differences in median progression free survival and overall survival were not
significant between the two arms, but that was not the endpoint of the trial50.
Table 1 Overview of the key clinical trials investigating 5-FU administration in constant or chronomodulated schedules.
17
• Abbreviations. Ref: reference number, m/f: male/female, PFS: progression-free survival, OS: overall survival, ORR: overall
response rate, chron.: chronomodulated administration, 5-FU: 5-fluorouracil, hfs: hand-foot syndrome, const.: constant
infusion.
In a recent meta-analysis study, data from three phase III randomised clinical studies were combined.
This study revealed that only male patients benefit from chronomodulated 5-FU administration65. A total
of 845 (346 females and 499 males) metastatic colorectal cancer patients were treated with chronoFLO
vs conventional 5-FU schedule50,64. No significant gender differences regarding response rate (RR),
progression-free survival (PFS) and overall survival (OR) were seen on the conventional therapy group.
However, on the chronomodulated arm, men had significantly higher RR, PFS and OS than men in
constant rate group. Contrarily, the chronomodulated administration presented similar or worse
efficacy compared to the conventional infusion in women. This may happens because of different
circadian genotypes between male and female patients or due to gender dependency of circadian
pharmacology of anticancer drugs. The study revealed significant gender differences regarding RR
(p=0.007), PFS (p=0.006) and OS (p=0.002) in response to chronotherapy and suggested that men with
colorectal cancer can benefit more from chronotherapy in comparison to conventional therapy of 5-FU.
Another study also showed that chronomodulated delivery of fluoropyrimidines may be beneficiary only
for men but not for women66. Therefore, more attention should be paid in gender differences for future
trials65.
6B. Oral administration
It has been previously discussed that capecitabine is designed to be preferentially converted to 5-FU in
the tumor tissue and therefore it is qualified as tumor selective11. The efficacy of capecitabine as single
drug was found to be similar to 5-FU/FA (folinic acid)67 and in combination with oxaliplatin (XELOX) was
found to be analogous to 5-FU/FA plus oxaliplatin (FOLFOX)68. Moreover, intravenously administrated
drugs, like 5-FU, have several drawbacks such as infections, bleeding, thrombosis, pneumothorax and
inconvenience for patients. Thus, capecitabine is a good alternative solution for chemotherapy.
Chronomodulated administration of capecitabine was tested in several phase I and II clinical trials. The
rationale to deliver a higher evening dose was driven by the circadian organization of the enzymes
involved in capecitabine metabolism in addition to the preceding data regarding 5-FU.
Santini et al. investigated the efficacy and toxicity of a chronomodulated schedule of capecitabine and
oxaliplatin (XELOX) in advanced colorectal cancer patients resistant to 5-FU chemotherapy69. When
oxaliplatin is co-administered with capecitabine, apart from the synergistic effect, it also improves
tumor-selective activation of capecitabine as oxaliplatin upregulates TP expression only in tumor
tissues70. In total, 36 metastatic colorectal cancer patients were included in the study. In this phase II
study, oxaliplatin was continuously infused from 08:00h to 20:00h on days 1 and 8. Capecitabine was
administered orally in 3 unequal doses every day: 25% of the dose at 08:00h, 25% of the dose at 18:00h
and 50% of the total dose at 23:00h on day 1-14 every 3 weeks. The daily dose of oxaliplatin was 70
mg/m² and for capecitabine 1,750 mg/m². The overall response rate estimated from this study was
30.6%, while disease stabilization was achieved in 36.1% of patients. The majority of toxic effects were
of grade 1 or 2, which is considered as mild or moderate. Few patients experienced grade 3 toxicities
18
including diarrhea, fatigue, mucositis, nausea, neurotoxicity and only two patients hand-foot syndrome.
In general, this combined continuous infusion of oxaliplatin with chronomodulated administration of
capecitabine demonstrated good overall survival and median time to progression69 (table 2). Results
from this trial show that this regimen is well tolerated and presents good safety profile69. Based on the
above evaluations, the group performed a phase I study with the goal to determine the maximumtolerated dose and the dose-limiting toxicities of chronomodulated capecitabine monotherapy. 27
patients with advance cancer were involved in this trial. The schedule of capecitabine administration
was equal to the previous study71. They observed that the chronomodulated administration of
capecitabine was safe and well tolerated in the majority of patients at the recommended dose of 2,750
mg/m/day. At this dosage the toxicities were prevalently hand-foot syndrome, fatigue and
gastrointestinal toxicity. However, almost all cases were reversible after dose modification. Only one
patient presented fatigue of grade 4. The tolerability of this schedule was confirmed after treatment of
five cycles71. The same research group conducted another phase II study using the same drug schedule
(fixed-rate infusion of oxaliplatin with chronomodulated capecitabine) including this time 46 chemonaive patients with advanced colorectal cancer72. Results from this study showed high tumor control,
and overall survival, good progression-free survival and very good safety profile. It is possible that the
favourable toxicity profile in these studies derives from the circadian organisation of the drug
metabolism, elimination and therapeutic targets. It can also be related to the new 3-times
administration schedule of capecitabine instead of the usual 2-times per day. Moreover, the 12h
continuous infusion of oxaliplatin partially coincides with its optimal administration time which can also
results in lower toxicity. It is particularly interesting that results were more promising than expected and
consistent with other studies in untreated patients72. However, it is not safe to extract solid conclusions
from these trials as there was no comparator arm.
Later, Qvortrup et al. performed a phase II study with chronomodulated XELOX (XELOX30chron). The
primary end point of this study was the response rate (RR) and secondary end points were toxicity,
progression-free survival (PFS) and overall survival (OS)73. The trial involved 71 patients with metastatic
colorectal cancer, pre-treated with 5FU and irinotecan. They designed a XELOX schedule that mimicked
a chronomodulated regimen but for practical, economical reasons and for patients’ convenience the
regimen was administered as follows: oxaliplatin was administered intravenously (30min infusion with
dose 130 mg/m²) on the first day between 13:00 and 15:00h. Oxaliplatin induces infusion rate
dependent neurotoxicity and a 2h infusion is recommended to reduce the incidence of neurotoxicity.
However, in a pilot study where oxaliplatin was delivered as 30 min infusion, it has not been observed
higher neurotoxicity74. A total daily dose of 2000 mg/m² of capecitabine was given twice per day in two
unequal doses. 20% of the dose was given in the morning (between 07:00 and 09:00h) and 80% was
delivered in the evening (between 18:00 and 20:00h). The main efficacy and toxicity results from
XELOX30chron can be seen in table 2. These were similar to what Santini et al. found for their
chronomodulated XELOX schedule used as second line therapy69 apart from neurotoxicity which was
lower for XELOX30chron.
19
The safety (mostly regarding the grade 3 toxicity) and efficacy of XELOX30chron were also comparable
with a previous study conducted by the same group, using this time a conventional schedule of
capecitabine in combination with oxaliplatin75. Again, in this phase II trial, 71 pre-treated patients with
advanced colorectal cancer were involved. Equal doses of capecitabine (1000 mg/m²) were administered
twice per day with an interval of 12h, from day 1 to day 14. Oxaliplatin was given as 30min infusion in a
dose of 130 mg/m²/day.
A multicenter phase II study was conducted comparing XELOX30 and XELOX30chron with the primary
objective of reducing toxicity76. The advantage of this study was that the results of the two different
therapeutic schedules could be compared directly. In this trial, 141 patients with metastatic
unresectable and untreated (except from previous 5-FU or capecitabine treatment ended 6 months
before the study) adenocarcinoma of colon or rectum were enrolled. Patients were separated in two
arms and randomly received XELOX30 (arm A) or XELOX30chron (arm B) as first line therapy. Both arms
received 30min infusion of 130 mg/m² oxaliplatin on the first day of each cycle. However, in arm A the
infusion time was between 08:30h and 16:00h but it was not systematically registered, while in arm B
oxaliplatin was given between 13:00h and 15:00h. A total daily dose of 2000 mg/m² of capecitabine was
delivered for 14 days to each group. In arm A, patients were treated with conventional capecitabine
schedule receiving half of the daily capecitabine dose in the morning and half in the evening. Patients in
arm B received 20% of the dose (400 mg/m²) in the morning between 07:00h and 09:00h, and 80% of
the dose (1600 mg/m²) in the evening between 18:00h and 20:00h. Treatment was repeated every third
week. The primary endpoint was to determine whether toxicity was reduced. The reduction of toxicity
from arm A to arm B was only 5% (p=0.47), as toxic effects > grade 2 were seen in 85% of the
chronomodulated arm and 90% of the conventional capecitabine therapy. Mild neurotoxicity was 15%
for the conventional therapy and 13% for the chronomodulated scheme. However, the incidence of
severe neurotoxicity was higher in group B that received chronotherapy. Furthermore, no significant
differences were found between arm A and B, regarding median progression-free survival (8.9 vs 8.8
months) and overall survival (17.6 vs 15.5 months)76. Thus, in this study, the chronomodulated XELOX
did not seem to reduce toxicity in comparison to the conventional XELOX schedule. On the other hand,
the chronomodulated administration of 5-FU with oxaliplatin reduced toxicity compared with fixed
infusion, according to previous studies50,64. Table 2 summarizes the main findings of studies with
chronomodulated capecitabine. In general, it has been observed that patients receiving more than six
cycles of chronomodulated XELOX tend to develop less severe neurotoxicity in comparison with other
XELOX regimens73. Overall, the trials described, using the oral chronomodulated administration of
capecitabine, were not able to demonstrate a clear time-dependency of safety and efficacy of the drug.
20
Table 2 Overview of the key clinical trials investigating the chronomodulated or conventional capecitabine administration.
• Abbreviations. Ref: reference number, m/f: male/female, PFS: progression-free survival, OS: overall survival, ORR: overall
response rate, chron.: chronomodulated administration, 5-FU: 5-fluorouracil, hfs: hand-foot syndrome, const.: constant
infusion.
7. Comments and perspectives
The circadian rhythm of TS and DPD has been studied in rodents and humans as well. According to these
findings, it has been hypothesized that treatment with fluoropyrimidines would probably be best
tolerated during resting phase. Based on that, chronomodulated intravenous administration of 5-FU and
oral administration of capecitabine have been both tested in several clinical trials. Great response
variability according to gender has been observed. Men will potentially benefit more from
chronotherapy compared to women. Moreover, results from 5-FU chronotherapy seem to be more
promising than capecitabine chronotherapy. This may have several explanations and one may be that
antitumor activity of 5-FU and capecitabine depends on different molecular markers. According to a
recent study performed in xenografts, there is great evidence that the sensitivity to capecitabine
depends more on mRNA expression levels of TP, TS and the ratio of TP to DPD, while 5-FU sensitivity
depends more on OPRT, UP and CDD expression6. This leads to the hypothesis that among
fluoropyrimidine metabolic enzymes that present circadian organization, only DPD is really important for
capecitabine sensitivity, while 5-FU sensitivity depends more on the circadian metabolic enzymes.
However, further investigation is needed in order to confirm these findings in humans, since the abovementioned study was performed in xenograft models. Moreover, the circadian pattern of OPRT and UP
has not been studied in humans.
21
An additional reason for which capecitabine chronotherapy did not show a clear time-dependency could
be the design of the trials regarding the kind of patients selected, the dosage and the administration of
the drug. Besides, capecitabine chronomodulated administration may not mimic 5-FU chronomodulated
administration because of extra metabolic steps required for capecitabine bioactivation77,78 and
absorption process58.
Two very important factors for the trial design are patient selection and the determination of
information deriving from each patient group. There are well documented differences in drug
metabolism between males and females regarding 5-FU, resulting in very different toxic pattern79.
Females presented much higher rate of severe toxic effects than males during treatment with
chronomodulated 5-FU/FA and oxaliplatin80 which may be explained by the lower 5-FU clearance in
females compared to males81. Moreover, the female circadian structure is somehow different from the
male one, presenting higher melatonin rhythm suppression by light at night82 and higher cortisol
response to stress83. However, all the above mentioned studies did not examine toxicity pattern
according to gender. The age is another variable that can influence the absorption, the metabolism as
well as the elimination of capecitabine and 5-FU. It has been recently reported higher capecitabine
Cmax and AUC in patients over 70 than under 60 years which may alter the pattern of toxicity84. Hepatic
and renal dysfunctions are a common problem in elderly and can affect both drug metabolism and
elimination. Blood flow is very important for drug transportation. According to the size and the
metabolic function each organ receives a certain fraction from the cardiac output. The higher the
amount of blood that an organ receives the higher the drug rate that can transfer to the extravascular
sites where the pharmacological receptors are. Blood flow and cardiac output decline with age by
almost 1% every year after the age of 25 years85. These and other phenomena of age, like several
possible comorbidities and the concurrent uptake of other medications, can modify the metabolism of
capecitabine/5-FU as well as the circadian pattern of the patient. Another drawback of the above
mentioned clinical trials is that the age range of the enrolled patients was very wide (ranging on average
from 28 to 76 years old). This means that, it is very difficult to draw safe results for efficacy/toxicity of
the drug from such a group. Inter-individual variations of circadian rhythms can affect drug exposure as
well. There are people that demonstrate opposite circadian patterns and present higher drug (5-FU)
concentration in the evening than in the morning86.
Dose and time of drug administration are also important determinants of the outcome of the trials
especially the chronomodulated ones. The recommended dose for capecitabine is 2500 mg/m²/day
orally given twice per day usually at the end of the meal. The proposed duration is 14 days with 1 week
rest period in 3-week cycles70. However, capecitabine trials that have been discussed used lower dose
than the recommended one and in none of these trials the dose exceeded 2000 mg/m²/day. The time of
capecitabine delivery may be crucial in a chronomodulated study. It has been reported that 5-FU
concentration reaches its peak about 2h after oral administration of capecitabine70 and besides that the
optimal time for 5-FU is at 04:00h. Therefore, early evening oral administration (18:00-20:00h) which
has been reported in most of the studies, will result in the higher concentration of the drug much earlier
than the optimal time. Furthermore, as it is already discussed, the infusion of oxaliplatin was not
accomplished at the same time for both arms.
22
The biggest difference between 5-FU and capecitabine chronomodulated administration is that 5-FU is
delivered intravenously while capecitabine is given orally. Oral drugs undergo an absorption process that
may exhibit circadian variation due to biological daily fluctuations58. Absorption of oral administered
drugs can be affected by several factors regarding the physicochemical properties of the drug as well as
patient-related factors like gastrointestinal mobility and gastric emptying, gastric pH, gastrointestinal
blood flow and the activity of protein-transporters and enzymes on gut surface87. As gastric pH,
emptying time and intestinal mucosa blood flow vary during the day, differences in drug absorption in
humans have been reported88. Gastric pH presents circadian rhythm modified by meals and nocturnal
duodenal-gastric reflux89. This can influence the ionization of drugs depending on day or night
administration. In general, lipophilic drugs are absorbed more rapidly when administered in the morning
compared to evening administration. This can lead to higher Cmax and shorter Vmax during morning
administration in contrast to evening administration58. Capecitabine is a lipophilic drug. Given that, the
dose delivered in the evening can result in very different 5-FU plasma levels compared to an equal
intravenously administered 5-FU dose. The rate of drug absorption can also be influenced from the
gastrointestinal perfusion as blood flow affects the passage of drugs through biological membranes,
especially lipophilic drugs that do not require active transportation58. Gastrointestinal blood flow is
increased in early morning78. This fact may explain a higher absorption of capecitabine in the morning
than in the evening. Furthermore, hepatic perfusion is higher in early morning as well78. The
bioactivation of capecitabine mainly happens in the liver11 which means that the higher the hepatic
blood flow is the more drug available for bioactivation.
Three enzymes are involved in the bioactivation of capecitabine: (a) carboxylesterase (CES) that is
mainly expressed in the liver and in the intestine90, (b) cytidine deaminase (CDA) that can be found in
many tissues including liver and tumor tissues and (c) thymidine phosphorylase (TP) which is also
expressed in many tissues and can be found in higher levels in some tumor tissues compared to the
normal ones90. It is not precisely known if there is no circadian variation in any of these three enzymes.
The pharmacokinetic parameters of capecitabine and its metabolites, 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'-DFUR) and 5-FU, present high inter-individual variability as also
happens with other cytotoxic drugs70. In a recently performed study, it has been reported that
coefficients of variance of the concentration in plasma of capecitabine and its metabolites ranged
between 36% and 142%91. Researchers believe that this is most likely because of variability of expression
and activity of enzymes involved in capecitabine bioactivation91. Mercier et al described a case of CDA
overexpression (180% higher activity compared to normal population) that led to unexpected and
severe toxicities because of extensive activation of capecitabine and overexposure to 5-FU92.
High inter-patient variability was also observed in TP expression levels in various tissues including liver
and tumor tissues93. This may also influence capecitabine pharmacokinetics.
Recent pharmacokinetic guided studies revealed that, among other factors, dose adjustment based on
pharmacokinetic monitoring may reduce the toxicity and increase the efficacy of capecitabine. Gamelin
et al performed a randomized phase III study where they noticed that pharmacokinetically guided 5-FU
dose adjustment increased the response and decreased the incidence and severity of toxicity94.
Moreover, Saif et al, after reviewing the various strategies developed to enhance 5-FU clinical activity,
23
mentioned that pharmacokinetically guided dose adjustments can improve the therapeutic index of 5FU33. Pharmacokinetics of capecitabine and its metabolites can potentially be useful for monitoring and
guiding the therapy with capecitabine or 5-FU. Nowadays, there are methods for the quantitative
determination of capecitabine and its metabolites which can be very helpful clinical studies95.
It cannot be excluded that a schedule of chronomodulated administration is possibly able to increase
efficacy and reduce toxicity of capecitabine. However, there is a need for further investigation of
capecitabine chronopharmacokinetics and better design of trials in order to validate if there is timedependency of capecitabine therapeutic index. Based on the above-mentioned observations and the
findings from the trials discussed, several adjustments can be done in order to receive more clear
information from future trials. It would be safer to compare chronomodulated to conventional
administration in untreated patients as their circadian pattern is not disrupted by previous anticancer
medications. Results should be compared separately in men and women as gender differences influence
both efficacy and toxicity of the drug. Safer conclusions would be drawn from a less wide age range of
enrolled patients. Moreover, we observed that three times delivery of capecitabine was less toxic from
that of two times per day. So, it would be better to use this schedule in future studies. The optimal time
for drug delivery can also be improved. Based on circadian DPD activity, the best tolerability of 5-FU is
near the peak of enzyme activity, around 01:00h, that has not been tested so far. However, there are big
circadian differences among individuals according to gender, age, polymorphisms and chronotype41. So,
to take full advantage of cancer chronotherapy and draw safe conclusions from prospective studies,
there is a need for patient-tailored chronotherapy. This can be done with the help of mathematical
models that take into consideration many parameters like circadian physiology, genetic background,
gender, age etc. Finally, the large progress in drug-development technologies can facilitate personalized
chronotherapy schedules.
24
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