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CHRONOTHERAPY OF CANCER:
A MAJOR DRUG DELIVERY CHALLENGE
by
William J.M. Hrushesky, M.D.1
Marek Martynowicz, M.D.1
Miroslaw Markiewicz, M.D.1
Reinhard von Roemeling, M.D.2
Patricia A. Wood, M.D., Ph.D.1
The evolution of life took place in a milieu influenced by cyclic interactions of
the sun, earth and moon. The existence of rhythmic changes in living organisms is
a sign of their adaptation to these relationships and serves as indirect evidence
for time-dependent variability of the response of the human body to many drugs,
including those used in the therapy of cancer. This latter possibility has been
confirmed for several classical chemotherapeutics in both murine and human trials.
Doxorubicin and cisplatin, as well as their analogs, 5-fluorouracil and FUDR have
been studied in the context of their circadian pharmacodynamics and toxicology. The
outcomes of these studies clearly show that proper timing of their administration
reduces drug toxicity and allows for substantial increases in the maximally tolerated
dose, which results in better treatment efficacy and greater comfort for patients.
Also, the first steps in investigation of optimal timing and scheduling of therapeutic
peptides and polypeptides (erythropoietin, TNF, IL-2) have been made. Preliminary
results suggest that these "natural drugs" may be considerably more circadian
time-sensitive than are classical chemotherapeutic agents.
The world of chronobiology provides a new dimension for drug delivery. Multi-agent
therapies, where each drug will be given in a time-dependent manner, will require
sophisticated computerized multiple reservoir drug delivery systems. Closed-loop,
implantable devices that stipulate optimal timing according to measures of internal
circadian timing are under development. Such systems will permit cancer patients to
become more active and productive. Finally, the adoption of such high-tech drug
delivery instruments will enable attention to be given to answering important
chronobiologic questions and so will help to turn the science of chronobiology into
what it truly is - a multidimensional and dynamic perspective on life and science.
INTRODUCTION.
Chronobiology is the study of the temporal relationships of biologic phenomena. All
living things evolved in a milieu characterized by constant change based upon the
cyclic relationships of the sun, earth, and moon. The early chemistry of life was
strongly helio-dependent. Organisms had to store energy during periods of daylight
for use during periods of darkness. Adaptability to the influence of the circadian
patterns of our planet was thus a sine qua non of life and it is apparent that all
organisms have incorporated and retained in their genetic make-up this essential
circadian periodicity. Circadian organization is such a basic property of life that
derangements may have lethal consequences, including for example, the severe effects
of sleep deprivation or the major schedule disruption during occurring transmeridian
travel in humans.
Life forms that have evolved and remained at those parts of the earth's surface where
day and night are of relatively equal duration throughout the year have developed
lower frequency patterns than those that had to cope with seasonal differences in
energy availability. Organisms have developed rather complex abilities to sustain
themselves through long seasonal periods of energy dearth - hibernation is the example.
During the millennia when life forms lived exclusively in the sea, the regular and
recurrent tidal forces generated by the moon and sun acting upon the earth also
required additional evolutionary adaptation of the vital chemistries of all creatures.
For example, the massive and regular movements of the fluid covering the planet have
defined the lunar day of 24 hours and 51 minutes, and the relationship of flood and
ebb tides with spring and neap tides have defined the 29 1/2-day lunar month.
Interestingly, a further rhythm having an endogenous periodicity of about 7 days (5-9
days) has been well-documented. This normally low amplitude rhythm in cytokinetic,
immunologic, and other variables may be markedly amplified when the organism is
perturbed. This approximately weekly rhythm is one of the most fascinating, because
there is no obvious exogenous geophysical timekeeper that has set it in motion. The
four biophysical rhythms - the solar day, the lunar month, the year, and the so-called
circaseptan rhythm - have left an indelible imprint upon all life forms. They have
created highly complex interacting temporal networks of biochemistry and genetics.
To help the reader realize how strongly they affect healthy mammalian organisms,
Figures 1 and 2 give some circadian patterns of such basic physiological variables
as temperature and blood pressure.
Chronobiology considers each of the above interacting time frames; it defines and
quantifies their biological effects; and uses the understanding of such phenomenon
to refine the way we ask scientific and biomedical questions as well as permits new
questions to be asked. Such questions may be asked more effectively and precisely
than can be done if chronobiological effects are ignored. Data will be reviewed here
which show that chronobiological considerations are important for understanding
cancer etiology, prevention, diagnosis and treatment. For example, in animals,
carcinogenesis is dependent upon the circadian timing of carcinogen application,
while disruption of the pineal-hypothalamic-pituitary-temporal balance will
influence the frequency of breast cancer development. Additionally, women at high
risk for the development of breast cancer have flatter circadian and circannual
prolactin rhythms than do women at lower risk. Rhythmic seasonal variations in death
from breast cancer and in average estrogen receptor content of human breast cancer
tissue each suggest the probable importance of the low frequency rhythmic balance
between host and cancer.
Physiological rhythms which could serve as a basis for the time-dependent drug
response of the organism.
A precondition for the improvement of therapeutic index by optimal circadian drug
timing is the ability to detect and quantify meaningful biologic rhythms [1], so
rhythmic changes in normal organ functions have been studied extensively in murine
models. A few examples of such changes follow:
Cytokinetics and nucleic acid metabolism.
In the mouse and rat liver, DNA synthesis, RNA synthesis, RNA translational activity,
mitotic index, weight, glycogen content, and activity of numerous enzymes are all
highly circadian stage-dependent and highly organized throughout the day. The
circadian rhythmicities of mitotic index and DNA synthesis in rat and mouse stomach,
duodenum, rectum, and bone marrow are also very well documented [2-3].
Mauer and more recently Mauer and Smaaland have shown circadian rhythms in DNA
synthesis and the mitotic index from bone marrow in normal human beings [4].
Polyamines, organic anions involved in the regulation of nucleic acid synthesis [4-7],
have been studied for circadian rhythmicity at the University of Minnesota's Clinical
Research Center. It was found that in normal volunteers the excretion of both
monoacetylputrescine and the N1/N8-acetylspermidine urinary ratio were predictably
rhythmic throughout the day (Figures 3,4).
These findings provide additional indirect evidence for overall circadian synchrony
in the cytokinetic activity of normal human tissues [8]. Preliminary results also
suggest that the circadian rhythms of polyamine excretion are disturbed in patients
with cancer, indicating that either cell division patterns are disturbed or the
temporal organizations of excretory organs are adversely affected.
Immunological rhythms of note.
The mammalian immune system is extraordinarily periodic. Circadian rhythms in all
circulating blood cell types have been well documented in both experimental animals
and human beings [9-10]. Numbers of total lymphocytes, B and T lymphocytes, and
natural killer cells demonstrate circadian periodicity [11].
Additionally, studies of immune functions along a 24-hour scale both in vivo and in
vitro have shown these endpoints to be equally circadian stage-dependent. Studies
of human beings by Cove-Smith and colleagues [11] have shown that both tuberculin
skin test reactivity and the incidence of human kidney rejections are circadian
stage-dependent. Tavadia et al. [12] have shown that tuberculin, pokeweed-, and
PHA-induced human lymphocyte transformation are circadian stage-dependent, and that
the peak ability to stimulate is antiphase with the peak of serum cortisol
concentration. Further, Fernandes and colleagues have demonstrated that the
plaque-forming cell response of spleens from mice immunized with sheep red blood cells
also has a marked circadian stage dependence [13-14].
Total RNA content of human lymphocytes has been found to have non-trivial circadian
dynamics. In our laboratory, six series of blood samples were obtained from healthy
volunteers and 19 series from ten women with advanced ovarian cancer. Each series
included one sample at each of six equally spaced circadian stages (4 hours apart).
The total RNA content per cell or per mg of cellular protein of circulating lymphocytes
from normal subjects differed predictably according to the circadian stage of blood
sampling. The time dependency of total RNA content of lymphocytes could best be
accounted for by a 12-hour bioperiodicity. Two populations of lymphocytes (as defined
by synchrony of total RNA content), or two populations of RNA, may be present in the
lymphocytes of normal individuals. The first peak of total RNA content occurs about
nine hours after sleep onset (time near highest circulating steroid concentration),
and the other peak occurs at 18 hours after sleep onset (near to the daily cortisol
low). The morphologic cell surface markers and functional activity of lymphocytes,
as well as the different RNA of these subpopulations obtained at different circadian
stages, need further scrutiny to clarify whether there are either two cell populations
or one cell population having a bimodal RNA distribution (Figure 5).
Ten women, 29-74 years of age, with metastatic ovarian tumors, and awakening daily
at around 0700 hours and retiring at about 2200 hours, were admitted at monthly
intervals for chemotherapy. They were studied in a manner similar to the volunteer
subjects one month after treatment during the 24-hour period before the next scheduled
dose of chemotherapy, on 19 separate occasions. A circadian rhythm in total RNA
content of lymphocytes with a single daily peak was present in these cancer patients.
The time of highest values of RNA content in the lymphocytes of these cancer patients
occurred 11 hours after sleep onset (about 10:15 hours) (Figure 6) near the usual
cortisol peak.
Others have shown that the total RNA content of leukocytes of five healthy volunteers
exhibited circadian rhythmicity [15]. The daily leukocyte RNA peak occurred at about
11:15 hours and corresponds roughly to the first daily peak in our normal control
subjects. The timing of peak RNA content rhythm of leukocytes from these volunteers
is very close to that of the lymphocytes of our patients. These data suggest a
molecular basis for the predictable circadian differences in lymphocyte sensitivity
to therapeutic manipulation. The differences in circadian lymphocyte RNA pattern
between ovarian cancer patients and normal control subjects require further
investigation.
Metabolic rhythms of importance in drug metabolism.
The reduced glutathione (GSH) content of heart muscle cells, which determine both
the redox potential and salvage from free oxygen radicals, maintains a significant
circadian rhythmicity [16]. This circadian organization has also been demonstrated
in the nucleated cells of human bone marrow, with timing of the highest daily levels
corresponding well with the daily timing of lowest doxorubicin (an important
oxygen-active anticancer antibiotic) clinical toxicity. Also, important metabolic
kidney functions exhibit circadian rhythmicity, and such rhythms, in part, determine
renal toxicity and the excretion pattern of certain anticancer drugs [17].
Hormonal rhythms of importance in cancer disease and treatment.
The activity and hormone secretion of the cells of the adrenal cortex undergo very
significant rhythmic fluctuations: concentration of corticosteroids in the gland as
well as the amount of these hormones in serum and 17-ketosteroids in urine show very
strong and well coordinated diurnal changes. Also the contents of ACTH in rodent
pituitary demonstrates a profound circadian periodicity. Cortisol concentration as
well as cortisol related phenomena (i.e., blood concentration of peripheral blood
eosinophils and mononuclear cells (PBM), mitotic activity of some tissues) may
rhythmically modulate immunity and cell-cycle phase- specific cytotoxic (cell cycle
specific) drug sensitivities of the organism. The menstrual cycle, like the circadian
cycle, also has profound effects upon the balance between host and drug toxicity as
well as host and development of cancer.
Chronopharmacokinetics.
The ability of the liver to detoxify, catabolize/metabolize a wide range of
xenobiotics is circadian-stage dependent. This has been described for the liver's
detoxification potential of various agents, including para-oxon, nicotine,
antimycine-A, phenobarbital, hexobarbital, and cytosine-arabinoside [18-20]. Such
rhythms profoundly affect the pharmacokinetics of many, if not most, useful drugs.
Circadian rhythmicity in anticancer drug pharmacokinetics has been described for
5-fluorouracil, cis-diaminedichloroplatinum II (cisplatin), oxaliplatine,
methotrexate, 6-mercaptopurine and doxorubicin [21-26], as well as many other agents
(more detail is provided later in this text).
Circadian organization of cytokinetics in tumors.
Another focus of attention for chronobiology has been whether tumor cells proliferate
either randomly or rhythmically. Mitotic index and/or DNA synthesis as usually
measured by tritiated thymidine uptake have been used to evaluate the proliferative
activity of many transplantable and some spontaneously arising tumors in laboratory
rodents. The data on fast or slowly growing hepatomas illustrate the fact that tumor
cell division may exhibit a more or less strong circadian organization, depending
upon the stage of tumor growth in this model. Thus, well-differentiated, slow-growing
tumors retain a circadian time structure, whereas poorly differentiated,
fast-growing tumors tend to lose it. Such a loss of circadian rhythmicity may also
be acquired during the course of tumor growth [27-28]. All in all, no consensus on
their critical points has yet been achieved for either transplantable or spontaneous
tumors in any species.
General methodology of chrono-oncological studies.
In order to interpret chronobiological data, an understanding of the methodology of
chronobiologic experimentation is required. Pre-clinical chronotoxicological
studies have tried to answer the question whether mice or rats tolerate the same dose
of the same anti-cancer agent differently depending upon when in the day or night
or throughout a 24-hour span it is given, and/or whether the LD10, LD50 and LD90 are
meaningfully different when the agent is given at different times of day. These
investigations are always performed in animals of the same strain, sex, and age, and
which have been synchronized for at least 2 weeks in a lighting regimen usually
consisting of an alternation of 12:12 hours of light:darkness in order to assure
reasonable inter-individual circadian synchrony. The most widely used endpoints to
evaluate the effect of dosing time upon chrono-tolerance have been survival rate,
mean survival time and overall survival pattern. In other studies, organ-specific
measures of lethal and sublethal toxicity have also been thoroughly investigated for
most common anticancer agents.
The kinds of chronobiologic study required for each agent depend upon the agent's
pharmacology and pharmacodynamics. Basic chrono-oncologic study includes bolus
chronotoxicology and bolus chrono-effectiveness. These types of studies determine
the effect of administration time upon drug toxicity and anticancer activity when
those drugs are given either by intravenous, intraperitoneal or oral bolus. For drugs
which have very short half-lives, or which have more favorable therapeutic indices
when given by infusion, both infusional chronobiological studies need to be performed
as well as bolus studies. Such studies compare the effect of the shape of
circadian-weighted infusions upon both drug toxicity and anticancer activity.
Whereas bolus studies are routinely performed upon mice, infusional studies are
usually performed upon rats because of size-related vascular access problems.
CHEMOTHERAPEUTICS AND CHRONOTHERAPY.
Doxorubicin and its analogs (preclinical data).
Anthracycline antibiotics are among the most active antineoplastic agents in clinical
use today. The most widely used anthracycline, doxorubicin, is a potent therapeutic
agent against a wide spectrum of malignancies, but it causes substantial acute and
chronic toxicity [29]. Profound myelosuppression, stomatitis, mucositis and
gastrointestinal disturbances are commonly observed acute toxic effects [30].
Chronic dosing causes a cardiomyopathy at cumulative doses exceeding 500 mg/m2 [31].
In an attempt to reduce doxorubicin toxicity, new anthracycline analogues have been
synthesized by slightly altering the molecular structure of doxorubicin. Among these,
epirubicin (4'-epi-doxorubicin) differs only from doxorubicin in the epimerization
of one hydroxyl group of the amino sugar moiety. Both the acute toxic effects and
the incidence and severity of cardiotoxicity are, on a molar basis, lower for this
analogue [32]. Despite their structural similarities, epirubicin and doxorubicin
differ in their temporal toxicity pattern as well as in their toxicity pattern. Both
molecules intercalate similarly between DNA base pairs [33], have both a similar
affinity for DNA and comparable cytotoxic effects in vitro [34]. Their
pharmacokinetics differ in that epirubicin is readily converted to epirubicinol,
glucuronides, and aglycone compounds [35], while doxorubicin is prominently
metabolized to doxorubicinol. The plasma clearance, tissue uptake and rate of
catabolism of epirubicin are greater than those for doxorubicin [36], and its
toxicities are proportionately lower on a weight for weight basis.
The first chronotherapy studies using doxorubicin, performed in 1977, revealed that
the rate of tumor shrinkage following doxorubicin treatment of a transplanted
plasmacytoma in rats is dependent upon the time of day that the drug is given. Fastest
shrinkage occurred when the animals were treated with doxorubicin toward the end of
their daily resting span and just prior to usual awakening [37-40]. A series of six
additional studies showed that the lethal toxicities of doxorubicin are circadian
stage-dependent. The circadian stage of maximum doxorubicin tolerance was
coincidentally shortly before normal awakening very near to the timing associated
with maximal anticancer efficacy.
Mormont and coworkers [41] found that administration of 25 mg/kg epirubicin as a
single i.p. injection given at one of six equally spaced circadian stages resulted
in 73% overall mortality from bone marrow and intestinal toxicity in mice. However,
significant differences in the proportion of survivors were found, depending on the
circadian stage of drug administration. Most survivors (54%) were found following
injection at 06 HALO (hours after light onset) and fewest survivors (11.4%) at 18
HALO. This optimal administration time is several hours earlier than for doxorubicin,
and occurs around usual mid-sleep. The same study was repeated four times during
different seasons of the year, and the results were analyzed for circadian and
seasonal variations in toxicity between studies. Significant effects of both
circadian time and season of treatment were noted (circadian timing: F=11.9, p<0.001;
season: F=24.7, p<0.001). Animals treated in late Spring and early Summer had a lower
mortality rate and survived longer than those injected in the Fall or Winter (p<0.01).
Best drug tolerance was calculated to be in July (Cosinor analysis; p<0.001). The
circadian dependency of epirubicin toxicity was observed during all seasons
regardless of the age of the animals used. Levi et al. have tested the impact of
circadian timing upon toxicity for another doxorubicin analog - THP-doxorubicin,
which turned out to be best tolerated in the late rest span [42-43] very near to the
best time for the parent compound. Overall, based upon these data, anthracyclines
should clearly be administered in the last half of the daily sleep span or just prior
to usual daily awakening.
Cisplatin and analogues.
Cisplatin is one of the most active drugs against a large spectrum of common solid
tumors. Its usefulness is limited, however, by serious toxicities including
gastro-intestinal, neurotoxicity, nephrotoxicity and myelosuppression at very high
doses. A variety of analogues have been developed and tested in an effort to avoid
certain cisplatin dose-limiting toxicities while retaining its anti-tumor activity.
Of the many cisplatin analogues developed, carboplatin has proven to be one of the
most clinically useful. Its toxicities differ from cisplatin in that myelosuppression,
especially thrombocytopenia, is dose-limiting while nephrotoxicity is minimal [44].
Another analogue, oxaliplatine, has proven antineoplastic activity in both
experimental models and phase I/II clinical trials, lacks cross resistance to
cisplatin, and demonstrates no significant hematologic or renal toxicity. Nausea and
vomiting are the major dose-limiting toxicities of oxaliplatine. A recently developed
analogue, B-85-0040 cells has reduced nephrotoxicity and lack of cisplatin cross
resistance [45]. Its clinical toxicity and usefulness are still to be determined.
Time-dependent pharmacokinetics.
Underlying mechanisms for circadian changes in cisplatin toxicity include
alterations in drug pharmacokinetics, with significant circadian based variations
in plasma binding and urinary excretion documented for rodents as well as for humans
[46-48]. However, no correlation between oxaliplatine tissue levels and toxicity has
been established [49]. It has been questioned whether circadian differences in stage
of cell division of target cells may play a role in the drugs' circadian toxicity
profile. However, the cell-cycle dependent sensitivity of cisplatin and its analogues
is poorly understood. It appears that some cells are most sensitive to cisplatin when
exposed during the G1 (intermitotic) phase of the cycle, possibly because of the delay
in cross-link formation, which then would be maximal during the following S phase
[50].
Clinical cisplatin pharmacokinetics were studied in patients bearing ovarian or
bladder cancer using an HPLC method for quantitative identification of urinary
cisplatin. The pattern of urinary excretion of cisplatin was studied after 51 courses
of 60 mg/m2 of this agent. Urine samples in which cisplatin was measured were obtained
immediately prior to and every 30 minutes after cisplatin infusion over 4.5 hours.
It was found that urinary cisplatin kinetics (peak concentration, time to peak, area
under the curve) were predictably different depending upon when the drug was infused,
with significantly higher concentrations, and subsequently much greater kidney
damage, arising following morning administration (Figure 7) [22-23].
Murine toxicity studies.
Another case in point is the pronounced circadian rhythm in cisplatin (DDP) lethal
toxicity, which was demonstrated in each of a series of 11 studies over the course
of about one year. Each study entailed injection of six groups of rats with toxic
doses (11 mg/kg) of cisplatin at one of six equispaced circadian stages, and
subsequent observation of the mortality. Each of these studies revealed that
cisplatin was tolerated better when given late in the animal's active phase (Figure
8) [51]. Mortality resulted from nephrotoxicity (Figure 9) and renal damage and was
most extensive in proximal convoluted tubules. A renal tubular brush border lysosomal
enzyme, ?-N-acetylglucosaminidase (NAG), is released into the urine in proportion
to the degree of histologically and chemically confirmed renal dysfunction induced
by cisplatin. This enzyme was present in the urine in normal animals with its baseline
concentration displaying a high amplitude circadian rhythm. When cisplatin was given
at its least favorable time of day, the circadian rhythm of urinary NAG was maintained,
but the mean and peak levels increased five-fold in direct proportion to the
subsequent rise in blood urea nitrogen (BUN). When cisplatin was given at a favorable
circadian time, these groups demonstrated a smaller NAG rise and had little histologic
renal damage and only a small rise in BUN [52-53].
The standard method of minimizing cisplatin nephrotoxicity is to give a pre-treatment
"flush" of saline. Thus, in another series of studies, an intraperitoneal saline load
of 3% body weight was given to or withheld from animals concurrently with cisplatin
at six separate circadian stages [54]. A marked circadian rhythm in the amount of
kidney protection achieved by the fluid load was noted (Figure 10). When cisplatin
or cisplatin-plus saline was given to the animals late in their activity span, a high
degree of protection was found. However, when the saline flush and cisplatin were
given to the animals at the circadian stages associated with early activity, less
effect resulted from the kidney protection regimen. These data indicated quite
clearly not only that the lethal nephrotoxicity of cisplatin was circadian-stage
dependent, but also that the standard method of renal protection (hydration) was
circadian-stage dependent in its ability to decrease cisplatin nephrotoxicity [53].
We have also tested whether cytotoxicity and anti-tumor activity of the cisplatin
analog B-85-0040 are circadian stage dependent. We treated 167 mice with a single
i.p. dose of B-85-0040 at a dose range between 300 and 525 mg/kg at one of six equally
spaced circadian stages. The administration of 300 mg/kg resulted in an overall
mortality of 5%. The best drug tolerance as gauged by weight loss was observed at
14 HALO (p<0.01) (Fig. 11). The administration of 525 mg/kg resulted in an overall
mortality of 84% (range 53-100%; X2=11.5, p<0.05) from bone marrow aplasia and
intestinal damage. The lowest mortality rate and longest survival times were observed
in the groups that had received treatment between 14 and 18 HALO (F=5.4, p<0.001;
Cosinor: p<0.004).
Subsequently, 46 mice were treated at one of 3 different circadian timepoints (0,
8, and 16 HALO) with a single i.p. dose of 300 mg/kg B-85-0040 five days after
inoculation of 1 x 106 L1210 leukemia cells. Kruskal-Wallis lifetable analysis
revealed highly significant differences between the treatment groups (w = 12.2, p
< 0.01). Cure rates were 67% for treatment at 8 HALO, 33% for 16 HALO, and 0% for
0 HALO (Fig. 12). Surviving animals had no evidence of leukemia when autopsied on
the 58th day post-treatment. As circadian stages of maximum toxicity and maximum
anti-leukemic activity differ, optimal drug timing may increase its therapeutic index
of B-85-0040. Studies on drug tissue distribution patterns at 1, 24, and 120 hours
after single dose B-85-0040 injection did not reveal circadian differences that would
explain the above observations. The underlying mechanisms are still being
investigated.
Combined therapy: Doxorubicin with cisplatin and their analogues.
Review of preclinical experiments.
Time-dependent synergistic effects of the anti-cancer drugs doxorubicin and
cisplatin have been demonstrated in tumor-bearing rats. Reduction in tumor size and
in the rate of renal excretion of the tumor marker, Bence-Jones protein, varied
predictably depending upon when these two drugs were given [55]. In these earlier
studies, however, the drugs were tested concomitantly at one of only two circadian
stages (late-rest and late-activity). It was found that animals treated with
cisplatin alone or concomitantly with doxorubicin died quicker than did either
untreated animals or rats treated with doxorubicin alone, indicating that the dose
of cisplatin (6 mg/kg) used for this study was too high. Even so, time dependent,
differential toxicity was clearly observed. Animals treated in late-rest tolerated
the drug treatment far better than did those injected in late-activity. The cause
of death in these studies was related primarily to the bone marrow toxicity of the
anthracycline which was consistent with other observations in the mouse.
Two more complete studies followed these initial investigations, using lower doses
of doxorubicin and cisplatin. Drug effects upon the host and tumor were tested at
6 different circadian stages. These experiments investigated whether circadian drug
timing can optimize the ability of the doxorubicin-cisplatin combination to cure
cancer in a rat model. Study 1 was primarily designed to test the effect of doxorubicin
as a single agent at each of 6 different circadian stages. By contrast, Study 2 was
designed to test the effect of doxorubicin administered only at the best circadian
time in combination with cisplatin at 1 of 6 different circadian stages, in order
to find the most effective circadian-timing of this drug combination. Optimal
doxorubicin/cisplatin timing tripled cure rate of this tumor.
These preclinical data suggested that dosing with doxorubicin and cisplatin should
be separated by about 12 hours, with doxorubicin given in the early morning (e.g.,
0600) and cisplatin given 12 hours removed from this (e.g., at 1800 ) for a patient
on a usual sleep-wake schedule (e.g., sleep from 2200 - 0600 ). It is critical to
point out that this suggested timing of the 2 drugs is by circadian stage, not clock
hour. Thus, a person on a consistently different sleeping schedule (i.e., sleep from
0800 - 1600 if a night-worker) might best receive these drugs at a different clock
hour (i.e., doxorubicin at 1600 and cisplatin at 0400 for the previous example). This
raises many questions about the circadian time structure of shift workers for which
there are less than clear answers. All clinical studies done to date have been
performed upon diurnally active and nocturnally sleeping individuals.
The relative contribution of drug sequence and the span between these two agents to
the schedule-dependent differences in therapeutic index was addressed in the two
studies described above. The pattern of therapeutic advantage across the day was very
similar in both studies, although the sequence of agents, the span between agents,
and the number of courses was different in the two studies. Regardless of these
schedule differences, the same doses of drug were substantially less toxic to the
host, and more effective in controlling the cancer, when doxorubicin was given just
prior to usual awakening and cisplatin was given in mid to late activity.
We have suggested that an appropriate rhythm marker (e.g., temperature, urinary
potassium) might be monitored before, during and after chemotherapy, in order to
ensure synchronization and proper circadian-stage timing of the therapy [56-57]. If
our results are relevant to human oncology, exploitation of circadian and other time
structures for optimal cancer chronotherapeutic schedules should lead to a
significant therapeutic improvement.
Review of clinical data: Studies in patients with ovarian and bladder cancer.
Doxorubicin and cisplatin are the most active drugs in treating several cancer types.
In ovarian cancer, the combination of these drugs has an advantage over single-agent
therapy when considering response rates and survival. Drug dose, to some extent,
determines tumor control [58]; however, only a third of patients with advanced disease
will have a complete clinical tumor response, and an additional third a partial
response. In only a few cases (< 20%) will complete clinical response result in the
absence of microscopic residual disease. Advanced disease is defined as ovarian
cancer metastatic in the abdomen without liver involvement, FIGO* Stage III (clinical
stage grouping for primary carcinoma of the ovary according to the International
Federation of Gynecology and Obstetrics), or distant metastases and/or liver
involvement (Stage IV). Most patients relapse and have only median survival times
between 10 and 36 months [59]. These disappointing results make any possible
improvement of therapy very urgent.
Metastatic bladder cancer is even more difficult to treat effectively. However,
chemotherapy combinations and schedules including the combination of doxorubicin and
cisplatin have emerged recently that can result in complete responses in some patients.
Response rates, response durations and survival patterns of the entire patient
population have, however, remained unsatisfactory. Higher dosages are associated
with better response rates but also with substantial toxicity; several adjuvant
studies have demonstrated an increase in length of disease-free survival for
chemotherapy-treated patients when compared to those who were observed following
operation without treatment [60].
Clearly, our goal in chronotherapy protocols for each of these diseases was to reduce
treatment-related toxicity and complication rates by optimal circadian drug timing,
allowing high-doses of drug to be administered safely and most effectively. With
optimal treatment timing, we also expected improved tumor control and patient
survival.
Toxicity study with crossover design.
Treatment plan: The first clinical study was performed to test two different circadian
time schedules of the same combination of doxorubicin and cisplatin with equal doses,
drug sequence
and interval, for possible pharmacokinetic and toxicity differences between the two
agents in the same patients treated at different times of day. More than 100 monthly
treatment courses consisting of doxorubicin at 60 mg/m2 and cisplatin at 60 mg/m2
were studied in 23 patients. This clinical protocol randomized initial doxorubicin
treatment time between 0600 and 1800. Cisplatin followed each doxorubicin infusion
by 12 hours. Each drug was infused over 30 minutes. A standard vigorous hydration
protocol of 4100 mL of normal saline (20 mEq KCl per liter) preceded and followed
each cisplatin infusion. Antiemetics and diuretics were not used. After the initial
treatment, the timing of doxorubicin for each subsequent cycle was alternated between
0600 and 1800, so that the drug timing was crossed-over throughout the study.
Twenty-one patients were considered evaluable since two patients refused further
therapy after the initial treatment. Each of these 21 patients had advanced malignancy
(12 had stage III and IV ovarian cancer and 9 had metastatic D2 transitional cell
cancer of the bladder). To assure precision, each patient was treated in a general
clinical research center metabolic ward.
Cisplatin-induced nephrotoxicity: A statistically greater per course drop in
creatinine clearance followed morning cisplatin administration compared to evening
administration (Figure 13). This difference was most striking following the first
course and then diminished as treatment time was alternated. There was either no
creatinine clearance decline or a permanent 30% fall following the first dose of
cisplatin depending upon when the cisplatin was given.
Bone marrow toxicity: When doxorubicin was given at 0600 and cisplatin at 1800, there
was less neutropenia and thrombocytopenia than when the doxorubicin was given at 1800
followed by cisplatin at 0600. The morning doxorubicin schedule resulted in
statistically significantly less depressed low counts and in full recovery of all
counts to pretreatment levels, usually within 21 days of treatment, while evening
doxorubicin led to less than full recovery, even after 28 days following therapy.
This is demonstrated in the pattern of the fall and recovery of leukocytes,
neutrophils, and platelets in an individual treated four times on one circadian
schedule and four times on an opposite circadian schedule (see Figures 14,15; shading
represents standard errors of counts). The clinical relevance of these findings is
demonstrated by the fact that treatments given with morning doxorubicin resulted in
statistically significantly fewer dose reductions and fewer treatment delays and
fewer serious treatment-related complications than found with the opposite circadian
drug schedule.
Cisplatin-induced nausea and vomiting: The most common reason for the discontinuation
of cisplatin treatment is the patient's refusal to accept further therapy because
of the severe nausea, vomiting and anorexia that it causes in nearly all cases. Until
recently, no antiemetic regimen had proven effective in eliminating this often
dose-limiting toxicity. Nausea and vomiting were studied quantitatively in 101
courses of combination doxorubicin and cisplatin chemotherapy administered without
antiemetics. Those patients who received cisplatin at 0600 had more vomiting episodes
(P < 0.01), which tended to begin sooner and last longer [61]. See Figure 16.
Randomized non-crossover study: Cumulative drug toxicities and efficacy.
Treatment plan: In the subsequent protocol, patients were randomized to receive each
of the nine planned doxorubicin-cisplatin treatments starting always at 0600 (morning)
or 1800 (evening). This fixed random assignment of circadian treatment stage allowed
analysis of the effect of drug timing upon all acute and cumulative drug toxicities,
as well as upon the effect of circadian schedule on quality of tumor response (partial
and complete response rate), time to response, response duration, patient survival,
and cure rate. Circadian Schedule A was morning doxorubicin followed by evening
cisplatin, and Schedule B was evening doxorubicin followed by morning cisplatin.
Bone marrow toxicity: Complete evaluation of the bone marrow toxicity of the first
37 patients who received all of 9 planned treatments revealed that the circadian stage
of chemotherapy administration determines whether or not this combination of drugs
induces cumulative bone marrow toxicity. Because of leukopenia, most patients treated
on Schedule B had to have greater than 33% doxorubicin dose reduction and many of
them had to have treatment delays of greater than two weeks as opposed to those on
Schedule A. Assessment by linear regression analysis of individual WBC decrease and
recovery (on days 1, 7, 14, and 28) after treatment revealed more cumulative bone
marrow toxicity for the majority of the patients treated on circadian Schedule B than
for Schedule A, despite substantial dose reductions.
Cisplatin-induced nephrotoxicity: Patients bearing cancer, or with other serious
illnesses, may not be precisely synchronized enough with regard to circadian rhythms
that are important in determining the amount of drug toxicity the patient might
experience. In order to investigate this finding more thoroughly, the circadian
rhythm characteristics of body temperature, neutrophil count, lymphocyte count,
heart rate, blood pressure and urinary volume, sodium, potassium and cortisol
excretion were studied. Forty-three patients were studied in this way prior to 295
separate treatment courses. Creatinine clearance fall after each treatment was then
compared. Less nephrotoxicity was seen when cisplatin was given at 1800, as compared
to 0600. For 24 to 48 hours prior to each treatment, urine was collected every two
hours and the rate of potassium excretion determined. Each individual's circadian
rhythm in urinary potassium excretion (expressed as mEq per hour) was calculated for
each course. The amount of subsequent renal damage was assessed by the creatinine
clearance decrease prior to the next course of treatment. Mean creatinine clearance
decrement results were also compared according to how far from the daily potassium
peak excretion that patient had, in fact, received the cisplatin. Creatinine
clearance results were analyzed according to whether cisplatin was received 0 to 6
hours or 6 to 12 hours after the daily peak in potassium excretion. This procedure
compared treatment times as gauged by a measure of internal, rather than external
time. Patients who were treated within three hours on either side of the span during
which their rate of potassium excretion was highest suffered no subsequent loss of
renal function, while those patients receiving cisplatin farthest away from the time
of highest potassium excretion had an average loss of 8 mL/min. in creatinine
clearance per treatment course. Since the standard treatment course of cisplatin for
this group of patients included nine courses of therapy, inopportune timing of
repeated cisplatin administration resulted in a substantial and preventable loss of
kidney function of more than 50%.
Other toxicities: neurotoxicity, chronic anemia, and transfusion requirement were
each statistically significantly different in favor of morning doxorubicin and
evening cisplatin [62].
Circadian schedule dependence of toxicity and dose intensity. Toxicity evaluation
following each of the 247 evaluable treatment courses included weekly 8 a.m. sampling
of hemoglobin, total and differential white blood cell count, platelet count and
creatinine clearance. These weekly laboratory values, combined with a monthly interim
history and physical examination, served to guide dose and schedule modifications.
Doxorubicin dose modifications or schedule delays were forced by three types of events.
These were1) a recovery (day 28) absolute granulocyte count below 1500 cells/mm3;
or 2) a recovery platelet count under 100,000 cells/mm3; or 3) interim infection or
bleeding If any of these conditions were present, a 25% doxorubicin dose reduction
or one-week treatment delay with subsequent re-evaluation was instituted.
Doxorubicin doses were more often reduced if an infection or bleeding complication
supravened, and treatment delays were more common with a poor recovery of blood cell
counts. No dose or schedule modifications were instituted on the basis of low counts.
Cisplatin was discontinued if creatinine clearance fell below 30 mL/min., but
otherwise given at full dose. Treatment complications were defined as: interim
clinical infections that required oral or parenteral antibiotics; interim bleeding
episodes of any kind, whether or not platelet transfusions were administered; and
anemia requiring a transfusion. Each transfusion episode usually required
administration of two or three units of packed red blood cells. The rates of
chemotherapy-related toxicity following either treatment schedule were calculated
per patient group and per treatment courses. The results are shown in Figures 17&18,
clearly indicating the profound influence of the time of day of chemotherapy upon
drug toxicity and maximum dose intensity.
Circadian dependence of tumor response and patient survival in ovarian cancer.
Sixty-three consecutively-diagnosed women (median age 60, range 29 to 87) with FIGO
Stage III [48] and IV [19] epithelial ovarian cancer were treated using one of 4
temporal schedules of the same two-drug protocol (60 mg/m2 of both doxorubicin and
cisplatin every 28 days for 9 months) to test whether drug timing affected tumor
control. Fifteen of these 63 women had optimal debulking operations and 48 had bulky
disease with residual masses (massive disease of > 10 cm masses in 40 patients). Sixty
of these 63 women were evaluable for response and survival. Each of the four treatment
groups was comparable with regard to patient age, FIGO stage, histological grade of
cancer, and quality of debulking surgery. Of these evaluable patients, 16 women had
received prior chemotherapy and 9 had prior abdominal pelvic irradiation. The four
different schedules were: U: (n=11) treatment with doxorubicin and cisplatin at
unspecified times of the day with no consistent sequence or interval between drugs.
A: Randomization to receive doxorubicin given at 0600, followed 12 hours later by
cisplatin. B: (n=20) Doxorubicin given at 1800, followed 12 hours later by cisplatin.
A/B: (n=12) Patients alternated monthly on A and B. Tumor responses are shown in Table
1.
Circadian scheduling significantly increased clinical complete response (CR) rates
(X2 = 38.8, P < 0.001) and survival. Median time of observation of all patients
exceeded 67 months (16-105 months). Pathological complete response rates were also
higher with chronobiological administration. All patients treated without regard to
drug timing (Schedule U) died within 3 years. Patients receiving Schedule A had a
5-year survival of 44%. This survival was exceeded by patients treated using the
crossover timing regimen. Patients treated on Schedule B had an 11% 5 year survival
(Figure 19).
Part of this schedule-dependent difference in patient survival may have been related
to differences in average dose intensity. Mean dose intensity (as % of planned dose)
of each agent for Schedule B (80% for doxorubicin and 82% for cisplatin) was
statistically significantly lower than for Schedule A (94% for doxorubicin and 95%
for cisplatin) and for the crossover regimen A/B (89% for doxorubicin and 90% for
cisplatin). However, patients on Schedule U received the drugs at the same or higher
dose intensity (94% for doxorubicin and 103% for cisplatin) as patients treated with
Schedule A or A/B, but their drug schedule failed very rapidly. While a difference
of 10-15% in dose intensity does not appear important, it must be kept in mind that
the relationship of drop in dose intensity to drop in disease control and cure rate
may not be linear. It can be expected from work by Levin and Hryniuk [63] that a drop
of 10% in dose intensity may result in a 50% drop in the ability of that regimen to
control cancer. Therefore, this statistically significant toxicity-related drop in
dose intensity may be important to our findings that timing of doxorubicin-cisplatin
administration markedly affects patient survival. The observation that unspecified
treatment timing results in relatively high dose intensity, but poor disease control,
also needs additional comment. That is, the dose intensity formula does not take into
account the possibility of early treatment failure. In reviewing the mean number of
courses for patients receiving treatment at unspecified times and those receiving
time-specified treatment, it is clear that early failures occurred much more
frequently in the group receiving treatment at unspecified times. In fact, the mean
number of treatment courses in this group is only a little more than three, whereas
8-9 treatment courses were given using all time-specified treatment regimens. The
other conclusion that we must reach about dose intensity and circadian timing is that
while dose intensity is important in determining how well a combination of treatment
controls cancer, there may also be effects of timing that are separate from and
additive to dose intensity effects. In any event, it is clear that dose intensity
can be maximized by appropriate circadian treatment timing, and that optimal
treatment timing results by whether mechanism(s) in prolongation of patient survival
in advanced ovarian cancer.
Circadian-stage dependent doxorubicin/cisplatin treatment for transitional cell
bladder cancer.
The same two-drug combination of doxorubicin and cisplatin was given to patients with
transitional cell carcinoma of the bladder. Patients were randomized to receive the
drugs at a dose of 60/m2 each at monthly courses following Schedule A or Schedule
B as outlined above. Forty-three consecutively-diagnosed patients with widely
metastatic cancer received up to nine monthly courses of the two-drug combination,
followed by cyclophosphamide, and 5-fluorouracil together with cisplatin as
maintenance for up to two years. Fifty-seven percent of the 35 evaluable patients
had objective response and 23% had complete clinical responses to the treatment.
Median survival from the first treatment for complete responders was more than two
years, and was one year for partial responders. Three of the complete responders were
alive without evidence of cancer more than two years after stopping all therapy.
Our chronotherapeutic approach safely allowed application of high dose intensity
treatment. The stipulation covering the order of the drugs, interval between them,
and the circadian time may have been favorable factors for this treatment's success,
and which compares very favorably to other chemotherapy regimens reported to date.
The fact that three patients with biopsy-proven metastatic transitional cell
carcinoma of the bladder have been taken off all therapy without disease recurrence
may suggest the eventual chemotherapeutic curability of this disease, which was not
possible before. Unfortunately, the numbers of patients per treatment group did not
allow interpretation of schedule-dependent differences in drug efficacy. Toxicity
evaluation confirmed, however, that Schedule A was superior to Schedule B with lower
toxicity in spite of higher dose intensity.
Adjuvant chronotherapy for transitional cell bladder cancer.
Finally, the same two-drug combination was given to 16 patients with transitional
cell carcinoma to the bladder, and who received chronotherapy monthly either on
Schedule A or Schedule B immediately after radical cystectomy. In 5 patients, cancer
had penetrated through the serosa of the bladder wall into the perivesical fat (stage
C). In 11 patients, cancer had spread further to other pelvic organs and pelvic lymph
nodes (stage D1). Eleven of these 16 patients showed no recurrence of the disease
after a median follow-up time of 3.5 years (range of 1 to more than 5.5 years). Two
of the 5 patients who ultimately failed the treatment becuase of its toxicity had
local tumor recurrence that developed much later than is usually the case (at 37 and
42 months). The circadian-timed drug regimen, given in full doses for nine courses
as adjuvant treatment, delayed and possibly prevented local and distant recurrence
of the stage C and D1 bladder cancer which can otherwise be expected in more than
90% of patients within two years of the surgery [64]. Again, similar differences in
schedule-dependent toxicity were observed, but the number of patients per treatment
was too low to properly test for differences in antitumor activity.
Circadian timing of THP-doxorubicin with cisplatin.
Levi and coworkers [65] reported the efficacy and toxicity of the new anthracycline,
4'-0-tetrahydropyranyl doxorubicin (THP) (50 mg/m2 intravenous bolus) in association
with cisplatin (100 mg/m2 as an intravenous 4-hour infusion) in 31 patients with
advanced ovarian carcinoma. Twenty-eight patients were assessable for toxicity, 25
for response. Nine patients had received prior treatment. Patients were randomized
to receive schedule A (THP at 6 hours, then cisplatin from 16 to 20 hours) or schedule
B (THP at 18 hours, followed by cisplatin from 4 to 8 hours). Schedule A was
hypothesized as less toxic since THP was best tolerated in the late rest span and
cisplatin near the middle of the activity span in experimental studies [42-43]. The
rate of clinical complete responses was 52%, that of partial response was 12%. The
overall clinical response rate was 64% (schedule A 73% and schedule B 57%). The
progression-free survival and overall survival times were 10 and 19 months,
respectively. Schedule A was associated with less neutropenia (p=0.1),
thrombocytopenia (p<0.01), anemia (p<0.01), and renal toxicity (p<0.05) than
schedule B. Of four patients withdrawn for toxicity, three were on schedule B (one
death). Mean dose intensities of THP and cisplatin decreased by 30% and 47% over the
five initial courses, respectively. Such decrease was significantly more pronounced
for schedule B than for schedule A in previously untreated patients (p<0.01). The
authors concluded that THP-cisplatin toxicities can be significantly decreased by
dosing THP in the early morning and cisplatin in the late afternoon as compared to
the opposite times of drug administration for both drugs. These findings confirm our
earlier results, described above.
Mechanisms of circadian drug pharmacodynamics of doxorubicin and cisplatin.
Doxorubicin-related heart muscle and bone marrow toxicities and pharmacodynamics are
believed to be a result of its NADPH-dependent single electron reduction to a reactive
metabolite. In both mice and man, the availability of a key free radical scavenger,
reduced glutathione (GSH), is circadian-stage dependent, with peak levels at the time
of lowest drug toxicity [16,66]. Tumor response, however, occurs mostly secondary
to drug binding to DNA intercalation and single strand breaks. Both circadian changes
in cell cytokinetics (e.g., rate of cells in S-phase DNA) and tissue-specific
differences between tumor and normal organs may have determined the observed
circadian differences in doxorubicin therapeutic index. Furthermore, circadian
changes in drug pharmacokinetics may have influenced the toxicity rhythm.
Both the normal kidney function and cisplatin pharmacokinetics are circadian-stage
dependent [67,68]. Pronounced circadian rhythmicity in lethal toxicity due to
cisplatin was demonstrated in each of a series of eleven studies over a course of
about one year. Each study entailed injection of six groups of rats with a toxic dose
(11 mg/kg) of cisplatin at different circadian stages and subsequent observation of
the mortality. It was found that cisplatin was tolerated far better when given late
in the animals' active phase [52]. It was also shown that mortality resulted from
nephrotoxicity. This was proven by monitoring blood urea nitrogen (BUN) and by
microscopic section of the severely damaged kidneys. Kidney damage was most extensive
in the proximal convoluted tubules. A brush border lysosomal enzyme, NAG, was found
to be released into the urine proportionately to the degree of renal dysfunction
induced by cisplatin. This enzyme was present in urine in normal animals, and its
baseline concentration was found to display a high amplitude circadian rhythm. When
cisplatin was given at the time it was most toxic, the circadian rhythm in urinary
NAG was maintained, but the mean peak levels increased 5-fold in direct proportion
to the subsequent rise in BUN. If cisplatin was given at a favorable circadian stage,
these rats did not demonstrate much NAG rise and had little renal damage with only
a small rise in BUN.
Mortality from doxorubicin, daunomycin, epirubicin and cisplatin is also influenced
by a non-trivial seasonality. For cisplatin, the extent of predictable seasonal
variation in the mean survival duration of rats after drug injection was about 40
percent [69]. These findings reflect the importance of circannual rhythms at all
levels of biologic organization. It is especially interesting that these circannual
susceptibility rhythms are found in laboratory animals kept under constant conditions
[70]. It is not unreasonable to expect that they may be greatly amplified in
free-living animals.
Fluoropyrimidines.
Trials of mechanistic importance.
DNA synthesis is interrupted because of the biding of FdUMP to thymidylate synthase
(TS). This binding is tightened by intracellular folate which complexes with the
FdUMP/TS. RNA production is affected because of the incorporation of the
fluoropyrimidine triphosphate into abnormal RNA.
Fluoropyrimidines anti-metabolites interfere with DNA, RNA, and protein synthesis.
They are widely used cytotoxic drugs. The toxicity pattern is dependent upon dose
and administration mode (e.g., bolus injection or continuous infusion).
Myelosuppression, stomatitis, and intestinal toxicity are dose limiting, and we have
found that circadian timing of these agents markedly affects their toxic/therapeutic
ratios.
Animal trials.
DPD-activity in liver cells.
The activity of dihydropyrimidine dehydrogenase (DPD), the initial, rate-limiting
enzyme in fluoropyrimidine catabolism to non cytotoxic products, was measured by
Harris et al. [71] at various times over a 24-hr period in the livers of male
Sprague-Dawley rats housed under standardized conditions of light and dark. Under
"normal" conditions, i.e. lights on from 6:00 AM to 6:00 PM and off from 6:00 PM to
6:00 AM, a circadian rhythm of DPD activity was observed (p<0.0001, cosinor analysis)
with the peak of activity at 4:00 PM or 10 HALO (2.96 nmol catabolites/min/mg) and
the trough at 4:00 AM (0.40 nmol catabolites/min/mg). Maximum enzyme activity
exceeded minimum activity more than 7-fold. Reversing the light-dark cycle resulted
in a corresponding shift in enzyme activity.
Catabolism of FU in the isolated perfused liver of the rat.
The same group subsequently measured the catabolism of 5-fluorouracil (FU) in an
isolated perfused rat liver model (IPRL) at various times of the day [72]. IPRLs were
prepared from rats sacrificed at 3-hour intervals and the elimination rate of FU and
FU-catabolites (i.e., rate leaving the IPRL in the effluent perfusate) following
infusion of FU was analyzed for circadian periodicity. Animals were housed under
standardized conditions of 12 hours of light and 12 hours of dark. A significant
circadian rhythm was observed in the elimination rate of FU and FU-catabolites, with
40-60% variation around the mean (p<0.001, cosinor analysis). Under "normal" light
conditions, the peak and trough elimination rates of FU were at 19 HALO and 7 HALO,
respectively. There was a reciprocal relationship between the elimination rates of
FU and FU-catabolites (Table 2). Under the reverse light-schedule conditions, the
rhythms were also reversed. Such a variation in the hepatic elimination rate of FU,
if also present in humans, could result in a variation in the systemic level of drug
during chemotherapy, thus affecting the therapeutic efficacy of FU.
Time dependent drug interconversion.
We adapted 60 female F344 Fischer rats to a 12:12 hour light:dark schedule. They were
kept in fully climatized, sound-proofed vaults with food and water freely available.
Continuous infusion was achieved by cannulation of the tail vein with a catheter,
by the method described by Danhauser et al. [73] They were randomized to receive 350
mg/kg FUDR as a 6-hour constant rate i.v. infusion by Intelliject pump from 10-16
or 22-04 HALO, the most or least toxic circadian stages found in our previous rat
toxicity studies. Blood was collected at 0, 2, 4, and 6 hours of infusion and at 5,
10, 15, and 30 minutes thereafter (4 samples per timepoint). FUDR plasma levels, as
well as FU plasma levels (as FUDR is converted into FU), were determined by
gas-chromatography/mass-spectrometry (GCMS) with a sensitivity limit of 1ng/ml
(Table 3).
The maximum plasma concentration (Cmax) of FUDR occurred at the end of infusion; Cmax
of FU was 10-15 min. delayed and was more than twice as high at 10 to 16 HALO as compared
to 22 to 04 HALO infusion (t=1.3, p=0.2). The proportion of FUDR converted into FU
was greater after infusion given between 10 and 16 HALO (48 vs 25%; t=1.4, p=0.2).
These findings suggests that the activity of the converting enzyme DPD is circadian
stage dependent. The half-life (t1/2) of FUDR was prolonged at 16 HALO (t=1.04, p=0.3)
(Figure 20). This pilot study suggested alterations in FUDR catabolism at two
different circadian stages. Possibly due to small sample size, they did not reach
statistical significance. Because only two circadian stages were tested in this study,
the maximum circadian variation may occur at different stages and may be far greater.
Human trials.
Circadian changes in DPD activity in human mononuclear cells.
Tuchman et al. [74] studied 3 healthy male and 4 female, 24 to 41 year old volunteers.
All 7 individuals had typical sleep and activity patterns and shared a similar diet
during testing. Peripheral blood was drawn every 3 hours (5 cases) or every 6 hours
(2 cases) for 24 hours. Enzymatic DPD activity in cytosolic preparations of
mononuclear cells was assayed with high performance liquid chromatography (HPLC) and
radioactivity flow monitoring. The specific activity of DPD was clearly
circadian-stage dependent. Each individual showed as little as a 38% and as great
as a 99% range of values throughout the 24-hour observation span. The peak enzyme
activity was consistently located around midnight for each subject and rhythmometric
analysis described a statistically-significant group rhythm with predicted time of
peak values occurring between 10 PM and 2 AM. The variation around mean ranged from
20 to 60.8%.
These findings were independently confirmed by Harris et al. [75], who simultaneously
determined the activity of DPD in peripheral blood mononuclear cells and plasma
concentration of FU in cancer patients receiving FU by protracted continuous infusion
(300 mg/m2/day over several weeks). Blood samples were drawn every 3 hours over a
24-hour period, after the patients had received continuous FU infusion for more than
2 weeks. The resulting DPD and FU values were
analyzed for circadian periodicity. In the 7 patients studied, a circadian rhythm
of DPD activity was observed (p < 0.00001, cosinor analysis) with the peak of activity
at 1 a.m. (0.20±0.01 nmoles/min/mg) and the trough at 1 p.m. (0.11±0.01
nmoles/min/mg). In addition, a circadian rhythm was observed for the plasma
concentrations of FU obtained over a 24-hr period (p < 0.00001, cosinor analysis)
with peak values (27.4±1.3 ng/ml) occurring at 11 a.m. and trough values (5.6±1.3
ng/ml) occurring at 11 p.m. The ratio of the maximum concentration of FU to the minimum
concentration observed was almost 5-fold. These studies demonstrated a circadian
variation of DPD activity in human peripheral blood mononuclear cells and a circadian
variation of FU plasma levels in patients receiving the drug by protracted continuous
infusion. They also revealed an inverse relationship between DPD activity in
peripheral blood mononuclear cells and plasma FU concentration.
Circadian variation of FU plasma level in patients receiving combination
chemotherapy.
Petit et al. [76] demonstrated a circadian rhythm in the plasma concentration of FU
in seven patients with bladder cancer receiving this drug as a continuous venous
infusion at a constant rate for 5 days in combination with cisplatin. Cisplatin (45-91
mg/m2) was administered on day 1 as a 30-minute i.v. infusion at 5 PM. The FU infusion
(450-966 mg/m2/day) was started on day 2 at 8:30 AM via a volumetric pump, and lasted
for 5 days (until day 6). Blood samples were obtained every 3 hours on days 2, 4 and
6 for each patient (20 samples/patient). FU plasma concentration was determined by
HPLC. Mean lowest and highest values (±SE) were, respectively, 254±33 ng/ml at 1
p.m. and 584±160 ng/ml at 1 a.m. (F=2.3; p<0.03). Because of large inter-subject
differences in 24-hour mean plasma concentration, data were also expressed as
percentages of each patient's 24-hour mean. Both analysis of variance and cosinor
analysis further validated (p<0.0001) a circadian rhythm with a double amplitude
(total extent of variation) of 50% of the 24-hour mean and an acrophase located at
1 a.m. (estimated time of peak). This peak occurred at a different time as compared
to Harris' [75] patients. However, drug schedules and doses differed greatly in the
two trials. The cisplatin chemotherapy, which preceded the FU infusion, may have
affected the rhythmic cycle of FU plasma levels in Petit's study [76]. Table 4
summarizes the clinical data on circadian changes in DPD activity and corresponding
FU plasma levels.
Review of preclinical chronotoxicological experiments.
5-Fluorouracil.
Lethal toxicity. Although fluoropyrimidines are now commonly used by long-term
infusion and, although the toxicity pattern of bolus and infusional administration
are disparate, most screening toxicology has been done using bolus administration.
Several investigators have tested whether FU toxicity is circadian stage dependent.
Burns and Beland [77] used a dose escalation protocol in male CD-1 mice standardized
to a 12:12 hour lights on:off schedule. The animals were randomized to receive the
treatment at one of two circadian stages: 5 HALO (mid-sleep); or 17 HALO
(mid-activity). They found drug tolerance to be significantly different at these two
times. The single intra-peritoneal (i.p.) bolus dose of FU killing 50% of the animals
(LD50) was significantly higher at 5 HALO (450-500 mg/kg) than at 17 HALO (250-300
mg/kg). Nearly a 2-fold difference in lethal toxicity, depending upon which time of
day the FU was given, was found.
Similar observations were made by Popovic et al. [78] who studied the tolerance of
female C3H mice for single i.p. FU bolus injections of 200 mg/kg at one of six different
circadian stages (lighting schedule not given). The best drug tolerance with 30%
mortality was at "mid-rest phase of the animals," corresponding to 5-7 HALO.
One-hundred percent mortality was observed for treatment during the late activity
phase of these nocturnally active animals' circadian cycle (approximately 20-22 HALO).
These two studies each showed that the best timing for FU was in the rest phase several
hours prior to usual awakening.
Gonzalez et al. [79] tested lethal toxicity of single i.p. FU bolus injections and,
concurrently in a second experiment under exactly the same conditions, that of
5-fluoro-2'-deoxyuridine (FUDR) i.p. bolus injections at one of six different
timepoints in Balb-C mice, standardized to a 12:12 hour lights on:off schedule. Lethal
toxicity at an FU dose of 150 mg/kg i.p. was significantly different dependent upon
its circadian treatment time. Maximum lethality was 100 % at 2 HALO; minimum mortality
was 13 % at 10 HALO (analysis of variance (ANOVA), F=11.9, p<0.001; Kruskal-Wallis
life-table analysis, w=38.9, p<0.001}. These 6-timepoint studies demonstrated that
the least toxic time of day for FU was two hours prior to usual daily awakening. These
3 studies performed at separate locations are quite consistent in their findings with
regard to the optimal time of bolus FU to lower serious toxicity. FU should be far
safer if given by bolus between mid-sleep and 2 hours prior to usual awakening.
Bone marrow toxicity and anticancer activity. The time-dependencies of specific
non-lethal fluoropyrimidine toxicities have also been investigated. Peters et al.
[80] tested the related anti-tumor and anti-bone marrow activity of FU in
transplantable mouse-colon carcinomas Colon-26 in Balb-C mice and Colon-38 in C57Bl/6
mice, standardized to a 14:10 hours lights on:off schedule. The animals were
randomized to receive multiple i.p. doses at either early rest or late rest phases
of their circadian cycle (2 or 12 HALO) (Balb-C: 100 mg/kg/day on days 0,7, and 14;
C57Bl/6: 60 mg/kg/day on days 0 and 7 for toxicity evaluation and on days 0,7,14,
and 21 for anti-tumor activity testing). Unfortunately each of these two test times
missed what would have been predicted to be the most and least toxic times of day
according to the work of the above 3 predecessors. Increased anti-tumor activity was
none the less reproducibly found at 2 HALO compared to 12 HALO, especially against
Colon-38. Hematotoxicity was also evaluated in C57Bl/6 mice for the same circadian
stages. Leukopenia was only observed after treatment at 12 HALO but not at 2 HALO.
Therefore, the therapeutic efficacy of bolus FU was markedly improved by treating
at 2 HALO. This is consistent with a safer and more effective treatment timing prior
to usual daily awakening.
Intestinal toxicity. Gardner and Plumb [81] used Wistar rats standardized to a 12:12
hour lights on:off schedule for toxicity studies at multiple circadian stages. Food
and water were freely available. The impairment of water absorption by small intestine
in vitro, and the incidence of diarrhea in vivo were assessed after the same single
i.p. dose of FU (187 mg/kg) as a function of circadian treatment time. Their findings
were correlated to deoxyribonucleic acid (DNA) content of small bowel epithelium
homogenate at the same circadian stage of treatment. Impairment of water absorption,
the incidence of diarrhea, and weight loss, were minimal after treatment at mid-daily
activity (19 HALO). The DNA content of mucosa of the small intestine also showed marked
diurnal variation, with a maximum at 20 HALO. During this stage, the maximum number
of cells are in the post-mitotic resting (G1) phase, and are less susceptible to the
effects from the antimetabolite FU. It is noteworthy that diurnal variations in
various intestinal absorptive and enzymatic activities and in the proliferative state
of the epithelium have been shown to be related to the feeding time in rats; the lowest
DNA synthesis rate appears to occur at or after feeding. Table 5 gives an overview
of FU circadian pharmacodynamics. The mid-activity optimum for the intestine to
tolerate bolus FU is several hours later in the day than is the optimum time for whole
animal toxicity and lethality. This observation requires further study in both fasted
and fed animals and in single and multiple dose regimens. It raises, however, the
possibility that optimal times may be different for different target tissues as well
as for different modes of fluoropyrimidine delivery.
Recent FU chronotoxicological study. In our recent trial we have studied the
time-dependent gastrointestinal, bone marrow and overall toxicity of 5FU in 150 CD2F1
female mice. We have administered 200 mg/kg FU i.v. as a bolus injection at one of
six equispaced circadian timepoints to each animal. We have found that toxic effects
were significantly dependent upon circadian treatment time.
Maximal mortality (70%) took place in mid-activity (considered as a time of maximal
DNA synthesis [77]), while 100% of animals treated in an early rest have survived
the FU treatment (p<0.001). These data correspond to the evaluation of toxicity by
body weight loss and recovery following FU administration, calculated as percentage
of initial body weight for each animal. The largest loss of body weight was observed
during mid-activity, while the most stable weights and the best recoveries were
observed at the mid-rest (considered as time of minimal DNA synthesis) [77] and at
an early rest time of a day (p<0.001) respectively.
Circadian dependency of gastrointestinal toxicity was evaluated on the basis of
weight of feces collected within 24-hour periods. Lowest values, suggesting lower
food consumption and impairment of the daily production of stool, occurred when
animals were treated during their late and mid-activity phases, while treatment
earlier in the daily activity span resulted in the lowest decrease in weight of stool
( p<0.001).
Analysis of data regarding bone marrow toxicity was based on an estimation of white
blood cells levels. This showed significant circadian timing-dependent differences.
White blood cells remained almost untouched after treatment in mid-rest and at
mid-activity spans. Recovery at these two timepoints was also excellent. The deepest
depression of white blood cell count was observed when the drug was given in the
late-activity span (22 HALO; p<0.001).
FUDR.
FUDR lethal toxicity. In a series of murine experiments we explored whether FUDR
toxicity and efficacy are circadian stage-dependent. Single bolus FUDR injections
of 1 of several doses between 1000 and 2000 mg/kg were given at 1 of 6 equally-spaced
circadian stages to more than 300 CD2F1 mice standardized to a 12:12 hours light/dark
schedule. Food and water were provided ad libitum. Temperature and humidity were held
constant in sound-proofed vaults. Animals were followed for survival every 4 hours.
Survival varied predictably by more than 50%, depending upon the circadian stage of
injection. Treatment during mid-to-late activity span (18-20 hours after light onset
= HALO) was consistently associated with best drug tolerance in three studies.
To identify the cause of death in these animals, we performed autopsies, macroscopic
inspections, and routine light microscopy of slides prepared from liver, small bowel,
colon, and bone marrow. Pathological changes were found in both small and large bowel,
explaining diarrhea and dehydration prior to death. Diffuse necrosis in the liver
and bone marrow aplasia were also present.
Gonzalez et al. [79] confirmed that lethal toxicity of 2000 mg/kg i.p. FUDR by single
bolus injection in female Balb-C mice is significantly different dependent upon the
circadian treatment time. In that study, minimum mortality was 13 % at 2 HALO (ANOVA,
F=2.69, p<0.03; Kruskal-Wallis life-table analysis, w=15, P<0.01); this is
approximately 6 hours later than the optimal time observed in the studies described
above in CD2F1 mice. However, it was also low with 20% mortality at 18 HALO, the least
toxic stage in our previous studies. Table 6 summarizes all the similarities and
difference in the results of all toxicity studies using FUDR by single bolus injection.
Figure 21 shows percent of mortality in six groups of animals treated in different
times of the day. Overall, drug tolerance was consistently better when FUDR was given
during the late activity/early rest phase of the recipient.
It must be concluded that the optimal circadian timing for bolus FU and bolus FUDR
is not identical. It is safest to give FU by bolus some time after mid-sleep and prior
to usual awakening, while it is safest to give bolus FUDR late in the daily activity
span prior to sleep onset. While the two drugs are related to one another and
inter-convertible it should not be unexpected that their optimal administration times
differ substantially. Different metabolic pathways are responsible for activation
of FU and FUDR, and the activity of regulating enzymes may follow different temporal
patterns.
Fluoropyrimidines are frequently given by continuous infusion. In order to test if
circadian drug timing is also important for toxicity and anti-tumor activity when
5-fluoro-2'-deoxyuridine (FUDR) is given by continuous infusion, we compared seven
equal dose FUDR daily infusion patterns in female Fisher 344 rats [82,83]. For the
variable rate infusion, the daily dose was divided into four 6-hour portions of 68,
15, 2, and 15% to achieve a quasi-sinusoidal pattern. Drug was delivered either by
constant rate infusion or by variable rate infusion with peak drug delivery at one
of six different times of day. At a dose level resulting in 50% overall mortality,
lethal toxicity differed substantially depending upon the circadian stage of maximum
drug delivery. Variable rate infusions were more, less, or equally toxic compared
to constant rate infusions, depending upon the circadian stage of maximum drug flow.
FUDR lethality was lowest when variable rate infusion peaked from 22-04 HALO during
the late activity/early rest span of the recipients (Figure 22). This is largely
consistent with earlier bolus studies, but the best time for drug tolerance was
slightly later. The circadian pattern of variable rate infusion also determined the
antitumor activity in adenocarcinoma bearing rats. At a therapeutic dose level, and
at identical dose intensity, the variable rate infusion pattern with peak drug flow
during the late activity/early rest span resulted in significantly greater tumor
growth delay than observed with either the flat infusion or other variable rate
patterns. In fact, this particular pattern was the only one associated with
significant objective tumor shrinkage. Therefore, FUDR toxicity and anti-tumor
activity each depend upon the circadian timing of the infusion peak when given by
variable rate. Since some of the studied circadian-stage related infusions are
toxicologically and therapeutically inferior to constant rate infusion, the
circadian pattern, and not the quasi-intermittency of circadian FUDR administration,
is primarily responsible for these pharmacodynamic differences. The optimal input
profile reduce FUDR toxicity and to maximize anti-cancer activity is daily infusion
given late in the daily activity span. These optimal anti-cancer peak FUDR infusion
times are, however, not the same as optimal FU bolus times for cancer control found
for the mouse model partially investigated by Peters [80]. The best anti-cancer
activity for FU in the mouse colon cancer was also close to its least toxic time of
day described before (between mid-sleep and awakening).
To summarize, the best time for FUDR by bolus or 48-hour infusion in terms of low
toxicity and high anti-tumor efficacy is late in the daily activity and in the early
rest span. The best time for FU by bolus is between mid-sleep and awakening. No
infusional preclinical data currently exist in this respect.
Clinical chronotoxicological experiments.
5-Fluorouracil.
Limited clinical experience is available with circadian stage specified FU
administration. Levi and coworkers [84] have extrapolated the quoted preclinical
findings to an assumption that FU is better tolerated during the mid-to late rest
phase of the recipients. There are no randomized clinical trial results yet to support
of this hypothesis, however, the following preliminary Phase I data are interesting.
High-dose intensity, circadian stage modulated 5-day FU infusion.
Levi et al. [84] have treated 35 ambulatory patients with metastatic colorectal cancer,
with 334 courses of FU by continuous intravenous infusion. A chronopump (Autosyringe,
USA) or a Intelliject pump (Ivion, Englewood, Colorado, American suppliers; Aguettant,
France, European suppliers) were used to automatically increase and decrease the
infusion rate of FU from 0% to 200% of the 24 hour mean. Maximum drug infusion occurred
at 4 a.m. and minimum at 4 p.m. The infusion was given for 5 days every 3 weeks.
Intra-patient dose escalation by 1 g/m2/course was planned from the starting dose
of 4 g/m2/course. Dose-limiting toxicities (WHO grade II or higher) included
stomatitis (in 9% of the treatment courses), diarrhea (5%), neutropenia (4%),
hand-foot syndrome (3%), and anemia (2%). No cardiac toxicity was encountered. Median
maximal tolerated dose per course was 8 g/m2 in patients with good performance status,
and 6.5 g/m2 in patients with impaired performance status (bed-ridden for 50% or more
during the day (p < 0.01)]. Ten partial responses (30%) and 15 stabilizations [44%)
occurred in 34 patients. In 22 previously untreated patients, 8 partial responses
(36%) and 9 stabilizations (41%) were observed. Median survival exceeded 20 months.
The authors concluded that compared to historical controls using 5-day constant-rate
infusion, automatic circadian chronotherapy with FU permitted an increase of dose
intensity by ~75% with fewer side effects. This may improve survival of patients with
metastatic colorectal cancer. Randomized time-specified comparisons of maximum
tolerated dose are clearly indicated.
Circadian patterned 5-day infusion of Fluorouracil+Leucovorin+Oxaliplatine.
Levi [85] has recently reported the highest objective response rate (66%) achieved
to date in patients with metastatic colorectal cancer treated with intravenous,
time-specified delivery of FU, tetrahydrofolate (Leucovorin = LV), and Oxaliplatin
(L-OHP). In a 5-day chronotherapy protocol, the infusion rates of FU, LV and I-OHP
were automatically increased and decreased along the 24 hour scale using a
multi-channel pump (Intelliject, Ivion Corp., Englewood, CO). Daily doses were: FU
= 500 mg/m2, LV = 300 mg/m2, and I-OHP 25 mg/m2 (3,500, 1,500 and 125 mg/m2 per course,
respectively). An admixture of FU and LV was infused between 10 p.m. and 10 a.m. The
infusion pattern had a bell-shape with peak at 4 a.m. Oxaliplatin was infused from
10 a.m. to 10 p.m. in a similar pattern, peaking at 4 p.m. Of 36 patients with
metastatic colorectal cancer accrued to this trial since January 1988, 32 are
evaluable for response (1 toxic death, 3 deaths before 2nd course in patients with
PS 3 III). Eighteen patients had received prior chemo and/or radiotherapy. In 120
courses, dose-limiting toxicities included moderate vomiting (3 2 episodes/day
despite 100 mg/day alizapride) and diarrhea (3 5 stools/day for > 7 days despite 10
mg/day loperamide). Reversible peripheral neuropathy with moderate functional
impairment was observed in 3 patients after 3 8 courses. Disease progressed in 6
patients (19%), was stabilized in 5 (16%), and regressed objectively by >50% (assessed
using computer-assisted tomography scans) in 21 (66%). If analyzed by pretreated and
untreated patients, the objective response rates were 57 and 72%, respectively.
Median survival has exceeded 18 months in previously untreated patients.
Time-specified randomized studies of this 3-agent combination are clearly indicated
in order to reproduce the results and determine what portion of the benefit of this
therapy derives from optimal circadian timing and what portion derives from the
combination of the agents irrespective of timing.
FUDR.
Randomized Phase I evaluation of circadian FUDR infusion shapes.
Continuous long-term FUDR infusion frequently causes severe and dose limiting
gastrointestinal toxicity when given by constant rate at commonly prescribed dose
levels. Based upon preclinical data reviewed above, we assumed that FUDR would be
better tolerated when most of the daily continuous infusion is given in the evening
(during the late activity phase). Therefore, we compared a circadian patterned
variable rate infusion with a maximal drug flow rate in the late afternoon/early
evening and minimum flow rate during the early morning hours with a constant rate
infusion in 54 patients with widespread cancer [86]. All FUDR infusions were
administered using a programmable implanted drug pump (Synchromed, Medtronic Inc.,
Minneapolis, MN).
In a pilot cross-over study and a second randomized trial, patients with metastatic
malignancies treated with equal dose intensities experienced less frequent and less
severe diarrhea, nausea and vomiting following variable rate infusion. In a third
study, the dose intensity of variable rate infusion was stepwise escalated to
determine the maximum tolerated dose. Patients receiving time modified FUDR infusion
tolerated an average of 1.45 fold more drug per unit time while experiencing minimal
toxicity. FUDR infusion was found to have activity against progressive metastatic
renal cell cancer: 23% of the patients responded objectively. These studies suggested
that increased dose intensity achieved by optimal circadian shaping may improve the
therapeutic index of infusional FUDR and may help control malignancies such as renal
cell cancer that are refractory to conventional chemotherapy [87].
Hepatic arterial FUDR infusion.
Liver metastases from colorectal cancer can be treated effectively with prolonged
intra-arterial infusion of FUDR. This treatment is, however, frequently accompanied
by severe and sometimes irreversible liver toxicity. Fifty ambulatory patients were
treated with intra-arterial infusion of FUDR at 0.1-0.3 mg/kg/day x 14 days every
four weeks by implanted pump [88]. Twenty-four patients received constant rate
infusion, and 26 received circadian modified infusion. Circadian-modified infusion
was technically as easily achieved as constant rate infusion by using programmable
pumps. Toxicity in the form of cholestasis and subsequent jaundice was less frequent,
less severe, and occurred later, when circadian infusion was employed.
Cholestasis gauged by alkaline phosphatase elevations occurred in 65% versus 44% of
the patients and jaundice in 26% versus 4% of those receiving flat or timed infusion,
respectively. Patients receiving the circadian-modified infusion tolerated 70%
higher average dose intensity. Nausea, vomiting, and diarrhea were mild and occurred
in 20% of the patients of each group. Gastric ulcers were equally frequent (12%).
More than half of the patients (52%) had no toxicity with circadian infusion, while
only one-fifth (22%) of those receiving constant rate infusions were free of toxicity
(p<0.03). The objective response rates were comparable at 35% and 32% for flat and
circadian infusion, respectively. Our study demonstrated that the maximum tolerated
dose intensity of intrahepatic arterial FUDR infusion can be increased substantially
by circadian modification of the continuous infusion rate, and debilitating liver
toxicity with jaundice can be significantly diminished. Incidentally, another group
of 7 patients with progressive metastatic renal cell cancer confined to the liver,
who were treated with the same hepatic arterial FUDR infusion schedule, had one
complete and three partial responses (objective response rate of 57%). This indicated
excellent anti-cancer activity of our treatment schedule in this highly
chemotherapy-refractory malignancy.
Mechanisms of circadian variations in fluoropyrimidine pharmacodynamics.
While much is known about fluoropyrimidine metabolism, little information is
available on circadian alterations in these complex biochemical processes. The
following description outlines the major metabolic pathways as a background for the
circadian studies presented below (Figure 23).
FUDR activation. After parenteral administration, FUDR utilizes the pyrimidine
nucleotide transport system to gain entry to cells [89]. FUDR is converted in a single
step reaction into the active metabolite FdUMP by deoxyuridine kinase (DUK), an enzyme
with limited capacity [90]. It is not known whether drug uptake, retention, or DUK
activation
vary
during
the
day.
The
intracellular
nucleotide
fluoro-deoxyuridine-mono-phosphate (FdUMP) has a relatively prolonged half-life and
persists in a profile characteristic of individual tissues [91]. FdUMP competitively
blocks the activity of thymidylate synthetase (TS), which converts
deoxy-uridine-mono-phosphate (dUMP) into deoxy- thymidine-mono-phosphate (dTMP),
thereby interfering with DNA synthesis. It is not known whether TS activity is
circadian stage dependent in most tissues relevant to FUDR toxicity or in tumor.
Cytotoxicity is a direct function of the amount of FdUMP and the length of time during
which it is present in the cell. The blockage of TS can be reversed by increased dUMP
levels, decreased FdUMP levels, decreased binding affinity to the enzyme, or
increased enzyme activity. Blockage of the enzyme is greater in the presence of
tetrahydrofolate excess. It is currently unknown whether tetrahydrofolate levels
vary with time-of-day. The complex formed between 5-FdUMP, thymidilate synthase, and
tetrahydrofolate is slowly dissociable with a half-life of 6 hours in intact cells.
The half-life of this dissociation may also be different depending upon the
time-of-day at which it is formed. A folate cofactor is an absolute requirement for
complex formation; severe depletion of intracellular reduced folates compromises
complex formation [92].
FUDR catabolism. The importance of FdUMP degrading enzymes in determining
cytotoxicity has not been fully explored. FdUMP may be dephosphorylated and converted
into FUDR by 5'-nucleotidase and phosphatase enzymes [93]. The formation of
fluoro-deoxy-uridine-di-phosphate (FdUMP) and -tri-phosphate (FdUTP) takes place
but seems to be quantitatively less important [94]. FUDR is split by the ubiquitous
enzyme pyrimidine nucleoside phosphorylase (PNP) into FU and deoxyribose-1-phosphate.
It is not currently known whether any of these enzymes are circadian rhythmic in their
activities.
FU activation. FU may be anabolized in a multi-step process, which results in the
formation of its main active metabolite, fluoro-uridine-tri-phosphate (FUTP),
interfering with RNA synthesis and to a lesser degree, to FdUMP which then interacts
with TS. The degree of activation and catabolism depends upon the regulating enzyme
capacity. Activation is promoted by ortate phosphori-bosyl transferase (OPRT),
pyrimidine nucleoside phosphorylase and uridine and thymidine kinases [93].
FU catabolism. The major FU-degrading enzyme, dehydropyrimidine dehydrogenase (DPD),
reduces FU to dehydrofluorouracil (DHFU). The activity of this enzyme is rate limiting
for FU degradation [95]. During subsequent degradation, alpha-fluoro-beta ureido
proprionic acid (FUPA), alpha-fluoro-beta guanido proprionic acid (FG-PA),
alpha-fluoro-beta alanine (FBAL), urea, carbon dioxide and ammonia are formed. None
of these products are cytotoxic. The main organ for fluoropyrimidine metabolism is
the liver. There is increasing information that FU catabolism is highly coordinated
in circadian time (Table 2).
Elimination of FU and FUDR from the plasma is a rapid process. The plasma half-life
of FU after IV injection in patients is anywhere between 6 and 20 minutes with
considerable inter-individual variation. Most of the drug is converted into carbon
dioxide. Less than 20% is excreted in the urine. The conjugation with bile acid has
been described [96], which may be of importance for the development of cholestasis
and cholangitis after hepatic arterial infusion of fluoro-pyrimidines. Bile acid
production has a profound non-meal dependent circadian-stage dependency, leading to
the possibility that conjugation dependent drug inactivation is also time-of-day
dependent.
Influence of drug administration mode, dose and duration on toxicology.
The metabolic pathway of FUDR depends on the method of drug administration. The
clearance of dose FU, given by a single bolus, is much slower than the clearance of
the same dose given over 24 hours, presumably due to saturation of the degradative
enzyme DPD [95]. If given in massive amounts over a short period of time as bolus
injection, most of it is split into FU and deoxyribose-1-phosphate. However, if given
at low dose over prolonged time periods, most of it is "salvaged" and converted into
FdUMP inside the cells. This is reflected by a profoundly changing pattern of drug
toxicity with increasing infusion duration. The dose limiting side effect for bolus
injection is bone marrow toxicity. This might correlate in part with direct FU
incorporation into bone marrow cells RNA via FUTP in addition to blockage of DNA
synthesis via FdUMP and impaired RNA synthesis via FUTP [97]. For continuous long-term
infusion, toxicity along the entire gastrointestinal tract becomes dose limiting with
the development of ulcerations and profuse diarrhea. There is no clinically
significant bone marrow toxicity when the drug is given by long-term infusion either
arterially or intravenously. Part of the explanation for this dramatic difference
in toxicity may be the accumulation of FdUMP, which is found at relatively higher
levels in epithelial cells of the gastrointestinal tract as compared to bone marrow
cells for the same plasma levels of FUDR [98].
Obviously, the circadian time structure of each of the regulating enzyme activities
in the anabolism and catabolism of fluoropyrimidines could be important in
determining the time dependency of FU and FUDR toxicities seen in listed bolus and
infusional preclinical and clinical experiments, eg. DPD described above.
Summary.
A growing number of fluoropyrimidine studies have addressed the issue of circadian
drug timing. They have demonstrated that 5-fluorouracil (FU) is better tolerated
during
mid-to-late
rest
span
of
the
recipients'
day,
and
that
5-fluoro-2'-deoxyuridine (FUDR) is less toxic during the daily late activity and
early rest spans. The circadian difference in maximum tolerated dose is substantial
and frequently exceeds 50% of the mean (i.e., the maximum average dose tolerated at
all circadian stages). Drug timing at a stage of reduced toxicity does not diminish
anti-tumor activity. On the contrary, tumors seem to be more sensitive at this stage.
In addition, because more drug may be given safely, the anti-tumor effect may be even
better if a positive dose-response relationship exists for the agent in question.
Initial explanations for these observations of circadian stage dependent drug effects
come from circadian studies of fluoropyrimidine pharmacokinetics and investigations
in cytokinetics for toxicity target tissues. The first careful attempts to clinically
utilize our preclinical experience have been successful in reducing toxicity and
allowing higher dose intensity.
Other chemotherapeutics and combined therapies.
Preclinical data.
Murine L1210 leukemia has been extensively evaluated at the University of Minnesota
and Albany Medical College with regard to the chronobiology of anticancer therapy.
A number of studies involving over a thousand mice [69,70,99] demonstrate that a
circadian sinusoidal modulation of the 3-hourly doses of cytosine arabinoside can
double the cure rate after the inoculation of L1210 leukemia cells and increase the
survival time by 60 percent when the highest doses were given near the time of best
circadian drug tolerance. A critical evaluation of additional data confirms these
findings [100].
Combining agents has been advocated to circumvent drug resistance [101], as
demonstrated for L1210 leukemia treated with cytosine arabinoside (ara C) and
1-(2-chlorethyl)-3-cyclohexyl-1- nitrosourea (CCNU) [102], resulting in what has
been called "therapeutic synergism" [103]. Using several model systems, we tested
one of the drugs present in each of several combination regimens
for circadian stage-dependent antitumor effectiveness. Thus, L1210 leukemia was
treated with a combination of cytosine arabinoside and cylophosphamide [104], or
doxorubicin and cyclophosphamide [105,106], a rat immunocytoma was treated with a
combination of doxorubicin and cisplatin [107,108]; and a rat mammary adenocarcinoma
was treated with doxorubicin and L-phenylalanine mustard. It was found that an
improved therapeutic effect was observed by giving chemotherapy at certain circadian
stages even when more than one drug was administered to an animal (Table 7).
Many other investigators have demonstrated cytokinetic circadian periodicity in
non-human systems [109-117]. It is highly likely that these findings seen in animals
are pertinent to the treatment of human beings with anticancer drugs, both in regard
to the efficacy of treatment and to the toxicity of the drugs.
Glucocorticoids and other hormones may attract the toxicity and the efficacy of
antineoplastic agents in either a favorable or unfavorable way. This is particularly
relevant since hormones are often administered concurrently with cancer chemotherapy.
Moreover, high doses of glucocorticosteroids are often used as anti-emetics, mostly
before and after cisplatin administration. Preclinical studies suggest that
concurrent high dose methylprednisolone and cisplatin administration may reduce the
therapeutic index of the latter [118]. Clearly, further work is needed to define the
temporal aspects of drug interactions and the effects on the time structures of the
treatment recipient.
Clinical experiments.
Based upon preclinical studies indicating a better tolerance for VP-16 during the
late rest/early awakening phase [119], a randomized, multi-center trial was started
in Belgium and France, offering chronotherapy with VP-16 and cisplatin to patients
with metastatic non-small cell lung cancer every 4 weeks [120]. Patients have been
randomized to receive VP-16 at 100 mg/m2/day on days 1-3 either at 6 or at 18 hours
and 100 mg/m2 of cisplatin on day 4 at 18 hours. An interim analysis of 76 cases has
not yet demonstrated significant differences in dose intensity or toxicity between
the treatment groups.
Focan [121] reported on 64 patients who received the same drug combination of
methotrexate, 5-fluorouracil, vinblastine, and cyclophosphamide at equal doses but
on two different time schedules. Significant differences in tumor responses were
found (58% versus 23% partial response rate).
Recently, the 5-year disease-free survival of children with acute lymphoblastic
leukemia was found to be different, depending upon the timing of their maintenance
chemotherapy (6-mercaptopurine + methotrexate); 80% (evening) versus 40% (morning)
[122].
Summary.
To date only a few metastatic solid tumors can be cured by chemotherapy alone. Most
malignancies are either primarily chemotherapy-resistant or will develop resistance.
This is especially true for the more frequent cancer types, including those of lung
and gastrointestinal origin. While various doses, routes, and modes of drug
administration have recently been studied more extensively, less attention has been
paid to circadian drug timing and dose intensity. While chemotherapy has reached a
plateau of low efficacy in these groups, every potential to make our currently
available tools more powerful must be used. Dose intensity and the tumor response
are often correlated [58,123]. This indicates the potential importance of any method
which can result in a safe increase of chemotherapy doses.
Optimal circadian timing of anti-cancer agents offers great promise in this respect.
While neither the existence of biological rhythms in all living beings nor the
preclinical chronotoxicity study results are seriously questioned, the clinical
application of chronochemotherapy is in its infancy, and progress is slow. Several
reasons can be cited for the reluctance of many oncologists to study or to adopt the
chronotherapy principles, including:
1. The limited understanding of underlying mechanisms
chronopharmacodynamic observations: "How does it work?"
that
explain
2. The complexity of preclinical study results and the apparent difficulty to make
simple extrapolations for the design of clinical studies: "What is the best schedule,
and how big is the gain?"
3. There is a paucity of convincing clinical data that chronotherapy is better than
conventional therapy. However, even well documented chronotherapy (e.g., the
time-specified combination of cisplatin with an anthracycline), seems impractical
and interfering with routine clinic or hospital schedules: "How can it possibly be
done?"
The first two issues are inter-related. Our understanding of the malignant processes
and the ability of chemotherapy to influence or reverse them is limited. For some
of the most frequently used chemotherapeutic agents, the ultimate mechanisms of
action are unknown. That makes the explanation of temporal variations even more
difficult. However, we can at least identify factors that are likely to play a major
role in causing rhythmic changes of drug pharmacodynamics.
Chronopharmacokinetics. In the mouse and rat liver, the activity of many enzymes
including the cytochrome P-450 system, are circadian stage-dependent, which affects
the detoxification capacity for various drugs [124,125]. Circadian differences in
blood flow to various organs and renal clearance may also affect drug distribution
and elimination [126]. Reproducible circadian differences of pharmacokinetics have
been found for cisplatin [48], oxaliplatine [127], carboplatin [128], doxorubicin
[129], 6-mercaptopurine, methotrexate [130] and some fluoropyrimidines.
Chronocytokinetics. Rhythmic changes in DNA and RNA synthesis, RNA translational
activity, and mitotic activity may determine the susceptibility to cell-cycle
specific cytotoxins. Such rhythms have been extensively documented in most murine
tissues including stomach, duodenum, rectum and bone marrow and used as explanations
for the observed rhythms in anti-cancer drug susceptibility [131-134]. Changing the
lighting regimen or feeding pattern of animals can predictably shift these rhythms,
especially in the intestinum, but not eliminate them [135-136]. Some tumor cells also
exhibit circadian-dependent growth while others do not. In general,
well-differentiated, slow growing tumors retain a circadian time structure, whereas
poorly differentiated, fast growing tumors tend to lose it [137-139].
Circadian-stage dependent cytokinetics of bone marrow cells and the intestinal
epithelium may for instance largely determine the susceptibility to
fluoropyrimidines. The circadian stages of maximum synthetic activity - and greatest
susceptible to an S-phase specific cytotoxic agent - differ between these tissues.
In the bone marrow, maximal activity occurs during the early- to mid-rest period [140];
however, it is not known to what extent rhythms in stem cell activity and proliferative
activity in the maturing cell compartment are coupled to each other. (Stem cells and
progenitor cells should be considered as the most important cells to protect with
a "time-shield".) In intestinal epithelium, maximal activity has been described
during the late-rest phase of the host [141].
As normal tissues and malignant tissues behave differently in terms of their temporal
organization, the circadian timing of optimal therapeutic efficacy and lowest host
toxicity frequently overlap. Haus and coworkers [142] have demonstrated with a mouse
L1210 leukemia model and Sothern et al. [143] with a rat solid tumor model that the
optimal timing of equal dose chemotherapy reproducibly results in a 2-3 fold
improvement of tumor cure rate. To date, the toxicities and anti-tumor activities
of at least 20 of the most commonly used chemotherapeutic agents have been documented
as being circadian stage-dependent both in animals and also for many in humans
[144,145].
The difficulty in making simple extrapolations from animal chronotoxicity findings
in order to improve clinical treatment schedules are illustrated by the
fluoropyrimidine example. FU and FUDR cytotoxicities are clearly circadian stage
dependent in animals. However, there does not seem to be one consistently less toxic
circadian stage across all studies. For example, the mid-activity optimum for the
intestine to tolerate bolus FU is several hours later in the day than is the time
optimum for whole animal toxicity and lethality. This observation requires further
study with fasted and fed animals in single and multiple dose regimens. It raises,
however, the possibility that optimal treatment times may be different for different
target tissues as well as for different modes of fluoropyrimidine delivery.
In addition, despite the chemical similarity of certain drugs, the circadian time
of best drug tolerance may be different. For example, the optimal circadian timing
for bolus FU and bolus FUDR is not identical. It is safest to give FU by bolus some
time after mid-sleep and prior to usual awakening, while it is safest to give bolus
FUDR late in the daily activity span prior to sleep onset. Different metabolic
pathways may be responsible for activation of FU and FUDR, and the activity of
regulating enzymes may follow different temporal patterns. Chronotoxicity
differences have also been found for other closely related drugs (e.g., doxorubicin
and epirubicin), while other related drugs (e.g., cisplatin and analogues) have very
similar or identical best and worst circadian times for treatment. The different
circadian stages for optimal drug timing, depending upon the endpoint studied, may
also come from differences in experimental model characteristics (e.g., age, sex,
species and strain of the animals, housing conditions, treatment dose, -mode, -route
and -schedules, season of experiment performance, etc.). In weighting the data for
analysis, one also has to recognize that not all investigators have tested the six
circadian stages that are necessary to define a circadian rhythm.
Clinical trials testing multiple (i.e., up to six) circadian stages in a randomized
fashion are not feasible, so one has to make a best estimate as to what to expect
in patients. In spite of the difficulties in extrapolation, clinical studies derived
from preclinical experience prove that chronotoxicity also exists in patients, and
circadian drug timing can result in less toxicity, which allows for a markedly higher
dose-intensity as described above. The ability to reduce toxicity by optimal
circadian drug timing, and to safely increase dose intensity, may ultimately
translate into improved anti-tumor efficacy, but further confirmation from
randomized trials is pending. Our ability to predict an individual's drug
susceptibility pattern must also be improved, as many treatment-related variables,
recipient related factors, and environmental and seasonal factors may each modulate
or shift the optimal circadian treatment time to some extent.
To facilitate the practical application of time specified drug delivery schedules,
programmable delivery systems have been developed. They are becoming increasingly
more powerful tools in terms of their versatility, precision, and reliability.
A still relatively small group of researchers and clinical investigators are working
to further explore and hopefully answer the major questions that have been raised
with chronotherapy. Circadian differences in drug toxicities challenge the concept
of standard drug screening and standard clinical phase I/II investigations that
consider only one circadian timepoint. These investigations may provide insufficient
or misleading information on toxicity, MTD, and anti-tumor activity for untested
circadian stages. While the issues remain difficult and complex for the time being,
and while no "cookbook recipes" can be offered, undeniable progress has been made.
We need to intensify basic research to further explore the mechanisms that determine
temporal changes. Clinical phase I/II studies should correlate circadian
pharmacokinetics and pharmacodynamics with individual marker rhythms. Randomized
multi-center studies should be performed to confirm the current clinical evidence
of chronotoxicity.
BIOLOGICAL AGENTS.
Progress in chronobiology depends on the development of basic knowledge, which
usually does not consider the periodicity of the phenomena being examined. Cancer
biotherapy is recent, and a chronobiological understanding of this nascent field is
rudimentary. Because of the modest amount of relevant work performed to date, this
section will focus upon the theoretical basis for anticipation of the circadian
organization of the relevant immune networks. Hopefully, the analysis of available
data will convince the reader that the response of the organism to the action of the
agents to be discussed (TNF, IL-2, interferon, erythropoietin) is, without exception,
highly dependent upon the circadian timing of their administration.
Cytokines: Tumor Necrosis Factor (TNF) and Interleukin-2 (IL-2).
Theoretical basis for time-dependence of cytokine/growth factors related
immunological phenomena.
Circumstantial evidence supporting the circadian coordination of cytokine/growth
factor/blood cells network.
The first biochemical circadian rhythm identified in human beings was the daily
glucocorticoid pattern, which was discovered by tracing its footsteps, i.e., the
circadian pattern of the concentration of certain cellular elements of blood,
especially eosinophils [146,147]. Since the concentration of these and all other
cells in the blood are profoundly influenced by growth factor availability, which
is in turn regulated by monokines and lymphokines, particularly IL-1, these early
observations of circadian rhythms in the cellular elements of blood tend to support
the likelihood that cytokine biology is under tight circadian coordination. Current
understanding of the details of how these cytokine/growth factor/blood cell networks
interact is incomplete. There are, however, many pieces of circumstantial evidence
indicating that these relationships are tightly coordinated in time with a prominent
circadian rhythmicity.
IL-2 dependent circadian stimulation of Natural Killer cell production.
Important components of cellular immunological response in which TNF and IL-2 play
an important role are coordinated in a circadian dependent manner. Lymphokine
Activated Killer cells (LAK) and Natural Killer cells (NK) are the two heterogenous
lymphocyte populations comprised, at least in part, of nonspecific cytotoxic T
lymphocytes produced in response either to an immune challenge, and/or to a subsequent
increase in IL-2 activity. These cells are able to lyse a variety of neoplastic and
non-neoplastic tissues [148-154]. NK cells, which have been the most well studied,
are an important factor inhibiting both the intravascular blood-borne phase of solid
tumor metastases [155] and the extravasation and/or early implantation phase of the
metastatic process [156]. Well documented circadian rhythms in NK cell activity both
in rodents and in humans are established [157-161]. If endogenous IL-2 significantly
influences the level of NK activity, this is evidence for either circadian
coordination of target lymphocyte IL-2 availability or sensitivity to IL-2.
Evidence for Cortisol-IL-1 Circadian Negative Feedback Loop.
Glucocorticoids affect IL-1 transcription, translation and cellular release.
Non-chronobiologic evidence for the likely chronobiological circadian coordination
of IL-1 regulation is mounting. Very recent attempts have been made to explain some
of the anti-inflammatory properties of steroids was made. Dexamethasone was shown
to inhibit the production and release of IL-1? by human monocytes [162], and soon
afterwards glucocorticoids were demonstrated to selectively inhibit the
transcription of the IL-1? gene and to decrease the stability of IL-1? mRNA [163].
Since glucocorticoids are exquisitely circadian rhythmic in their production and
release, such a profound glucocorticoid effect upon the production of such a central
molecule as IL-1 suggests that cytokine/growth factor networks may be tightly
coordinated in a circadian manner by the hypothalamo/pituitary/adrenal axis.
IL-1 influences cortisol effects. It has been reported that IL-1 decreases cytosolic
binding of glucocorticoids in the liver, leading to reduced steroid induction of
gluconeogenic enzymes. It has been also found that human recombinant IL-1? affects
intracellular glucocorticosteroid binding in rat hepatoma cells. Binding assays
revealed that IL-1? significantly reduced dexamethasone binding within 2-4 hours
following interleukin administration. Analysis of data indicated that IL-1?
treatment decreases the number of available cytosolic receptors. It may, in turn,
affect the downstream action of glucocorticoid upon gene expression of IL-1, so
closing the loop of negative glucocorticoid/IL-1 interaction.
TNF/glucocorticoid interactions at a level of their cytotoxic effects.
Physiologic cortisol concentrations influence TNF cytotoxicity. It has been
attempted to clarify the regulation of tumor necrosis factor biology by examining
the effects of glucocorticoids upon the toxicology of TNF. Purified human recombinant
TNF incubated at concentrations of 400 U/mL at 37°C for 40 hours with L929 fibroblasts
resulted in 25% survival of these cells. Glucocorticoids added 1 hour before TNF
treatment [Hydrocortisone at 1x10-7M (molar concentration) and Dexamethasone at
1x10-7M] substantially inhibited TNF toxicity, resulting in 88% and 89% fibroblast
survival, respectively. Normal human plasma contains 1.3-5.5x10-7M of hydrocortisone.
Therefore, these data indicate that hydrocortisone may potentially act as an
endogenous in vivo modulator of TNF activity, which may, in part, help explain the
extremely variable TNF toxicity between patients. Since corticosteroid levels are
highly circadian stage dependent, large circadian stage dependent differences in the
toxic/therapeutic ratio of this drug can be expected.
Further evidence for possible glucocorticoid/cytokine interaction: glucocorticoid
mediated cytotoxicity. Cytotoxic T lymphocytes (CTL) induce apoptosis, a cytolytic
process in target cells which effects a rapid and characteristic degradation of
chromosomal DNA. Glucocorticoid-mediated cytolysis of immature thymocytes resembles
this process to a high degree [164]. The possibility has been explored that these
two lethal processes share the common pathway by studying the susceptibility of
glucocorticoid-resistant mutants to CTL-mediated killing. He reports that an unusual
thymoma mutant (having normal glucocorticoid hormone receptor activity) is resistant
to both glucocorticoid and CTL. What is more, a single-step genetic reversion can
restore sensitivity to both glucocorticoid and CTL. The genetic locus thus identified
reveals one element of the endogenous cell suicide pathway that can be triggered by
different effectors, corticosterone and CTL. These data, together with those
presented in the former paragraph, indicate how the profound circadian dependency
of cytokine-induced cytotoxicity of glucocorticoid and/or CTL sensitive cells may,
in principle, be directly modulated by an endogenous circadian rhythm in the
glucocorticoid concentration.
Circadian dependent modulation of glucocorticoid production by IL-2.
Sanchez de la Pe?a [165] has attempted to discover whether the interaction between
neuroendocrine and immune networks, as mediated by IL-2, is circadian stage dependent
light/dark standardized. Hence, rats were killed at one of 6 circadian times of day
and quarted adrenals or isolated adrenocortical cells were then incubated for 60 min
with or without IL-2. It was found that IL-2 stimulated corticosterone production
in adrenal cells harvested from rats at usual daily arousal (p=0.02), but not those
harvested at other times of day. Analysis revealed a circadian rhythm in IL-2 effect
upon cortisol production (cosinor p=0.014). These results show that the in vitro
action of IL-2 upon the adrenal cortex is circadian stage dependent.
Role of IL-1 in circadian regulation of body temperature and fever.
Early observations by Dinarello raising the possibility that IL-1 may be an important
molecular mediator of fever, independently raise the likelihood of tight cytokine
circadian control [166]. Febrile reactions secondary to a wide variety of infectious,
inflammatory and malignant processes do not occur at random. They almost always occur
in the evening, at the time of the day usually associated with the daily nadir of
cortisol. In fact no one, as yet, has attempted to explain the molecular basis of
the normal daily temperature rhythm. If IL-1 does influence temperature regulation,
this prominent circadian rhythm which serves as one of the main circadian markers
for chronobiologic studies, peaking near the usual circadian cortisol nadir, may be
viewed as circumstantial evidence for temporal cytokine coordination. Extrapolating
Dinarello's reasoning, the normal circadian rhythm in body temperature may represent
cortisol/IL-1 counterbalancing one another and a resultant temporally coordinated
cytokine circadian biology.
Cell cycle-specific effects of TNF.
Each important target tissue of TNF toxicity including immunocytes, nucleated bone
marrow precursors, the gut epithelium and the skin, undergo DNA synthesis and traverse
through all other phases of the cell cycle non-randomly (rhythmically) throughout
the day. Experiments by Darzynkiewicz [167] demonstrate that either the lophase or
some very early postmitotic cell cycle stage appears to be the specific point in the
cytokinetic cycle at which TNF treated cells undergo lysis. This conclusion is also
supported by earlier observations of cells in culture, indicating that, in the
presence of tumor necrosis factor, many cells divide and subsequently lyse. As cell
cycle is a process showing a prominent circadian coordinated periodicity these data
strongly suggest that TNF cytotoxicity will also undergo strong diurnal rhythmic
modulation.
Central circadian coordination of cytokine biology.
Lotz, et al. [168] have recently demonstrated prominent effects of neuropeptides upon
the production of IL-1, TNF ??and IL-6 by monocytes. Two groups of mediators, the
neuropeptide substance P and K and the monocyte-derived cytokines interact in the
neural regulation of immunological and inflammatory responses. Both substance P and
K as well as the carboxyl-terminal peptide SP (4-11) induce the release of the
cytokines mentioned above. Since monocyte-derived cytokines regulate multiple
cellular functions in inflammation and immunity, and since neuropeptides can be
released from peripheral nerve endings into surrounding tissues, these findings
identify a potent mechanism for nervous system circadian regulation of host defense
responses [168]. All plasma and spinal fluid concentrations of neuropeptides studied
chronobiologically participate in high amplitude circadian rhythms which are
coordinated through the interaction of the suprachiasmatic nucleus - pineal neurophysis - pituitary - adrenal networks. It would be attractive to think of a
circadian IL-1 rhythm balanced in time by cortisol suppression and substance P and/or
K induction (see Figure 24).
IL-1 - Central molecule in cytokines-growth factors network.
Interleukin 1 (? and ?) seem(s) to be in a central position in chronobiologic
coordination of cytokine production, release, toxicity and activity. IL-1 shows
anticancer activity, TNF-like hemodynamic activity and growth factor activity. IL-1
alpha has in vivo antitumor activity against Meth A sarcoma, colon 26 adenocarcinoma,
B16 melanoma and Lewis lung carcinoma [169]. Both IL-1 molecules also induce a
shock-like state in rabbits and this state can be synergistically worsened by
concurrent TNF administration, indicating an in vivo interaction between these
cytokines [170]. As well as having anticancer and pro-inflammatory activity,
interleukin-1 alpha and beta also enhance the release of bone marrow neutrophils and
activate circulating tissue phase phagocytes. They also increase the bone marrow
production of neutrophils and monocytes by stimulating fibroblasts to synthesize the
growth factors GM-CSF and G-CSF [171]. It seems as though most cytokines have multiple,
complementary and/or synergistic effects with one another. If the regulation of these
factors is coordinately arranged anatomically, they are probably also dynamically
coordinated in circadian time.
The IL-1 dependent IL-2 synthesis. In the process of immunostimulation, the T-cell
networks are affected by Interleukin-1. This cytokine, which can be produced by many
cell types, but usually the macrophage, must interact with the T-cell as the
macrophage is presenting the antigen to which this T-cell will ultimately respond.
One of the most critically important responses following this IL-1 dependent
initiation of the cellular immune response is the quick (6-9 hour) expression of the
IL-2 gene, production of IL-2 mRNA and production of the IL-2 protein. Subsequently
and relatively quickly, IL-2 receptor begins to be expressed in high concentrations.
These dynamics subsequently result in an amplification of the local and systemic
immunocyte response.
Experimental evidence for circadian stage dependency of TNF and IL-2 effect.
Early experiment with E. coli endotoxin.
Indirect evidence for non-trivial reproducible circadian variation in cytokine
toxicology was first presented in 1960, when work in Halberg's laboratory connected
the chronobiological studies to all of these early and more recent observations. These
studies showed large magnitude circadian stage dependence of the lethal toxicities
of E. coli endotoxin (Figure 25) [172]. It has been also shown that TNF is an important
mediator of endotoxin-induced septic shock. This, interpreted in light of these 1960
findings, would indicate that the lethal and nonlethal toxicities of this and perhaps
other cytokines are likely to be very different at different times in the circadian
cycles of treated animals. Our recent results with TNF support this hypothesis.
TNF studies.
The advent of an ever-increasing number of genetically-engineered biological
substances having anticancer activity has been tempered by unpredictable and poorly
reproducible toxicities. Recombinant human tumor necrosis factor (TNF) has shown both
toxicity and anticancer activity in transplantable tumor models [173]. In a series
of experiments, we examined the effect of administration timing of TNF on toxicity
and therapeutic effectiveness [174,175]. We gave a single IV injection of either 250,
500, 750 or 1000 μg/kg of TNF to 8-10 week old female Balb/C mice and 1000 or 1500
μg/kg to 15-16 week old mice, kept under a 12:12 lights on/off schedule. The 240
mice were time-randomized to receive their injection at one of 6 4-hourly circadian
stages and followed for survival. Mortality across the 6 test times was compared by
Chi-square analysis with the following results:
Less than 50% overall mortality was reached in each of the first four doses. Except
for the lowest dose, mice treated during mid to late activity (18-22 HALO) lived longer
and died less frequently than those injected during the resting phase. For the highest
doses (1000-1500 μg/kg), mice injected during their activity span had less than 30%
mortality as compared to 70 to 100% for those treated during the resting phase (02-10
HALO) (Figure 26). The circulatory collapse and shock may represent a final common
pathway of endotoxin mediated lethality, and, indeed, the mortality from TNF displays
a circadian pattern strikingly similar to that reported in mice from E. coli endotoxin
nearly 3 decades ago [172].
In a preliminary study on tumor-bearing mice, 75 female Balb/C mice with Meth A sarcoma
were treated daily for seven days, or every other day for 14 days, with 1 μg/mouse.
The tumor growth was much slower in animals treated with TNF then in those treated
with saline, our control group. The effect of TNF was highly dependent on the circadian
stage of its administration (F =2.2, p = 0.07 from analysis of variance). Fewest cures
were observed in the daily activity span, just after the daily peak in corticosterone
(Figure 27). We concluded that the toxicity of TNF and its antitumor activity was
highly circadian-stage dependent and in need of further study to deduce the proper
timing to result in its most effective use for clinical success.
IL-2 study.
In another trial, we investigated dependence of IL-2 effects upon the time of its
administration. To achieve this goal we injected 60 female C3H mice with 25,000 U/24h
or the same volume of excipient (control) during 3 consecutive days i.p., at 6
circadian stages. Mice were killed 48 hours after the last IL-2 injection, and cell
suspensions from spleen and bone marrow were prepared. The endpoints of IL-2 effect
were: cell counts and proportion of different lymphocyte subsets, spleen weight and
wet-dry lung weight. Analysis of data showed an increase in the number of spleen cells
by 16% overall, but up to 3-fold more when IL-2 was given during the activity span
of the animals (p<0.02). Spleen weight was also correspondingly dependent upon timing
of IL-2 administration. The capacity of this molecule to modify the proportion of
lymphocyte subsets was very different, depending upon when in the day it was given,
and an increase in number of NK cells by >50% took place only in those animals treated
during their daily activity span. Toxicity, as gauged by wet-dry lung weight, was
also circadian stage dependent. If the circadian timing of IL-2 and TNF is ignored
it can be expected that experimental and clinical results will be highly variable
and (apparently) irreproducible.
Interferon.
Alpha-interferon is active against a variety of malignancies in phase II studies,
including leukemia (especially hairy cell leukemia), malignant lymphoma, multiple
myeloma, melanoma, colon cancer, renal cell carcinoma, some AIDS-related
malignancies, and chronic hepatitis B and C. It is being used both as a monotherapy
and in combinations with other biological agents (e.g., IL-2) or classic
chemotherapeutic agents. Its optimal mode, schedule and time of day for delivery are
unclear.
The rather limited efficacy of this drug needs to be improved, likewise serious
attempts to diminish interferon side effects, including flu-like symptoms (fever,
myalgia, athralgia, malaise, fatigue), nausea, vomiting, diarrhea, anorexia, renal
and liver injury, bone marrow suppression, somnolence and mental deterioration which
will often force physicians either to diminish the dose of this drug or to stop therapy
should be encouraged.
Theoretical basis for better efficacy and fewer side effects after early night
interferon treatment.
Bocci has described a theoretical basis for expecting better efficacy and fewer side
effects if night time interferon administration is prescribed [176]. He also gives
that there are significant and highly reproducible diurnal variations of several
lymphocyte subsets, with the lowest levels of many occurring daily at 8 to 10 hrs
in the early part of the diurnal activity span, and the highest daily levels occurring
just prior to nocturnal sleep onset [177-180]. Levels of circulating monocytes are
also low upon arising and high in the evening. On the other hand, cortisol
concentration remains at a low level during most of the night, increases rapidly in
the early morning hours, and reaches maximum at about 8 a.m., just at the time of
the lowest levels of circulating PBM (peripheral blood mononuclear cells). This
negative relationship between cortisol and PBM blood concentration has also been
demonstrated in many strains of mice [181]. It is very likely that the early morning
increase in plasma cortisol causes a disappearance of PBM, while the decrease in the
cortisol level between 8 a.m. and midnight may favor their return into the
circulation.
On the other hand, plasma levels under physiological conditions are negligible in
the morning and tend to increase in the late afternoon [182,183]. Following exogenous
administration, IFN peak plasma levels are reached within an hour after IM injection
[184]. IFN is removed very rapidly from the blood, thus when it is administered in
the morning, its plasma peak coincides with the lowest PBM levels, thereby decreasing
interaction of the IFN with effector cells. Finally, interferon administration causes
a rise in total serum 11-hydroxycorticosteroids, with a peak some 8 hours after
injection [185-187]. So when IFN is given daily, the cortisol level, which ebbs
normally during the day, will rise, and thus diminishes the expected diurnal increase
of PBM. Interestingly, the in vitro responsiveness of PBM to mitogens varies inversely
with the level of plasma cortisol and is maximal between 8 p.m. and 2 a.m. in subjects
that usually sleep between 11 pm and 7 am [188,189].
Thus, it appears that IFN administered in the evening should cause a minimal systemic
disturbance by reinforcing natural diurnal patterns of biovariability, and by
preserving the natural circadian coordinate function of the hypothalamus-pituitaryadrenal axis. Moreover, evening administration will permit maximal IFN plasma
concentration to coincide with the usual daily maximal concentration of PBM - its
effector cell population. Lymphopenia which often follows interferon injection,
would also be minimized or would not take place when IFN is given at the time of the
day when the most effective immunological response is anticipated.
Clinical report about the advantage of night-time interferon administration.
A preliminary communication [190] has reported experience of a group of patients
treated with recombinant ?-interferon either at night or during the day. Seventy-five
percent of the patients (90% of those treated with a low dose) noticed an improvement
in their energy level when they received IFN at night, as compared with that
experienced when IFN was given during the day. Many patients taking interferon at
night for several months have reported completely normal work tolerance and feelings
of well-being, which is a rare finding when IFN is administered in the morning.
Both the toxicity and efficacy of interferon are very likely to depend upon the
circadian stage of their administration. So far, circadian rhythms of endogenous
levels of these molecules have been totally disregarded. As a starting point, it seems
appropriate to at least fix daily administration timing of the agent to the time of
the daily physiological peak. Because the pharmacological administration of
cytokines is able to perturb the normal oscillation of hormonal secretion, it should
also be given at times that tend least to alter important circadian rhythms of hormonal
secretion and circulating pools of effector cells. Improvement of efficacy is
probable if the agent is given at times when effector cells are more readily available
for activation. Describing an optimal schedule of time-dependent IFN-delivery,
however, is at an embryonic stage and intensive preclinical and clinical research
is required to explore more fully the probable advantages offered by optimal
chronobiological IFN scheduling.
Erythropoietin.
Human recombinant erythropoietin, the first commercially available growth factor,
is effective in ameliorating the anemia of end stage renal failure and pre-dialysis
renal insufficiency. Its utility extends to the treatment of anemias caused by
malignancies and chemotherapy, as well as is both certain of the anemias associated
with AIDS and to chronic anemias caused by inflammatory disorders. It will also be
widely used to decrease transfusion requirements, and to allow preoperative banking
of larger amounts of autologous blood prior to elective surgery.
Evidence for circadian rhythmicity of erythropoiesis.
Erythropoiesis is known to be highly rhythmic on the circadian (24 hour) time scale
with respect to serum levels of erythropoietin [191], erythroid progenitor cell
numbers, the resultant number of reticulocytes and mature erythrocytes [192], and
the susceptibility of red cell precursors to myelotoxic drugs [193]. The biologic
and biochemical basis for the coordination of these endogenous hematopoietic rhythms
remains to be fully explained.
There is increasing evidence for the participation of glucocorticoids in coordinating
the circadian rhythm in erythropoiesis. The circadian variation in serum
glucocorticoid levels in man and animals is well known. These highly circadian-stage
dependent steroids have been shown at physiologic concentrations to promote the
survival and proliferation of human erythroid and myeloid precursors in vitro [194,
and Chikkappa and Pasquale, unpublished]. The role of these steroids in coordinating
the circadian-based hematopoietic rhythms has not been defined. The stimulation of
human myeloid precursor cell growth by glucocorticoids in vitro has been shown to
be a result of an indirect effect on accessory (non-precursor cell) cells, and can
be reproduced by supernatants of such cells [194]. It has been hypothesized that this
effect could be due to the production or activation of positive growth-promoting
factors (eg. IL-3, GM-CSF), or the withdrawal or inhibition of negative growth factors.
Glucocorticoids are known to decrease the production of several cytokines that are
inhibitors of erythropoiesis, such as TNF [195] and interferon [196]. This inhibition
of cytokine production by glucocorticoids may occur at the level of a 3' UA-rich
regulatory region in these mRNAs which regulate their stability and may control
translational efficiency of these RNA species [197,198].
Experimental evidence for circadian character of erythropoietin biology.
Circadian rhythm of endogenous EPO concentration in human serum.
When a sensitive radioimmunoassay for serum erythropoietin level (s-EPO) (using
labelled rHuEPO) and antiserum to urinary rEPO were used, Wide, et al. [191] noticed
a large intra-individual variation. In order to investigate whether a circadian
rhythm in endogenous serum erythropoietin levels was the cause of this variability,
they studied serum EPO level in 27 hospitalized adult patients suffering from various
illnesses, but all of whom had a level of s-EPO at 8-12hrs within the range of healthy
individuals, and had normal cortisol levels during the 24 h. A well-marked circadian
rhythm of serum EPO levels was found. Its peak was placed at 20 hrs, and its trough
at 8 hrs. Although the mechanisms behind the observed rhythm are unknown, these
results confirm a circadian character of erythropoietin biology.
Circadian-dependent response of the bone marrow upon EPO administration.
Erythropoietic effect of EPO may differ in healthy and anemic organisms as well as
in different anemias. To investigate these possibilities, we have used three
different animal models: a) intact mice, b) mice made anemic by bleeding, and c) mice
anemic as a result of bearing the transplantable tumor. In the course of the studies
presented in this chapter, intensity of EPO-induced hematopoiesis was estimated on
the basis of hematocrit and reticulocyte determinations.
Circadian stage dependent erythropoietin response in intact female CD2F1 mice. Our
first EPO trial was performed on 90 intact CD2F1 female mice [199]. They were injected
subcutaneously with a single dose of recombinant human erythropoietin (rHuEPO; 300
IU/kg of b.w.) at one of six different circadian times. Erythropoietic response was
evaluated by hematocrit and reticulocyte determinations after exsanguination at 24
hour intervals over five days following injection.
It was observed that baseline hematocrit and reticulocyte concentrations each varied
rhythmically as a function of the circadian stage of measurement. The overall increase
in reticulocytes was found to be maximal 48-72 hours after injection. For this single
fixed dose of rHuEPO, the rise in reticulocytes varied from a 50% increase when
injected at 23 or 1 HALO (early light, beginning of rest in nocturnal animals, usual
cortisol low), to a 310% increment when injected at 15 HALO (early dark, beginning
of activity) (Figure 28). This time of maximal reticulocyte response roughly follows
the known time of maximal corticosterone level in these animals by 4 - 5 hours.
Over the 5 days following injection of rHuEPO, the hematocrit response varied from
a 9% increase at 72 hours when given at 23 or 1 HALO, to the lack of a significant
elevation when given at four other intermediate HALOs (Figure 29). This dissociation
of reticulocyte and hematocrit response requires additional study and may relate to
the relatively weak effect of a single EPO injection and could also reflect
non-hematopoietic changes in hematocrits related to volume status. Erythropoietin
injection also resulted in an early (24-48 hours) decrease in hematocrit. The
magnitude and timing of this initial rHuEPO-associated hematocrit fall was markedly
circadian stage dependent.
Circadian stage dependent erythropoietin response in anemic female CD2F1mice. In the
next experiment, 60 CD2F1 female mice had been bled for 4 days, so that within this
time their HCT levels fell by 10%. The next day, half of the mice were injected with
300IU/kg of EPO, the other half remained untreated and were used as the control group.
After erythropoietin administration, hematocrit and reticulocytes were estimated for
all animals each day during the 8 following days.
Analysis of data revealed peak hematocrit response to the 3 days after EPO
administration: it was largest in those mice injected in late rest (10 HALO), and
weakest when injected within the activity span (Figure 30). The two-way interaction
between EPO effect and time of its injection was significant (p<0.001). Also analysis
of reticulocyte response upon rHuEPO injection showed its strong dependence upon
timing of drug administration: significant response of young red blood cells appeared
during animals' rest period only, with the peak at 10 HALO. The lowest response took
place in the mid- to late activity span (Figure 31). The EPO effect and injection/time
interaction, however, did not reach statistical significance (p=0.084). Low dose of
erythropoietin did not influence murine bone marrow sufficiently to make this
interaction significant. Two-way ANOVAs for each HALO, however, showed large
differences in EPO effect upon reticulocyte counts.
Circadian stage dependent erythropoietin response in anemic, female C3HeB/FeJ mice
bearing transplantable mammary carcinoma. Another experiment was performed in order
to investigate changes in HCT and RTC levels when EPO was administered at different
times of the day in the model of tumor bearing mice. Twenty-five female C3HeB/FeJ
mice were inoculated subcutaneously with mammary carcinoma which occurs
spontaneously in this strain of mice. Two weeks later, tumor was palpable by 85% of
animals, with a corresponding 14% fall in hematocrit (compared with the values on
day of tumor innoculation). Fourteen days after injection of malignant cells, EPO
was administered to half of the mice (1200 IU/kg s.c.), and an equal volume of diluent
to the other half (control). Within the following days, blood was examined for
hematocrit and reticulocytes levels.
Analysis of data revealed a significant influence of the time of EPO injection upon
both hematocrit (p<0.001) and reticulocyte counts (p=0.022). Hematocrit was
significantly improved only in those animals injected at 10 HALO (p=0.014). In both
other groups of mice (those injected at 2 and 18 HALO), the influence of EPO upon
hematocrit was not significant (Figure 32). Levels of reticulocytes were
significantly higher only in those animals injected at 18 HALO (r=0.013). Those
injected at 2 and 10 HALO did not show any significant change in RTC count.
At some circadian stages a lower erythropoietin dose has a greater effect upon
hematocrit than higher doses. A further experiment was carried out at two timepoints,
i.e., 2 and 18 HALO, in order to test for circadian dependence of the effect of three
different doses of recombinant human erythropoietin (300, 600 and 1200 IU per kg of
body weight) upon hematocrit levels in CD2F1 female mice. Results showed that the
greatest HCT rise in mice injected at 2 HALO appeared three days after administration
of the hormone. A dose of 1200 IU/kg turned out to be the most effective in this group.
However, in mice injected at 18 HALO, the peak HCT response was observed to be 2 to
4 days after treatment, and was greatest in those animals injected with the lowest
dose of EPO. The influence of both time and dose of EPO administration upon hematocrit
levels were statistically significant (ANOVA: p<0.001 and p<0.01 respectively).
This series of preliminary findings confirms a time dependence for the effect of
erythropoietin. The final pattern of proper circadian timing of EPO administration,
however, needs further study. Also, its interpretation in the context of such factors
as age, stage of estrus cycle, and season of the year has to be performed. If these
results can help to describe the most effective schedule for EPO delivery, it could
radically reduce the required amounts of EPO, which is very expensive. For example,
in the USA, there are about 100,000 patients annually who would benefit from dialysis
combined with EPO treatment. Given that basic EPO treatment costs are $5,000 per
patient annually, it can be seen that if circadian modulation of drug delivery can
reduce the amount of EPO required by 30% (which, on the grounds of our results, seems
to be highly possible) then this would result in a potential reduction in cost of
EPO by $150 million per annum!
The intelligent use of biological therapy.
While it has been adequately demonstrated that the therapeutic ratio of toxicity to
anticancer drugs is dependent to a large extent upon the timing of drug delivery (i.e.,
relative to circadian time, time between doses, and drug sequence), these variables
are much more relevant to the effective use of biological response modifiers. The
time of day when interferons, tumor necrosis factors, IL-2, LAK cells or growth
factors are given - relative to the patient's circadian cycle, repeat dosages, their
sequences among one another, and standard cytotoxic treatment - ultimately will
determine how effective these approaches are.
It is clear that the standard Phase I and II approaches to the study of biological
agents will not adequately define either their activity or even the relevant
toxicities of these agents, even though they have profound biological effects in
picogram quantitates while the relationship between host and tumor is elegantly and
temporally complex. Administering milligram quantities of biologicals without regard
to this complexity can only result in excessive expense, higher toxicities than
necessary and finally, great frustration. The studies described above give that the
availability of instruments able to stipulate sequence, interval, circadian stage
and infradian pattern of immune modulation is a sine qua non to optimal biotherapy.
FUTURE PERSPECTIVES OF CHRONOTHERAPY OF CANCER.
The suggestion that chemotherapy schedules adjusted to circadian rhythms will result
in better drug tolerance and higher efficacy dates almost 20 years, to when Haus et
al. [99] reported the circadian stage-dependence of arabinosyl-cytosine tolerance
and efficacy in leukemic mice. Since then, experimental evidence documenting the
critical importance of circadian timing of cytotoxic treatment has been mounting
steadily [200]. The most commonly used anticancer drugs have been shown to be either
substantially less toxic, more effective, or both, at certain circadian stages. More
recently, genetically engineered biological response modifiers and growth factors
have entered clinical trials. They form a broader basis for hope in the fight against
the solid tumors that most commonly afflict humans. Initial chronobiologic studies
showed that these new therapeutic agents can be several-fold more exquisitely
circadian-stage dependent in the amount of toxicity and anticancer activity [184].
Proper circadian timing will, therefore, be of growing, rather than diminishing,
importance as we learn to use these agents. An entirely independent, yet related,
literature documents the sequence and interval dependence of both conventional
chemotherapeutic agents and biological response modifiers. As treatment regimens are
becoming more complex, further progress against cancer will depend in part upon a
more sophisticated (technological) approach to drug administration.
Careful extrapolation from our extensive preclinical investigations has allowed us
to design time-qualified clinical treatment schedules for doxorubicin and cisplatin.
Optimal drug timing resulted in less toxic, higher dose intensity levels being
tolerated safely, with improved tumor control and patient survival. Since then,
similar clinical studies have been performed elsewhere, with results confirming and
emphasizing the importance of circadian drug timing [202].
As the extent of host-tolerance for each anticancer agent tested in humans differed
markedly as a function of its dosing time, the extension of circadian-timed therapy
is logical [203]. Since inter-individual differences may determine host
chronotolerance, and to some extent tumor susceptibility, circadian monitoring of
marker variables must be considered and developed.
To date only a few metastatic solid tumors can be cured by chemotherapy alone. Most
malignancies are primarily chemotherapy-resistant or will develop resistance. This
is especially true for the more frequent cancer types. We argue that more attention
must be paid to circadian drug timing, mode, route, drug sequence and intervals, and
dose intensity to make the currently available pharmacological tools more effective.
Priority setting for future study.
There are obviously too many anticancer agents to study each of them thoroughly. Even
if that could be accomplished, it is not desirable to put the same effort into a
chronobiologic study of each and every agent. Clinical considerations must, therefore,
determine which drugs are to be studied with respect to chronobiology. Thus, highest
priority must be given to agents that have:
1) known clinical activity, or (based upon screening information obtained in cell
lines of human tumors in nude mice or standard murine screening systems) have a high
likelihood of being active against the commonest solid tumors;
2) apparent predictable (and hopefully steep) dose intensity/response relationships;
3) a narrow therapeutic index;
From this, murine studies on bo lus toxicity and therapeutic efficacy of
cyclophosfamide show this drug to be highly indicated. This drug has just about the
broadest spectrum of clinical activity of any available anticancer agent. It has
severe toxicities and a narrow therapeutic index. Its dose intensity response
relationship is clear and steep. It is also used in very high doses for a growing
number of indications. There are some studies documenting a circadian rhythm in this
drug's therapeutic index, but additional intravenous bolus studies are required to
clarify the best circadian stages for its clinical use.
Automatic time-specified drug delivery systems.
Chronobiology introduces to medicine a new factor - time. It makes us not only think
differently about human physiology, pushing us out of old stereotypes that result
in treating patients' bodies in the same way when they sleep and when they are active,
but also brings with it many technical challenges. For example, in the case of cancer
treatment, it might appear rather difficult to deliver multi-agent therapy when each
drug should be administered as a long-lasting infusion and each in a different
circadian-dependent manner. Fortunately, the renaissance of interest in biologic
rhythmicity coincides with a period of unparalleled technological advance. Complex
devices with one or multiple reservoirs (each reservoir independently, complexly
programmable, with many steps which can also be completely coordinated by the program)
can now be used for optimized chronotherapy [204,205]. First and second generation
devices are currently available. These work reasonably well, and newer generations
of computerized drug delivery systems are becoming available. Most infusional therapy
is still monotherapy. However, infusion regimens for metagents will need to be
eventually be combined with both one another and with timed bolus treatments, as well
as with carefully sequenced applied cytokines and growth factors, etc.. Therefore,
further generations of automated drug delivery will be multi-channel devices. Each
channel will be independently programmable, allowing for concurrent or sequential
administration of drugs. All single or multiple channel devices used to deliver timed
bolus and or continuous infusion chemotherapy should have flexible programmability
and an internal clock and calendar capability.
Although wearable devices are most commonly employed for mono- or multi-agent
chemotherapy, implanted programmable pumps also work excellently. Unexpectedly, the
costs associated with the use of implanted programmable devices may be lower than
the use of wearable devices rented from home-care agencies when the treatment exceeds
a certain duration (usually 3-4 months). No implantable system is, however, yet
available with more than a single reservoir. The next individual step in circadian
optimized cancer therapy is the development of closed-loop systems, which are
self-regulated by sensors of circadian time-structures and respond with optimal
individual drug timing.
It is of practical value to describe these instruments currently available for
circadian time-based anti-cancer therapy. The first implantable programmable system
is the Synchromed pump (Medtronic Inc., Minneapolis, MN, USA). This system is filled,
and then programmed transcutaneously by telemetry after surgical implantation. The
system can deliver variable-rate infusion cycles or timed bolus injections either
intravenously, intra-arterially, or intra-thecally. The pump reservoir has a maximum
usable capacity of 18 mL. This system can administer either a single drug or a mixture
of two or more drugs which are compatible both with one-another and with the pump
components and catheter.
Single channel, wearable devices which can be flexibly programmed to deliver
time-shaped infusion are not currently available commercially, though a prototype
Autosyringe device used by Levi was designed to his own specifications (personal
communication with Dean Kaman, DEKA Laboratories).
The Strato Medical Corporation (Beverly, MA, USA) produces the only commercially
available single channel device with a clock and calendar. This device may be used
to increase and decrease the drug flow over time in a very limited circadian cycle.
Ivion (Englewood, CO, USA) has produced a 4-channel, flexibly programmable device
that can deliver optimal time-specified treatments for up to 4 drugs. The maximum
infusate volume is 30 mL per channel. However, the programming procedure requires
some skills, and once programmed, on-site schedule modifications are not possible.
Such modifications require the programming of a new micro-chip which stores the
information and controls the pump's actions. The device is also fairly bulky compared
to other portable pumps.
Devices in development which should be able to deliver time-based infusion of at least
one agent include the new Parker (Strato Medical, Beverly, MA, USA) micro-pump which
has up to 24 steps per program cycle. I-Flow Co. (Irvine, CA, USA) will offer a
4-channel wearable device for poly-chemotherapy. None of these companies currently
have a closed loop delivery system available, although several are developing this
technology. Thus, in the near future, we will be technically equipped to easily
administer time-specified mono- or combination therapy.
How these systems will be used.
It is interesting to speculate on how the findings of chronobiology will translate
into clinical practice. Automatic programmable, implanted, wearable, and bedside
systems will have uses in virtually every speciality of medicine and surgery.
Initially, open-loop and, subsequently, closed-loop multiple drug systems will
pervade medical practice.
Modern oncology will demand circadian timing-stipulated, multi-drug regimens,
biological therapies, and hybrid chemobiotherapies. Each of these types of therapy
will not only stipulate the time of day of each dose, but how many hours or days between
repeat cycles, as well as the order in which the agents or patterns of agents are
to be used. Using numerous drugs in the treatment of cancer is common, making multiple
reservoir systems very useful. Implantable systems will be used for loco-regional
therapy, for quasi-continuous systemic cell cycle synchronization, and for longterm
immune modulation. These systems will be complemented by the use of extracorporeal
devices for short-term therapy in the same patients.
Protocol design will no longer be constrained by questions of protocol compliance.
National cooperative groups and Cooperative Clinical Oncology Programs (C-COPs) will
be able to effectively test and apply complex basic findings to the more immediate
benefit of the patient with cancer.
Endocrinology will someday rely upon implanted, closed-loop, artificial hypothalmus,
pituitary, adrenal, and pancreas technologies. In the interim, many advances will
be made possible by simple, closed-loop systems. Right now, ultradian (high frequency)
and circadian patterned therapy is essential for treating fertility and growth
problems, and it will soon be required to alleviate sleep and emotional disorders.
Effective biotherapy will require complex administration of a wide variety of drugs
and biologicals. Their application in rheumatology, transplantation, cancer therapy,
and treatment of immunodeficiency syndromes (including AIDS) will be expedited by
these kinds of delivery systems. Also, in hematology, desferoxamine chelation
treatment of iron overload may be improved by automatic delivery systems.
Cardiologists and nephrologists treating arrhythmias or hypertension soon will be
assisted by closed-loop automatic systems, in which these new technologies will
"sense" the events that prompt therapeutic interventions. Even before this, however,
programmed, circadian-modified, transcutaneous, or intravenous delivery of
nitroglycerine, nitroprusside, dopamine, xylocaine, and other drugs may make
clinical sense.
Rheumatologists, allergists, and pulmonary doctors may use anti-inflammatory and
bronchodilators that are best given after careful consideration of their circadian
timing, using automatic programmable devices; while gastroenterologists will use H-2
blocking agents which are bioavailable at that time of day associated with highest
basal and stimulated acid secretion (i.e., 2-4 a.m.).
Intensive care unit acute care medicine may be the first place where multi-agent
therapy will be used in a closed-loop manner, administering diverse drugs to patients
with multiple organ failure, according to the physiologic - biochemical signals fed
to a device directly from the patients' own monitoring systems. These and many other
applications will result as these devices mature, become easier to use, and are
expanded to do more, more simply.
What should the use of these devices mean for the practice of medicine, and how will
their use affect chronobiology?
The adoption of "intelligent" automatic, programmable, drug delivery devices will
make medicine both intrinsically more complex and extrinsically more simple. It will
complex drug and biological regimens safer, less error-prone, and less expensive.
The therapy of serious diseases will be more uniform. Protocols developed in
university centers and by clinical research groups will be widely available to all
medical practitioners and, once instituted, will be carried out more exactly. Better
medicine will ultimately be more widely available to more citizens.
The clinical research to establish these protocols should be funded by collaboration
between government, industry, and academics. The pressure to produce and to market
new drugs may decrease and might well be replaced by a more rational pressure to
discover and effectively protect the best way of giving drugs. A wide variety of
devices and competing protocols will keep each system and protocol cost-effective,
and this competition will both assure a return on investment and hold down costs.
This cost-accounting of clinical applications research will result in less expensive
medical care.
Finally, the adoption of programmed automatic drug delivery will bring attention to
temporal chronobiologic questions which have been, until now, unanswerable. This
attention will turn chronobiology into what it truly is - a multidimensional and
dynamic perspective on life science. This movement toward considering the temporal
aspects of drug action (and administration) will lead to chronobiology becoming an
integral part of the make-up of biologists and physicians alike.
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