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.