现将朱彬先生的“肿瘤时间生物学研究进展”和 William J.M. Hrushesky 的“CHRONOTHERAPY OF CANCER:A MAJOR DRUG DELIVERY CHALLENGE”发表如下，供参考，相信本文对我院临床应用时间化疗有一定指导意义。
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肿瘤时间生物学研究进展

朱彬

     众所周知，生物体的生理活动、生化过程以及外在行为均存在着周期节律的特征，从系统、器官、组织水平到单个细胞，乃至各种亚显微结构其物质代谢的变化均有一定的节律性，这种节律性同样也表现在肿瘤细胞之间，从基因表达到细胞功能活动等均与正常组织细胞存在着明显的差异性，对肿瘤时间节律性的研究，为肿瘤的发生、发展及诊断治疗提供了新的思路和方法，并已成为近年来国内外肿瘤研究领域内的热点。

一．近日节律的分子结构

     生物界存在着周期不同的节律，其中，近日节律使有机体能适应每日外界环境如光照、温度、社会活动等的变化，使机体生命代谢中生理、生化、社会行为等功能活动之间能协调一致的进行。哺乳动物细胞内已经鉴定克隆出十种与近日节律相关的生物钟基因，它们参与调节着与细胞生理功能相关的其它基因的转录及转录后的过程，从而使细胞生理功能呈现出24小时的变化。现已证实，下丘脑视交叉上核的生物钟结构是机体生物节律的中枢性震荡器，即主要起搏点，它通过白天光照，夜间松果体分泌的褪黑素调控外周震荡器使机体生物节律与外周环境同步，生物体内最主要的近日节律就是休息—活动周期(rest-activity cycle)，机体正常组织细胞生长、增殖代谢均表现出一定生物节律，且该节律受机体rest-activity cycle影响。

二．近日节律与肿瘤发生的分子机制

     近年来研究发现，近日节律与肿瘤的发生密切相关。

1．近日节律与细胞周期

     近日节律基因通过调控细胞的增殖和死亡在肿瘤发生与抑制方面具有重要作用。
生物体另一个具有明显节律的生命现象就是细胞周期。通过对细胞周期与近日节律的共同研究发现，二者不能等同，如在增生不活跃或停止分裂的组织细胞，甚至体外培养诱导分化阻滞的纤维母细胞，虽然细胞停止了分化增生，但近日节律依然存在。进一步的研究发现，对于更新较快的组织细胞，与细胞分裂有关的基因、蛋白、酶等均表现出近日节律性，且众多在体研究实验证实，参与细胞增殖的许多基因均属于钟控基因，如研究发现，近日节律异常可引起细胞周期的时相发生改变，在小鼠，mper1基因表达发生位移，可引起小鼠肠道、骨髓等细胞周期的时相发生相应改变，mper2基因缺失的小鼠，与细胞周期相关的基因如cyclin D1, cyclin A, mdm-2或gadd45a的表达失去调控，表现出明显的患癌倾向。

2．近日节律与癌基因、抑癌基因

     研究发现，近日节律可通过调控原癌基因与抑癌基因的表达，在肿瘤发生中具有直接或间接的作用。
国外学者对正常人皮肤及口腔粘膜组织学检查，发现P53表达具有近日节律性，进一步提示依赖P53的细胞凋亡也具有近日节律性。Grundschober等人研究发现：基因产物BMAL1-Rev-erb β可直接调控原癌基因c-myc的表达。Levi等进一步证实，mper2突变小鼠c-myc基因过度表达，肿瘤发生率明显增高。

3．近日节律与血管内皮生长因子

血管生成与肿瘤生长、侵袭、远处转移密切相关，肿瘤生长需要有新生血管生成以供给其营养及其转移的途径，血管内皮生长因子(vascular endothelial growth factor,VEGF)是目前已知作用最强的肿瘤血管生成诱导因子，在恶性肿瘤，VEGF表达明显升高，且与肿瘤不良预后直接相关。最近的研究发现，植入到小鼠体内的肿瘤细胞VEGF mRNA水平表现出近日节律性，而这种节律性与近日表达的Per2及Cry1抑制了VEGF基因启动子的活性有关，表现出小鼠血浆中VEGF的浓度的白天升高，夜间下降，这为临床抗血管生成因子的择时治疗提供了参考。
肿瘤形成、生长的节律性

三．肿瘤生成和生长节律性

    了解肿瘤细胞以及正常组织细胞的增殖分化、生长代谢的节律性对于肿瘤的临床治疗具有重要意义，将DNA合成作为参数，各种肿瘤细胞分裂增殖的节律性不同，如卵巢癌的高峰时相位于11：00—15：00，乳腺癌为13：00—15：00，子宫颈癌为12：00，头颈部癌为10：00；而正常组织如口腔粘膜为20：00，直肠粘膜为7：00，骨髓为12：00—16：00等，提示选择恰当时间应用DNA合成抑制剂可提高药物疗效减轻毒副作用。另外，Hori等研究发现，实体瘤组织在机体休息期的血供是其活动期的3倍，提示单位时间内药物到达瘤体内的浓度也具有昼夜节律变化。

    近年研究还发现，宿主内分泌近日节律的改变可影响肿瘤的发生、发展。动物研究显示，损害下丘脑视交叉上核扰乱小鼠rest-activity cycle以及血清皮质酮激素的节律性，可使荷瘤小鼠的肿瘤生长速度较对照组快2—3倍，生存期明显缩短。流行病学调查也显示，轮换班的妇女乳腺癌的发病率明显增高。

四．肿瘤的时间药物治疗

   据报道，机体对大约30余种抗癌药物的耐受性或疗效随昼夜节律的改变而不同，其波动范围可达50%以上，因此，根据人体的生物节律作用用药具有明显的科学性。抗癌药物的细胞毒作用在24小时中随给药时间不同的有明显差异，其中的机制涉及细胞代谢、增殖过程及药物动力学的时间节律变化，对一些药物的作用机制研究发现，抑制细胞生长的药物其药物代谢动力学参数随给药时间的不同而改变，主要是由于体内快速增殖组织细胞对烷基化合物、铂复合物的细胞毒保护作用以及体内药物吸收代谢的酶、谷胱甘肽代谢酶活性的周期性变化所致。

    虽然对于机体、肿瘤与抗癌药物三者之间作用的时间节律机理尚不明确，但这种现象是客观存在的，它表明，同一剂量的药物，在机体rest-activity cycle的不同时间给药，产生不同的疗效，在周期的某一位相对机体产生有益作用，而在另一位相则产生有害作用，甚至可以致死，因此，择时用药具有重要的临床意义。

目前临床常用抗癌药物主要有以下几种：

    诺维本：是长春碱类细胞毒抗肿瘤新药，研究显示其血液毒性及临床疗效与用药的时间有密切关系。对接种P388白血病细胞的小鼠研究发现诺维本在19时或23时给药，小鼠平均生存时间最长，而在7时组最短，且在19时药物的耐受剂量明显高于7时组；而对正常细胞的研究显示，诺维本在19时或23时用药组P53表达的荧光指数高2倍，提示骨髓细胞与诺维本接触的时间位相不同，其修复能力也有差别。

     卡铂：对荷S180瘤小鼠腹腔注射给药，2时给药组动物死亡率最低，平均存活期最长，且外周血白细胞下降最不明显，而14时给药组动物死亡率最高。5-FU是近40多年治疗胃肠道肿瘤的常用药物之一，如何提高5-FU的抗肿瘤效果一直是人们关注的问题，研究表明卡铂能够调节5-FU的细胞毒作用，增强其抗肿瘤作用，同时发现昼夜节律对卡铂、5-FU的药动力学有一定影响。根据生物节律用药，可以通过与具有调节细胞自身节律性的某些酶如二氢嘧啶脱氢酶(dihydropyrimidine dehydrogease,DPD) 的变化而改变5-FU及卡铂的药代动力学，从而提高药物临床疗效，减低药物毒性反应的发生率及程度。
泰素帝：是一种紫杉醇类抗癌新药，抑制细胞的微管系统，对肺癌、乳腺癌等多种恶性肿瘤具有良好的杀伤作用，研究发现，泰素帝的治疗指数与其用药时间位相有一定关系，Granda等通过对小鼠乳腺癌的研究发现，无论泰素帝使用的剂量如何，最大疗效和耐受剂量则出现在活动期，休息期的肿瘤完全缓解率为81%，根据人类昼夜节律与小鼠相反的现象，从而推测对癌症患者泰素帝的最佳应用时间应该在午夜休息期间。

     生物免疫反应调节剂：已经证实人体免疫功能水平的高低具有一定的节律性，因此对一些生物免疫反应调节剂采用昼夜节律性用药，其疗效和毒性均会产生明显的差异。动物实验表明，小鼠脾脏细胞对IF-2、IFN的反应与接触时间呈强烈相关性，通过节律性用药，可以在一定程度上提高IFN的用药剂量而使毒性增加不明显或降低，实验表明，IFN的使用剂量可以提高到常规用药剂量的2～4倍，因此，通过昼夜节律性变化应用生物免疫反应调节剂是一种有效的优化用药方式。

     造血生长因子：粒子细胞集落刺激因子等造血生长因子的开发成功在很大程度上解决了化疗所致的粒细胞低下的毒性反应，使得大剂量应用细胞毒性抗癌药物成为可能。已有研究表明，不同时间用药影响到造血生长因子的效果。

五．临床研究

    目前，肿瘤的时辰化疗已广泛应用于临床，取得了较为可喜的成果，疗效较肯定的肿瘤主要有以下几种：
直肠癌：欧洲3个国家9个研究中心报道，116例转移性直肠癌，时间化疗和常规化疗各93例，两组有效率分别为51%与29%，严重粘膜毒性反应的发生率分别为14%和76%。外周神经炎的发生率分别是16%和31%，肿瘤无进展时间分别为6.4个月和4.9个月。因此目前认为，对晚期结肠癌患者采用近日节律性用药方案与传统以5-Fu为主的联合化疗方案相比，客观有效率可提高2～6倍，中位生存期可提高50%，不良反应率降低2～10倍左右。

    乳腺癌：研究结果发现，时辰化疗组与常规化疗组的临床完全缓解率分别为38.98%及23.5%，有效率80.7%与70.6%，病理完全缓解率26.5%、13.2%，二者具有明显差异。时辰化疗组III、IV度白细胞下降显著低于常规化疗组。

    卵巢癌：基础研究显示ADM、DDP的药力学与昼夜节律性有关，其剂量强度可由13%提高到45%，而且即使同一剂量强度，时辰用药疗效也明显优于常规治疗组，大户等采用ADM+DDP治疗卵巢癌患者，随访5年，结果早晨用ADM，而夜间用DDP的18例患者，5年生存率50%，而不考虑用药时间安排的17例患者，在2.5年随访时间内全部死亡。

    肺癌：Focan等采用5-Fu+CF+CBP(卡铂)时辰化疗32例晚期非小细胞肺癌患者，结果显示总的毒性反应均可耐受，4.6%出现III、IV度白细胞下降，7%出现III、IV血小板下降，7.8%出现恶心、呕吐，不到3%出现粘膜炎、腹泻、脱发等，较常规化疗者毒性反应均明显降低。

    胰腺癌：以往研究显示对胰腺癌的化疗效果较差，而且常规多药联合化疗的效果并不优于单用5-Fu，但近年来一些研究结果研究显示，时间化疗在一定程度上有助于提高胰腺癌患者的生存期。

六．肿瘤时间放疗学研究

    目前针对肿瘤放疗的时间治疗研究开展较少。基础研究已经发现，细胞对射线的敏感性随细胞自身的周期而有差异，当干细胞集团是同步的情况下，在昼夜节律的不同时相以X射线照射，动物致死量因放射时间不同而异。

    由于人类癌细胞的增殖周期有明显的时间特征，而且与正常的增殖周期有显著的差异，因此选择适当的放疗时间，可以使放射线对癌细胞的杀伤力最大而对正常细胞的杀伤力最低，即使放疗疗效最高而副作用最小，该研究具有远大前景。

七．近日节律与肿瘤诊断及预后

    近年对与激素相关的肿瘤流行病学的研究中发现，体内激素水平近日节律的变化可以预示肿瘤的发生，如激素近日节律改变的妇女较节律正常者乳腺癌的患病率明显增高。

    目前，已经有许多生理指标如肿瘤细胞的分化程度、有无转移或肝脏转移后肿瘤占位的大小等相结合用于临床判断恶性肿瘤患者的预后。近年来研究发现，近日节律可作为一个独立的指标用于一些肿瘤预后的判断。国外学者对rest-activity cycle及皮质激素的近日节律性与肿瘤患者的生存质量及预后的相关性进行了研究。

    Mormont等对200名转移性直肠癌患者rest-activity cycle节律性的研究发现，该节律明显的患者较节律幅度降低或节律发生改变的患者生存期长，前者4年存活率是后者的2倍。另外对104名乳腺癌患者皮质激素近日节律的研究，也有同样发现，即皮质激素节律正常的患者其4年存活率是节律有变化患者的2倍。上述资料显示，肿瘤患者近日节律的改变可加快肿瘤的生长，虽然其机制仍需进一步探讨。

展望

八．展望

    通过对肿瘤时间节律的研究，可以了解与肿瘤发生、发展有关的分子机制，对肿瘤的诊断、判断肿瘤的预后及转归，以及指导临床治疗的最佳时间具有重要意义，同时时间化疗的实施，还将有助于推动“老药新用”的临床研究和新品种的开发和利用。相信对肿瘤的时间生物学的研究，将为人类最终攻克癌症作出巨大贡献。

参考文献

［1］ Levi. F. Cancer chronotherapy: principles, applications, and perspectives. Cance, 2003,97:155—169
［2］ Rosbash.M., Takahashi. S. Circadian rhythm: the cancer connection. Nature, 2002,420(6914):373—379.
［3］ Coudert B, Focan. E. Donato. P etc.It’s time for chronotherapy! European J of Cancer. 2002,38:s50—s53

CHRONOTHERAPY OF CANCER:
A MAJOR DRUG DELIVERY CHALLENGE

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)