**5. Human beings**

*Chronobiology - The Science of Biological Time Structure*

reduces septic death in Bmal1-deficient mice [47–49].

**NSCLC**

**4.1 Animal data**

antibody or supplementation with recombinant interleukin-7 (IL-7) facilitates

**4. Chronopharmacology of anticancer drugs active against human** 

the circadian chronoefficacy of cyclophosphamide (CPA) in LLC [51].

marrow division activity (around 9 HALO) [52].

(i.e., ERCC1 and AP1) [53].

in human NSCLC [53].

downstream factors increase [55, 56].

Chronotolerance to an experimental radioimmunotherapy with 131 I-anticarcinoembryonic antigen (CEA) IgG was reported [52]. A 30% increase in maximum tolerated dose was possible when the drug was given at the trough of the bone

Clock, as a member of histone acetyltransferases, controls acetylation of histone 4 required for repair of DNA double-strand breaks thanks to several repair genes such as excision repair cross-complementing group 1 (ERCC1) or activator protein 1 (AP1) [6]. Expression of histone acetyltransferase genes is associated with cisplatin resistance [6, 53]. Histone acetyltransferase inhibition (i.e., by vorinostat) may increase carboplatin and paclitaxel activity in NSCLC cells [54]. The acetyl-CoAbinding motive is found in clock and shows sequence similarity with MYST members, i.e., Tip 60. Tip 60 which is overexpressed in human epidermoid cisplatin-resistant cancer cells [53] exerts a control regulation on several genes implicated in DNA repair

Furthermore, the promoter region of the Tip 60 gene contains several E-boxes, and its expression is regulated by the E-box-binding circadian transcription factor clock! Thus, clock and Tip 60 regulate not only transcription but also DNA repair, through periodic (diurnal) histone acetylation in cell populations that can be found

Finally, of interest, diurnal-varying pharmacokinetics of erlotinib (a largely used tyrosine kinase inhibitor (TKI) for treating human NSCLC) has been reported

both in xenograft-bearing nude mice [55] and in Lewis tumor-bearing mice. [56]. Circadian rhythm plays a critical role in the pharmacokinetics of erlotinib in mice, and the mechanisms may be attributed to gene expression rhythms of drug-metabolizing enzymes in liver tissues [56]. The inhibitory effect of erlotinib on phosphorylation of EGFR, AKT (type of serine/threonine protein kinase, also called protein kinase B), and mitogen-activated protein kinase (MAPK) varies with its administration time. The results indicate that the antitumor effect of erlotinib is more potent when the drug is administered when the activities of EGFR and its

microbial clearance, inhibits T cell apoptosis, reduces multiple organ dysfunction, and

Circadian variation in pharmacokinetics (PK) has been observed in rodents for all the drugs routinely administered to LC patients, i.e., pyrimidine derivatives, anthracyclines, vinca-alkaloïds (vinorelbine), topoisomerase inhibitors, taxanes, platinum derivatives, gemcitabine, other antimetabolites, etc. [1, 3–5]. These chrono-PK were expressed though circadian-varying metabolization, detoxification, excretion, and also maximal concentration (CMax) or area under the curve (AUC) [1, 3–5]. Chronotolerance has been observed in rodent studies long time ago for any chemotherapy agents routinely used for NSCLC patients [1–5]. As a recent example, best tolerance and chronoefficacy of gemcitabine alone or in combination with cisplatin were observed with best antitumor efficacy when both drugs were given around their least toxic time schedule, respectively, 11 and 15 hours after light onset (HALO) in animal facility [50]. In an older study, Flentje et al. had also documented

**80**

In human as well, pharmacokinetics of some major drugs used in NSCLC have been reported to be circadian varying [1–5, 28, 44]. Circadian variation of plasma 5-fluorouracil (5 FU) concentration has been repeatedly observed when the drug is infused for a few days at a constant rate [57, 58]. This was reported when the drug was given either as a single agent or with a platinum derivative [58]. Similarly, plasmatic concentration of vindesine, a semisynthetic vinca-alkaloïd derived from vinblastine, exhibit circadian variation with peak between 9 am and 3 pm, when infused at a constant rate for 48 h [59]. Also the fixation of platinum ion to plasma proteins was shown to be circadian varying with an acrophase during late afternoon [60]. More recent assessment of circadian variability of cisplatin pharmacokinetics confirmed that cisplatin clearance was 1.38- and 1. 22-fold higher for total and unbound drug with administration at 06:00 pm vs. 06:00 am [61].

Host chronotolerance to anticancer drugs used in NSCLC patients has also been observed in clinical practice. Pyrimidine derivatives such as 5 FU are less toxic when infused during nighttime sleep [1, 3–5, 57, 58]. Also platinum derivatives such as cisplatin, carboplatin, and oxaliplatin are better tolerated between 3 and 6 pm while anthracyclines are less toxic in the morning [1, 3–5, 57, 58]. The first reported chronotherapy randomized trial, based on diurnal cell kinetics, treating mostly NSCLC patients, compared a 40-h sequential chemotherapy beginning either at 10 am or at 10 pm [28]. In this study, patients who received the sequential chronotherapy from 10 am experienced significantly greater granulocyte toxicity [14, 28].

Focan et al. also reported on host chronotolerance of 124 chemotherapy-naïve advanced NSCLC patients, receiving randomly etoposide for 3 days either at 6 am

#### **Figure 3.**

*Programs of ambulatory chronotherapy with 5 FU, folinic acid (leucovorin-LV), and carboplatin (CBDCA). The reference schedule is compared to two others with varying peaks (−8 h; +8 h). Daily delivery is automatically repeated by a chrono-programmable pump (Melodie) five times every 21 days (FFC5\_16).*

(group A) or 6 pm (group B) and cisplatin at day 4 at 6 pm [62]. A lesser degree of hematological toxicity was documented in group A, while cisplatin was better tolerated in group B [62]. Similar results were reported by Krakowski et al. [63].

Focan et al. also performed a randomized phase I–II trial comparing as first-line treatment, a complex sequential chronotherapy with 5 FU, folinic acid (FOL), and carboplatin with 24-h sinusoidal variation of drug delivery [64] (**Figure 3**). The reference schedule (peaks of 5 FU and FOL at 4 am, peak of carboplatin at 4 pm vs. two other schedules peaking, respectively, at + or – 8 hours, repetition for 5 days every 3 weeks) appeared to be the least toxic one with an overall excellent clinical tolerance [14, 64] (**Figure 4**). Toxicity data were reviewed in order to detect a possible gender effect as had been observed in metastatic colorectal cancer [3–5, 65]. Despite of no significant difference in treatment adaptation or dose intensity between men and women, overall increased serious toxicities were recorded in women versus men. Severe leukopenia and mucositis occurred more than twice as frequently in women than in men (grade 3–4 leukopenia per course, 7.7 vs. 3.2%; grade 3–4 mucositis, 6.6 vs. 1.2%) [14].

According to the results of the phase I–II trial described above [64], a phase II study was further performed on 68 advanced NSCLC previously untreated patients

#### **Figure 4.**

*Tolerability of chronomodulated 5 FU-LV-CBDCA infusion (grades 3–4 toxicities; in green, leucocytes; in red, granulocytes; in yellow, mucositis). Reference schedule was clearly the least toxic one (p < 0.007–0.025).*


**83**

immune function [72, 73].

be the advancing stage of disease [71–76].

*Integration of Chronobiological Concepts for NSCLC Management*

**6.1 Hormones, immune functions, and tumor markers**

with the best circadian schedule [3, 14]. An excellent therapeutic index with a maximum of 17%, grades II–IV toxicities were observed and a gender effect was

varying expression was quite different in men and women in oral mucosa [44].

**6. Circadian biological and behavioral determinants in LC patients**

A number of groups studied tumor-marker (CEA-carcinoembryonic antigen, alpha-fetoprotein, and others) rhythms but with similar disappointing results [1, 2, 67]. If in controls, a clear group circadian rhythmicity with an afternoon peak around 03:00 pm was evident, in cancer patients, individual variability or absence

Hormonal, hematological, and rest-activity cycles perhaps might constitute more promising markers of the host's internal circadian time structure. The most prominent hormonal circadian rhythms are cortisol (peak time occurs early in the morning in diurnally active persons) and melatonin (peak time occurs during the first half of the dark period in diurnally active people) [3–5, 67]. Important alterations of the normal circadian profile of these rhythms have been described in lung and other cancer patients with low performance status and high tumor burden [67]. Bartsch and colleagues [68] reported peculiarities in the cortisol and melatonin circadian rhythms in LC patients. Experimental data suggest interactions between interleukin-2 (IL-2; antitumor immune response is an IL-2-dependent phenomenon) and the pineal gland, which also may play a role in the control of immunity and cancer growth [69, 70]. The melatonin rhythm was evaluated in a group of LC patients receiving subcutaneous IL-2 treatment [70]. Prior to IL-2 therapy, none of the patients showed a normal 24-h rhythm of melatonin; IL-2 administration induced a normalization of the melatonin circadian rhythm with a nighttime peak in the majority of cases [70]. This observation suggests that abnormal pineal function in some lung cancer patients might arise in part from altered endogenous IL-2 production [70]. On the other hand, IL-2 administration induced a rise in cortisol with maintenance of 24-h rhythmicity [70]. Melatonin, tryptophan, and 6-sulfatoxymelatonin circadian profiles in blood and urine were compared in 30 advanced NSCLC cancer versus 63 healthy volunteers [66, 71]. All three molecule concentrations were significantly lower in cancer patients with a significant inverse correlation between melatonin and tryptophan levels [71]. Circadian rhythmicity in growth hormone (GH) and insulin-like growth factor-1 (IGF-1) was studied in control subjects and LC patients [72]. GH stimulates IGF-1 production in the liver and other tissues, while IGF-1 can promote cell cycle progression and apoptosis inhibition. GH and IGF-1 also stimulate lymphopoiesis and

In LC patients, a progressive increase in GH and a steady decline of IGF-1 serum levels with loss of circadian rhythmicity were observed. This loss of diurnal rhythms could play a role in carcinogenesis and tumor growth regulation [71–75]. All profiles of time-related neuroendocrine-immune system components seemed to

Further analyses in advanced colorectal cancers have shown that the pattern of chronotolerance in men was rather sinusoidal with an optimal time corresponding to the reference modality; conversely in women the pattern was damped with optimal peaks of delivery possibly located 6 h later than in men [66]. The male subgroup showed a mean clearance (CL) value twice larger than the value observed in the female subgroup [66]. On the other hand, one has also to remind that the distribution of genes with circadian-

*DOI: http://dx.doi.org/10.5772/intechopen.85710*

confirmed (**Table 1**) [3, 14].

of rhythm were evidenced [1, 67].

*Chronobiology - The Science of Biological Time Structure*

(group A) or 6 pm (group B) and cisplatin at day 4 at 6 pm [62]. A lesser degree of hematological toxicity was documented in group A, while cisplatin was better toler-

Focan et al. also performed a randomized phase I–II trial comparing as first-line treatment, a complex sequential chronotherapy with 5 FU, folinic acid (FOL), and carboplatin with 24-h sinusoidal variation of drug delivery [64] (**Figure 3**). The reference schedule (peaks of 5 FU and FOL at 4 am, peak of carboplatin at 4 pm vs. two other schedules peaking, respectively, at + or – 8 hours, repetition for 5 days every 3 weeks) appeared to be the least toxic one with an overall excellent clinical tolerance [14, 64] (**Figure 4**). Toxicity data were reviewed in order to detect a possible gender effect as had been observed in metastatic colorectal cancer [3–5, 65]. Despite of no significant difference in treatment adaptation or dose intensity between men and women, overall increased serious toxicities were recorded in women versus men. Severe leukopenia and mucositis occurred more than twice as frequently in women than in men (grade 3–4 leukopenia per course, 7.7 vs. 3.2%; grade 3–4 mucositis, 6.6 vs. 1.2%) [14].

According to the results of the phase I–II trial described above [64], a phase II study was further performed on 68 advanced NSCLC previously untreated patients

*Tolerability of chronomodulated 5 FU-LV-CBDCA infusion (grades 3–4 toxicities; in green, leucocytes; in red, granulocytes; in yellow, mucositis). Reference schedule was clearly the least toxic one (p < 0.007–0.025).*

ated in group B [62]. Similar results were reported by Krakowski et al. [63].

**82**

**Table 1.**

**Figure 4.**

*Gender effect in NSCLC.*

with the best circadian schedule [3, 14]. An excellent therapeutic index with a maximum of 17%, grades II–IV toxicities were observed and a gender effect was confirmed (**Table 1**) [3, 14].

Further analyses in advanced colorectal cancers have shown that the pattern of chronotolerance in men was rather sinusoidal with an optimal time corresponding to the reference modality; conversely in women the pattern was damped with optimal peaks of delivery possibly located 6 h later than in men [66]. The male subgroup showed a mean clearance (CL) value twice larger than the value observed in the female subgroup [66]. On the other hand, one has also to remind that the distribution of genes with circadianvarying expression was quite different in men and women in oral mucosa [44].

## **6. Circadian biological and behavioral determinants in LC patients**

#### **6.1 Hormones, immune functions, and tumor markers**

A number of groups studied tumor-marker (CEA-carcinoembryonic antigen, alpha-fetoprotein, and others) rhythms but with similar disappointing results [1, 2, 67]. If in controls, a clear group circadian rhythmicity with an afternoon peak around 03:00 pm was evident, in cancer patients, individual variability or absence of rhythm were evidenced [1, 67].

Hormonal, hematological, and rest-activity cycles perhaps might constitute more promising markers of the host's internal circadian time structure. The most prominent hormonal circadian rhythms are cortisol (peak time occurs early in the morning in diurnally active persons) and melatonin (peak time occurs during the first half of the dark period in diurnally active people) [3–5, 67]. Important alterations of the normal circadian profile of these rhythms have been described in lung and other cancer patients with low performance status and high tumor burden [67].

Bartsch and colleagues [68] reported peculiarities in the cortisol and melatonin circadian rhythms in LC patients. Experimental data suggest interactions between interleukin-2 (IL-2; antitumor immune response is an IL-2-dependent phenomenon) and the pineal gland, which also may play a role in the control of immunity and cancer growth [69, 70]. The melatonin rhythm was evaluated in a group of LC patients receiving subcutaneous IL-2 treatment [70]. Prior to IL-2 therapy, none of the patients showed a normal 24-h rhythm of melatonin; IL-2 administration induced a normalization of the melatonin circadian rhythm with a nighttime peak in the majority of cases [70]. This observation suggests that abnormal pineal function in some lung cancer patients might arise in part from altered endogenous IL-2 production [70]. On the other hand, IL-2 administration induced a rise in cortisol with maintenance of 24-h rhythmicity [70].

Melatonin, tryptophan, and 6-sulfatoxymelatonin circadian profiles in blood and urine were compared in 30 advanced NSCLC cancer versus 63 healthy volunteers [66, 71]. All three molecule concentrations were significantly lower in cancer patients with a significant inverse correlation between melatonin and tryptophan levels [71].

Circadian rhythmicity in growth hormone (GH) and insulin-like growth factor-1 (IGF-1) was studied in control subjects and LC patients [72]. GH stimulates IGF-1 production in the liver and other tissues, while IGF-1 can promote cell cycle progression and apoptosis inhibition. GH and IGF-1 also stimulate lymphopoiesis and immune function [72, 73].

In LC patients, a progressive increase in GH and a steady decline of IGF-1 serum levels with loss of circadian rhythmicity were observed. This loss of diurnal rhythms could play a role in carcinogenesis and tumor growth regulation [71–75]. All profiles of time-related neuroendocrine-immune system components seemed to be the advancing stage of disease [71–76].

Mazzocoli et al. assessed altered time neuroendocrine-immune system function in lung cancer patients [75]. Circadian rhythmicity with night acrophases was validated in the control group for hormone serum level (melatonin, TRH, TSH, GH,) and for lymphocyte subset variation (CD3-, CD4-, HLA DR-, CD20-, and CD25-expressing cells). Cortisol, CD6, CD8 bright, CD8 dim, CD16, TcR-delta-1, and delta-TcS1 presented circadian rhythmicity with acrophase in the morning/at noon. In LC patients cortisol, TRH, TSH, and GH serum level and all lymphocyte subsets (except for CD4) showed some altered circadian rhythmicity. Mesor of cortisol, TRH, GH, IL-2, and CD16 was increased, whereas that of TSH, IGF-1, CD8, CD8 bright, TcR-delta-1, and delta-TcS1 was decreased [75]. Peak times however are related similar to those of control subjects [75]. The melatonin/cortisol mean nocturnal level ratio was also decreased in LC patients [75, 76]. Taken together, these results suggested that lung cancer is associated with alteration in the proportions and 24-h profiles of various lymphocyte subsets; this may be related to disease stage and probably altered immune function [73–75, 76].

Lissoni et al. also observed in NSCLC treated by chemotherapy (+/− melatonin) that lymphopenia and altered cortisol rhythmicity were associated with worsened quality of life (QOL), loss of psychosexual identity, and lower spiritual and faith scores [77, 78].

#### **6.2 Host marker rhythms**

The persistence of a circadian time structure like that of control normal subjects seems to be an independent prognostic factor, at least in advanced breast or colon cancers [10, 18, 79, 80]. As stated earlier, the strongest circadian rhythms are those of cortisol (with the clinical interest being the morning-afternoon gradient) and melatonin (with a nighttime peak) [2, 4]. Noninvasive easy-to-repeat assessment techniques have been validated for the determination of the 24-h time structure: titrations of cortisol and melatonin in the saliva and 6-hydroxymelatonin sulfate in the urine [81]. Nocturnal urinary 6-sulfatoxymelatonin is also correlated with the proliferation of cell nuclear antigen (PCNA) in lung tumors [68]. Thus, its determination might constitute a noninvasive tool to estimate circadian tumor cell proliferation in lung or other tissues.

Cortisol diurnal rhythm and slope have been related to survival in metastatic breast cancer patients by Sephton et al. [18]. Similarly, the same authors together with Chinese counterparts could also link the quality of persistent diurnal cortisol rhythm to the prognosis (survival) in LC patients [19, 20].

Assessment of the rest-activity circadian cycle by actometry measurements also seems an easy way to estimate the general circadian profile of individuals [1, 4, 5, 82–84]. In advanced colorectal cancer, it was demonstrated in two studies that patients retaining a prominent rest-activity circadian rhythm will enjoy better quality of life and sleep, less fatigue, less depression, and improved survival [82–84]. Such evaluations were proposed to lung cancer patients and validated [82–84]. Focan et al. evaluated rest-activity rhythms in 28 advanced NSCLC before chronotherapy [83]. Better general physical activity and circadian rhythmicity were recorded in those patients receiving also corticosteroids [83]. Hrushesky and his group also studied circadian function in NSCLC patients by actigraphic recordings [82, 84]. They tried to correlate the rest-activity rhythms with sleep disturbances, quality of life (European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30)) and mood (anxiety; depression—HADS—hospital anxiety and depression scale) [82, 84]. All patients suffered from severe disturbances of daily sleep-activity cycles, but each patient also maintained some degree of circadian organization. QOL measurements were correlated with circadian destructors, fatigue prominent during daytime, and altered moods [82, 84].

**85**

*Integration of Chronobiological Concepts for NSCLC Management*

Before initiation of their radiotherapy, a high percentage (30–50%) of 185 patients experienced significant disturbances in sleep-wake circadian rhythmicity;

Recent clinical observations have shown that elevated levels of TGF-alpha are associated with fatigue, flattened circadian rhythms, loss of appetite, and depression in patients with metastatic cancer [46]. These data support the hypothesis that a symptom cluster of fatigue, appetite loss, and sleep disruption commonly seen in cancer patients may be related to EGFR ligands released either by the cancer itself or by the host in response to the stress of cancer and suggest that further examination of their role in the production of symptom clustering is warranted [46]. Those observations also suggest to consider the central clock as a possible target for restoration of normal circadian rhythmicity in cancer

During the last decade, the management of NSCLC has evolved [86–97]. Platinum-based chemotherapy remains the standard front-line in treatment of advanced unresectable NSCLC in which cisplatin or carboplatin are combined with another chemotherapeutic agent such as taxanes, pemetrexed, or gemcitabine [87]. However the results in terms of response rate, progression-free survival, and

Thus, progresses confirmed by phase III trials came from targeted and immunotherapeutic biological approaches. Targeted therapies against EGFR mutations and anaplastic lymphoma kinase (ALK) gene rearrangement have improved the survival in a small proportion of patients whose tumors were expressing these molecular

Also the recent development and success of immune checkpoint inhibition of programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) for treating metastatic cancers seemed to

Unfortunately to the best of our knowledge, despite precise theoretical observations depicted related to circadian expression, on targets of TKI inhibitors, EGFR blockers, antiangiogenic agents, and immune active-agents (lymphoid system; PD1; PDL1), by now no study has been launched to take into account any

As a matter of fact, in NSCLC, only a few studies deal with considerations on

Studies from Focan et al. have already been mentioned and reviewed elsewhere [14]. In the first pioneered study [14, 28], a significant better tumor outcome was observed in the group treated at the better dosing time, thus from 10 am for a 40-h sequential schedule [14]. In the second phase III trial using etoposide and cisplatin, despite varying toxicities, there were no differences in overall drug dose intensities nor in tumor outcome gauged by the frequency of tumor responses as well as survival [62]. On the contrary, Krakowski et al. observed an increased dose intensity of

median overall survival remained stable over time [86–97].

open a new pavement for fruitful research [86, 92–97].

drugs when given at their best circadian schedule [63].

these perturbations occurred in both sleep initiation and maintenance [85].

*DOI: http://dx.doi.org/10.5772/intechopen.85710*

patients!

**7. Clinical trials**

**7.1 General considerations**

abnormalities [88–91].

**8. Chemotherapy**

chronobiological considerations!

temporal dimension for drug delivery.

*Integration of Chronobiological Concepts for NSCLC Management DOI: http://dx.doi.org/10.5772/intechopen.85710*

Before initiation of their radiotherapy, a high percentage (30–50%) of 185 patients experienced significant disturbances in sleep-wake circadian rhythmicity; these perturbations occurred in both sleep initiation and maintenance [85].

Recent clinical observations have shown that elevated levels of TGF-alpha are associated with fatigue, flattened circadian rhythms, loss of appetite, and depression in patients with metastatic cancer [46]. These data support the hypothesis that a symptom cluster of fatigue, appetite loss, and sleep disruption commonly seen in cancer patients may be related to EGFR ligands released either by the cancer itself or by the host in response to the stress of cancer and suggest that further examination of their role in the production of symptom clustering is warranted [46]. Those observations also suggest to consider the central clock as a possible target for restoration of normal circadian rhythmicity in cancer patients!
