Circadian chemotherapy for gynecological and genitourinary cancers

The circadian timing of surgery, anticancer drugs, radiation therapy, and biologic agents can result in improved toxicity profiles, tumor control, and host survival. Optimally timed cancer chemotherapy with doxorubicin or pirarubicin (06:00h) and cisplatin (18:00h) enhanced the control of advanced ovarian cancer while minimizing side effects, and increased the response rate in metastatic endometrial cancer. Therapy of metastatic bladder cancer with doxorubicin–cisplatin was made more tolerable by this same circadian approach resulting in a 57% objective response rate. This optimally timed therapy is also effective in the adjuvant setting, decreasing the expected frequency of metastasis from locally advanced bladder cancer. Circadian fluorodeoxyuridine (FUDR) continuous infusion (70% of the daily dose given between 15:00h and 21:00h) has been shown effective for metastatic renal cell carcinoma resulting in 29% objective response and stable disease of more than 1 yr duration in the majority of patients. Toxicity is reduced markedly when FUDR infusion is modulated to circadian rhythms. In a multicenter trial in patients with metastatic renal cell cancer, patients were randomized to a flat or a circadian-modified FUDR infusion. This study confirmed a significant difference in toxicity and dose intensity, favoring the circadian-modified group. Hormone refractory metastatic prostate cancer has been treated with circadian-timed FUDR chemotherapy; however, without objective response. Biological agents such as interferon-α and IL-2 have shown low but effective disease control in metastatic renal cell cancer, however, with much toxicity. Each of these cytokines shows circadian stage dependent toxicity and efficacy in model systems. In summary, the timing of anthracycline, platinum, and fluoropyrimidine-based drug therapies during the 24h is relevant to the toxic–therapeutic ratio of these agents in the treatment of gynecologic and genitourinary cancers.     

Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics

Even though 5-fluorouracil (FU) is one of the oldest anticancer drugs, its use in cancer chemotherapy continues to increase. Fluorouracil is a pro-drug that requires intracellular activation to exert its effects. This makes it difficult to associate blood drug concentration with cell toxicity directly, although data from the literature show the existence of such a relationship. The relationship between FU pharmacokinetics and patient response has been explored extensively and reports attest a link between systemic drug exposure and response and survival. This has led to the concept of maximal tolerated exposure, and strategies to achieve this rely on pharmacokinetic follow-up and individual dose adjustment. More than 80% of the administered FU dose is eliminated by catabolism through dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme. Dihydropyrimidine dehydrogenase activity is found in most tissues but is highest in the liver. Peripheral blood mononuclear cells (PBMC) are used to monitor clinically DPD activity. A significant, but weak correlation between PBMC and liver DPD activity has been observed. The relationship between PBMC-DPD activity and FU systemic clearance is weak (r²=0.10); thus, simply determining PBMC-DPD is not sufficient to predict accurately FU clearance. Population pharmacokinetic analysis identified patient co-variables that influence FU clearance; drug kinetics is significantly reduced by increased age, high serum alkaline phosphatase, length of drug infusion, and low PBMC-DPD. Autoregulation of FU metabolism also is suggested; inhibition of DPD activity was observed after FU administration in both colorectal cancer patients and an animal model. Circadian rhythmicity in DPD activity is suggested from both human and animal investigations. In patients receiving protracted low dose 5-FU infusion, the circadian rhythm in FU plasma concentration peaks at 11:00h and is lowest at 23:00h, on average. The inverse relationship observed between the circadian profile of FU plasma concentration and PBMC-DP activity in these same patients suggests a link between DPD activity and FU pharmacokinetics. The impact of the biological time of drug administration was also studied with short venous infusions; clearance was 70% greater at 13:00h than at 01:00h. Similarly, peak drug concentration occurred in the first half of the night in patients receiving constant rate 5-FU infusion for 2-5 d. Several studies describe wide interindividual variation in the timing of the peak and trough of the 24h rhythm in DPD activity. The rational for FU chronomodulated therapy has been the circadian rhythm in host drug tolerance, which is greatest during the night time when the proliferation of normal target tissue is least. A randomized study of chronomodulated FU therapy with maximal delivery rate at 04:00h was shown clearly to be significantly more effective and less toxic than control flat FU therapy. Future research must focus on easy-to-obtain markers of specific rhythms to individualize the chronomodulated FU delivery.     

Clinical pharmacokinetics of 5-fluorouracil with consideration of chronopharmacokinetics

Even though 5-fluorouracil (FU) is one of the oldest anticancer drugs, its use in cancer chemotherapy continues to increase. Fluorouracil is a pro-drug that requires intracellular activation to exert its effects. This makes it difficult to associate blood drug concentration with cell toxicity directly, although data from the literature show the existence of such a relationship. The relationship between FU pharmacokinetics and patient response has been explored extensively and reports attest a link between systemic drug exposure and response and survival. This has led to the concept of maximal tolerated exposure, and strategies to achieve this rely on pharmacokinetic follow-up and individual dose adjustment. More than 80% of the administered FU dose is eliminated by catabolism through dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme. Dihydropyrimidine dehydrogenase activity is found in most tissues but is highest in the liver. Peripheral blood mononuclear cells (PBMC) are used to monitor clinically DPD activity. A significant, but weak correlation between PBMC and liver DPD activity has been observed. The relationship between PBMC–DPD activity and FU systemic clearance is weak (r²=0.10); thus, simply determining PBMC–DPD is not sufficient to predict accurately FU clearance. Population pharmacokinetic analysis identified patient co-variables that influence FU clearance; drug kinetics is significantly reduced by increased age, high serum alkaline phosphatase, length of drug infusion, and low PBMC–DPD. Autoregulation of FU metabolism also is suggested; inhibition of DPD activity was observed after FU administration in both colorectal cancer patients and an animal model. Circadian rhythmicity in DPD activity is suggested from both human and animal investigations. In patients receiving protracted low dose 5-FU infusion, the circadian rhythm in FU plasma concentration peaks at 11:00h and is lowest at 23:00h, on average. The inverse relationship observed between the circadian profile of FU plasma concentration and PBMC–DP activity in these same patients suggests a link between DPD activity and FU pharmacokinetics. The impact of the biological time of drug administration was also studied with short venous infusions; clearance was 70% greater at 13:00h than at 01:00h. Similarly, peak drug concentration occurred in the first half of the night in patients receiving constant rate 5-FU infusion for 2–5 d. Several studies describe wide interindividual variation in the timing of the peak and trough of the 24h rhythm in DPD activity. The rational for FU chronomodulated therapy has been the circadian rhythm in host drug tolerance, which is greatest during the night time when the proliferation of normal target tissue is least. A randomized study of chronomodulated FU therapy with maximal delivery rate at 04:00h was shown clearly to be significantly more effective and less toxic than control flat FU therapy. Future research must focus on easy-to-obtain markers of specific rhythms to individualize the chronomodulated FU delivery.     

Circadian Rhythms of Renal Hemodynamics in Unanesthetized, Unrestrained Rats

Catheters were placed in the jugular vein and femoral artery of male Sprague-Dawley rats and connected to a specially designed perfusor for continuous constant infusion of 0.9% NaCl and a syringe to perform simultaneous and intermittent blood collections. This permitted continuous 24-h study of renal hemodynamics, estimated by inulin (C_in) and p-amino-hippuric acid (C_PAH) clearances; C_in represents glomerular filtration rate and C_PAH renal plasma flow. Animals were individually housed in metabolism cages in a controlled environment with light/dark 12:12 h. Urine was collected every 4 h (12:00, 16:00, 20:00, 24:00, 04:00, and 08:00) and blood sampled at the midpoint of urine collection periods. Urine and plasma sodium, potassium, inulin, and PAH were spectrophotometrically assessed. During continuous infusion of isotonic saline, C_in exhibited circadian changes with large decrease between 12:00 and 20:00 h (0.9 ± 0.2 ml/min) and acrophase at 00:30 h. Rhythmicity in C_PAH was similar with the minimum between 16:00 and 20:00 h (2.5 ± 0.3 ml/min) and peak between 00:00 and 04:00 h (acrophase at 00:25 h). Water and electrolyte excretion were also circadian rhythmic with a similar nighttime enhancement and daytime minimum. Such circadian changes persisted during continuous 0.9% NaCl infusion for several consecutive days. The unanesthetized, unrestrained rat model enables investigations in renal chronopharmacology and chronotoxicology.     

Weekly Taxol (Paclitaxel) administration in the second-line treatment of breast cancer

Based on phase II and III trials Taxol administered in the form of three times/week 1 or 3 hr infusion as mono-or combined chemotherapy of breast cancer is an effective treatment option. These studies proved that this form of drug delivery is effective and well tolerated and the overall response rate is around 50%. The 1 hr weekly infusion of Taxol is an effective second-line treatment in metastatic breast cancer and is better than the 3-weekly infusion since the decreased toxicity increases the therapeutic index.     

Time-Dependent Efficacy of Antihypertensi Agents in Spontaneously Hypertensive Rai

The efficacy of antihypertensive agents was compared when given at different time points in the circadian rhythm. Spontaneously hypertensive rats (SHRs) were kept on a 12/12-h cycle with lights on/off at 07:00/19:00 h. A computerized system was used to measure intraarterial blood pressure and heart rate continuously. Agents or vehicle were intravenously injected at two time points. One at the beginning of the sleeping period, at which low efficacy was expected (T = 10), and one at T = 16, which is 3 h before the circadian peaks in blood pressure (BP) and heart rate (HR), aimed at reducing the rise in BP and HR at awakening. The hypotensive effect of propranolol, metoprolol, labetalol, prazosin, clonidine, and rilmenidine was greater when injected at T = 16 than at T = 10 (p < 0.05 for propranolol, metoprolol, and rilmenidine). In contrast, the renal vasodilators cap-topril and tertatolol were more potent after injection at T = 10. Felodipine was equally effective at both time points. Thus, the effects of antihypertensive agents are related to the phase of the circadian rhythm. The data on the sympatholytic agents in general and β-blockers and centrally acting agents in particular support antihypertensive regimens with timed administrations.     

Circadian Rhythms of Renal Hemodynamics in Unanesthetized,Unrestrained Rats

Catheters were placed in the jugular vein and femoral artery of male Sprague-Dawley rats and connected to a specially designed perfusor for continuous constant infusion of 0.9% NaCl and a syringe to perform simultaneous and intermittent blood collections. This permitted continuous 24-h study of renal hemodynamics, estimated by inulin (C_in) and p-amino-hippuric acid (C_PAH) clearances; C_in represents glomerular filtration rate and C_PAH renal plasma flow. Animals were individually housed in metabolism cages in a controlled environment with light/dark 12:12 h. Urine was collected every 4 h (12:00, 16:00, 20:00, 24:00, 04:00, and 08:00) and blood sampled at the midpoint of urine collection periods. Urine and plasma sodium, potassium, inulin, and PAH were spectrophotometrically assessed. During continuous infusion of isotonic saline, C_in exhibited circadian changes with large decrease between 12:00 and 20:00 h (0.9 ± 0.2 ml/min) and acrophase at 00:30 h. Rhythmicity in C_PAH was similar with the minimum between 16:00 and 20:00 h (2.5 ± 0.3 ml/min) and peak between 00:00 and 04:00 h (acrophase at 00:25 h). Water and electrolyte excretion were also circadian rhythmic with a similar nighttime enhancement and daytime minimum. Such circadian changes persisted during continuous 0.9% NaCl infusion for several consecutive days. The unanesthetized, unrestrained rat model enables investigations in renal chronopharmacology and chronotoxicology.     

Sex and dosing-time dependencies in irinotecan-induced circadian disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.     

A phase I trial of recombinant tumor necrosis factor (rTNF) administered by continuous intravenous infusion in patients with disseminated malignancy

rTNF was administered to 28 patients with advanced metastatic cancers by continuous intra venous infusion for 5 consecutive days every 2 weeks. The dose levels were 30, 40, 70, 110, 180 and 290 µg/M²/day. Groups of 3 patients were started at each successive dose level and then on subsequent courses treated with the next dose level through 4 escalations as tolerated. Tumor types were: colon cancer 14; adenocarcinoma of unknown primary, 2; renal cancer, 2; leiomyosarcoma, 2; lung cancer, 1; prostate cancer, 1; thymona, 1; bladder cancer; 1; parotid, 1; Kaposi's sarcoma 2; ovarian 1. Toxicities included fever and chills (usually within the first 8 hours of infusion), fatigue, headache, decreased performance status, hypotension and CNS. All patients experienced leukopenia and thrombocytopenia within 24 hours or less after start of infusion with return of baseline by 72 hours after rTNF was stopped. The fall in these counts averaged 50% and was not dose related. No major changes in liver or renal function, coagulation or blood lipids were seen. Major dose limiting toxicities were fatigue, confusion, thrombocytopenia, seizures, hypotension and decreased performance status. NK cell activity measured against K562 target cells was augmented from about 30% target cell lysis to about 70% target cell lysis over the first 7 days of treatment. Two patients, both with metastatic colon cancer showed transient, objective tumor regression which did not qualify as a partial response. One patient with ovarian cancer had a stable partial response but progressed after 13 courses of treatment. Continuous infusion of TNF can be safely administered to patients with a maximum tolerated dose of only between 30 and 40 µg/M²/day. In addition, the MTD with continuous infusion seems to be highly variable and unpredictable from patient to patient. These data suggest that continuous infusion will not be an optimal way to administer TNF.     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

Circadian rhythm in prostatic acid phosphatase (PAP): a potential tumor marker rhythm in prostatic cancer (PCa)

In order to define circadian states for an earlier diagnosis and for optimal response to treatment, the possibility of a circadian rhythm in serum PAP was investigated in subjects with and without prostatic cancer. Two groups of subjects were investigated: a. 12 patients affected by PCa, further subdivided in two subgroups: 1. without metastasis (6 patients) and 2. with metastatic disease (6 patients); b. 9 age-matched healthy control subjects. Controls and PCa patients were synchronized before starting the study with standardized meal times and nocturnal rest (22(00) to 06(00) ). Venous blood samples were drawn at prearranged hours (00(00), 04(00), 08(00), 12(00), 16(00), 20(00) ) for 24 consecutive hours. Each serum sample was assayed for PAP. Data on each group and subgroup were evaluated by conventional statistical analysis and by 'single' and 'population mean cosinor' to define rhythm parameters. PCa patients, as a single group, did not show a significant circadian PAP rhythm. A statistically significant circadian PAP rhythm was however detected in the subgroup without metastasis, on the contrary no rhythm was detected in the subgroup with metastatic disease. The potential of these rhythms as marker of cancer is noted.     

Circadian rhythms of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), cortisol, and melatonin in women with breast cancer

Background: Circadian rhythms in plasma concentrations of many hormones and cytokines determine their effects on target cells. Methods: Circadian variations were studied in cortisol, melatonin, cytokines (basic fibroblast growth factor [bFGF], EGF, insulin-like growth factor-1 [IGF-1]), and a cytokine receptor (insulin-like growth factor binding protein-3 [IGFBP-3]) in the plasma of 28 patients with metastatic breast cancer. All patients followed a diurnal activity pattern. Blood was drawn at 3h intervals during waking hours and once during the night, at 03:00. The plasma levels obtained by enzyme-linked immunoassay (ELISA) or radioimmunoassay (RIA) were evaluated by population mean cosinor (using local midnight as the phase reference and by one-way analysis of variance (ANOVA). Results: Cortisol and melatonin showed a high-amplitude circadian rhythm and a superimposed 12h frequency. bFGF showed a circadian rhythm with an acrophase around 13:00 with a peak-to-trough interval (double amplitude) of 18.2% and a superimposed 12h frequency. EGF showed a circadian rhythm with an acrophase around 14:20, a peak-to-trough interval of 25.8%, and a superimposed 12h frequency. IGF-1 showed a high value in the morning, which is statistically different t test) from the low value at 10:00, but a regular circadian or ultradian rhythm was not recognizable as a group phenomenon. IGFBP-3 showed a low-amplitude (peak-to-trough difference 8.4%) circadian rhythm with the acrophase around 11:00 and low values during the night. Conclusions: (1) Circadian periodicity is maintained in hospitalized patients with metastatic breast cancer. (2) Ultradian (12h) variations were superimposed on the circadian rhythms of the hormones and several of the cytokines measured. (3) Studies of hormones and cytokines in cancer patients have to take their biologic rhythms into consideration. (4) The circadian periodicity of tumor growth stimulating or restraining factors raises questions about circadian and/ or ultradian variations in the pathophysiology of breast cancer. (Chronobiology International, 18(4), 709-727)     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

A randomized multicenter study of optimal circadian time of vinorelbine combined with chronomodulated 5-fluorouracil in pretreated metastatic breast cancer patients: EORTC trial 05971

Studies in animals synchronized with an alternation of 12 h of light and 12 h of darkness have showed that hematological and systemic toxicities could be reduced if vinorelbine were administered 19 or 23 hours after light onset (HALO), corresponding to 17:00 and 21:00 h in diurnally active humans. This trial aimed to define the least toxic time of vinorelbine administration in metastatic breast cancer patients. Initially, the study treatment consisted of three courses of vinorelbine of 30 mg/m(2)/d on D1 and D6 and chronomodulated 5-fluorouracil of 850 mg/m(2) from D2 to D5 every 21 days. Ninety metastatic breast cancer patients were randomized to receive vinorelbine at one of the eight possible dosing times. Further to the recommendations of the Independent Data Monitoring Committee, the vinorelbine dose was reduced to 25 mg/m(2)/d midway through the study. The primary objective of the study was detection of the least toxic time based on the incidence of grade 3-4 (G3-4) neutropenia. To show a significant result, the 90% confidence interval width of the least toxic time had to be<6 h. The least toxic time detection based on the incidence of other toxicities was also analyzed. The time of least drug toxic was estimated using a logistic regression model assuming that the logit transformation of the toxicity rate follows a sinusoidal distribution over 24 h. The bootstrap technique was used to obtain the 90% confidence interval. The least toxic time of G3-4 neutropenia was observed at 21:00 h with a non-significant 90% CI. Secondary endpoint analyses indicated the least toxic time could differ when based on other toxicity parameters (e.g., a significant least toxic time of 17:00 h was observed for G3-4 leucopenia), in agreement with animal data. The least toxic time of 10:30 h was estimated for any G3-4 gastrointestinal toxicity. This results of this study do not allow us to recommend an optimal time for vinorelbine administration. It has highlighted, however, the inherent methodological difficulties in the conduct of such a trial in the human setting. It indicates that future optimal time-finding trials should have tolerability and/or activity as the primary endpoint in place of a particular toxicity. The randomized optimal time-finding design may be used to identify the best time of chemotherapy administration.     

Sex and Dosing-Time Dependencies in Irinotecan-Induced Circadian Disruption

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50?mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2?h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers. (Author correspondence: francis.levi@inserm.fr)     

Twenty-four-hour variation of vestibular function in young and elderly adults

ABSTRACT

Animal and human studies demonstrate anatomical and functional links between the vestibular nuclei and the circadian timing system. This promotes the hypothesis of a circadian rhythm of vestibular function. The objective of this study was to evaluate the vestibular function through the vestibulo-ocular reflex using a rotatory chair at different times of the day to assess circadian rhythmicity of vestibular function. Two identical studies evaluating temporal variation of the vestibulo-ocular reflex (VOR) were performed, the first in young adults (age: 22.4 ± 1.5 y), and the second in older adults (70.7 ± 4.7 y). The slow phase velocity and time constant of the VOR were evaluated in six separate test sessions, i.e., 02:00, 06:00, 10:00, 14:00, 18:00, and 22:00 h. In both studies, markers of circadian rhythmicity (temperature, fatigue, and sleepiness) displayed expected usual temporal variation. In young adults, the time constant of the VOR showed variation throughout the day (p < .005), being maximum 12:25 h (06:00 h test session) before the acrophase of temperature circadian rhythm. In older adults, the slow phase velocity and time constant also displayed temporal variation (p < .05). Maximum values were recorded at 10:35 h (06:00 h test session) before the acrophase of temperature circadian rhythm. The present study demonstrates that vestibular function is not constant throughout the day. The implication of the temporal variation in vestibular system in equilibrium potentially exposes the elderly, in particular, to differential risk during the 24 h of losing balance and falling.     

Downregulation of HIPK2 Increases Resistance of Bladder Cancer Cell to Cisplatin by Regulating Wip1

Cisplatin-based combination chemotherapy regimen is a reasonable alternative to cystectomy in advanced/metastatic bladder cancer, but acquisition of cisplatin resistance is common in patients with bladder cancer. Previous studies showed that loss of homeodomain-interacting protein kinase-2 (HIPK2) contributes to cell proliferation and tumorigenesis. However, the role of HIPK2 in regulating chemoresistance of cancer cell is not fully understood. In the present study, we found that HIPK2 mRNA and protein levels are significantly decreased in cisplatin-resistant bladder cancer cell in vivo and in vitro. Downregulation of HIPK2 increases the cell viability in a dose- and time-dependent manner during cisplatin treatment, whereas overexpression of HIPK2 reduces the cell viability. HIPK2 overexpression partially overcomes cisplatin resistance in RT4-CisR cell. Furthermore, we showed that Wip1 (wild-type p53-induced phosphatase 1) expression is upregulated in RT4-CisR cell compared with RT4 cell, and HIPK2 negatively regulates Wip1 expression in bladder cancer cell. HIPK2 and Wip1 expression is also negatively correlated after cisplatin-based combination chemotherapy in vivo. Finally, we demonstrated that overexpression of HIPK2 sensitizes chemoresistant bladder cancer cell to cisplatin by regulating Wip1 expression.

Conclusions

These data suggest that HIPK2/Wip1 signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of bladder cancer.     

A Randomized Multicenter Study of Optimal Circadian Time of Vinorelbine Combined with Chronomodulated 5‐Fluorouracil in Pretreated Metastatic Breast Cancer Patients: EORTC Trial 05971

Studies in animals synchronized with an alternation of 12 h of light and 12 h of darkness have showed that hematological and systemic toxicities could be reduced if vinorelbine were administered 19 or 23 hours after light onset (HALO), corresponding to 17:00 and 21:00 h in diurnally active humans. This trial aimed to define the least toxic time of vinorelbine administration in metastatic breast cancer patients. Initially, the study treatment consisted of three courses of vinorelbine of 30 mg/m²/d on D1 and D6 and chronomodulated 5‐fluorouracil of 850 mg/m² from D2 to D5 every 21 days. Ninety metastatic breast cancer patients were randomized to receive vinorelbine at one of the eight possible dosing times. Further to the recommendations of the Independent Data Monitoring Committee, the vinorelbine dose was reduced to 25 mg/m²/d midway through the study. The primary objective of the study was detection of the least toxic time based on the incidence of grade 3–4 (G3–4) neutropenia. To show a significant result, the 90% confidence interval width of the least toxic time had to be<6 h. The least toxic time detection based on the incidence of other toxicities was also analyzed. The time of least drug toxic was estimated using a logistic regression model assuming that the logit transformation of the toxicity rate follows a sinusoidal distribution over 24 h. The bootstrap technique was used to obtain the 90% confidence interval. The least toxic time of G3–4 neutropenia was observed at 21:00 h with a non‐significant 90% CI. Secondary endpoint analyses indicated the least toxic time could differ when based on other toxicity parameters (e.g., a significant least toxic time of 17:00 h was observed for G3–4 leucopenia), in agreement with animal data. The least toxic time of 10:30 h was estimated for any G3–4 gastrointestinal toxicity. This results of this study do not allow us to recommend an optimal time for vinorelbine administration. It has highlighted, however, the inherent methodological difficulties in the conduct of such a trial in the human setting. It indicates that future optimal time‐finding trials should have tolerability and/or activity as the primary endpoint in place of a particular toxicity. The randomized optimal time‐finding design may be used to identify the best time of chemotherapy administration. (Author correspondence: bcoudert@dijon.fnclcc.fr)     

Treatment-time-dependent difference of ketamine pharmacological response and toxicity in rats

Circadian rhythms impact many physiological functions that may affect drug pharmacological response. Ketamine is a dissociative agent commonly used for surgical anesthesia in rats. The aim of the present study was to analyze the central nervous system (CNS) depression and lethality of ketamine injected intraperitoneally at different times during the 24 h. The study was conducted in October 2001, spring in the Southern hemisphere. Female prepuberal Sprague-Dawley rats synchronized to a 12h light:12h dark cycle (light, 07:00h-19:00h) were studied. Ketamine (40 mg/kg) was administered to one of six different clock-time treatment groups (n=6-7 rats each). Duration of latency period, ataxia, loss of righting reflex (LRR), post-LRR ataxia, and total pharmacological response were determined by visual assessment. To investigate acute toxicity, ketamine lethal dose 50 (148.0 mg/kg) was also administered as a single injection to six different treatment-time groups of rats. Significant temporal differences and circadian rhythms were detected in drug-induced post-LRR ataxia and total pharmacological response duration. The longest pharmacological response occurred in rats injected during the light (rest) phase and the shortest response in the dark (activity) phase. No circadian rhythm was detected in acute toxicity. The study findings indicate that the duration of CNS depression of ketamine in rats exhibits circadian rhythmic variation.     

Circadian response of annual weeds to glyphosate and glufosinate

Five field experiments were conducted in 1998 and 1999 in Minnesota to examine the influence of time of day efficacy of glyphosate [N-(phosphonomethyl)glycine] and glufosinate [2-amino-4-(hydroxymethyl-phosphinyl)butanoic acid] applications on the control of annual weeds. Each experiment was designed to be a randomized complete block with four replications using plot sizes of 3×9 m. Glyphosate and glufosinate were applied at rates of 0.421 kg ae/ha and 0.292 kg ai/ha, respectively, with and without an additional adjuvant that consisted of 20% nonionic surfactant and 80% ammonium sulfate. All treatments were applied with water at 94 L/ha. Times of day for the application of herbicide were 06:00h, 09:00h, 12:00h, 15:00h, 18:00h, 21:00h, and 24:00h. Efficacy was evaluated 14 d after application by visual ratings. At 14 d, a circadian response to each herbicide was found, with greatest annual weed control observed with an application occurring between 09:00h and 18:00h and significantly less weed control observed with an application at 06:00h, 21:00h, or 24:00h. The addition of an adjuvant to both herbicides increased overall efficacy, but did not overcome the rhythmic time of day effect. Results of the multiple regression analysis showed that after environmental temperature, time of day was the second most important predictor of percent weed kill. Thus, circadian timing of herbicide application significantly influenced weed control with both glyphosate and glufosinate.