Introduction

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Vinorelbine is a new Vinca alkaloid modified on the catharantine part of the molecule. It displays high antitumor activity as a single agent or combined therapy in patients with advanced non-small cell lung and breast cancer (Cvitkovic and Izzo, 1992). It exerts its cytotoxic effect upon rapidly proliferating tumors through the prevention of mitotic spindle formation (Zhou and Rahmani, 1992). Although adverse effects are generally manageable, an improvement in the efficacy/toxicity ratio could result from a safe delivery of higher doses.

Changing the timing of administration along the 24-h time scale can profoundly modify the extent of dose-limiting toxicities of anticancer agents (Lévi, 1997). The adaptation of several cancer chemotherapy regimens to circadian rhythms improved their safety as well as their antitumor activity in patients (Hrushesky, 1985; Lévi et al., 1994, 1997). It was reported previously that the toxicity of vinblastine, a Vinca alkaloid closely related to vinorelbine, varied according to dosing time in mice (Mormont et al., 1986). The highest tolerance to this drug was found in the middle of the nocturnal activity span. A previous study demonstrated a circadian rhythm in vinorelbine toxicity in healthy male B6D2F₁ mice. Lethal toxicity and body weight loss were, respectively, three times and twice as large in the mice receiving injections during the light span (rest) as compared with those treated in the second half of the dark span (end of nocturnal activity) (Tampellini et al., 1995a).

In the present studies, we investigated the main target organs involved in vinorelbine toxicity rhythm and the relevance of dosing time for vinorelbine efficacy in mice inoculated with P388 leukemia. The high reproducibility and rapid growth of this tumor model previously allowed documenting dose- and schedule-dependent effects of vinorelbine antitumor activity (Vendetti, 1975; Cros et al., 1989).

	Materials and Methods

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Animals and Synchronization

Female mice were 4 weeks of age upon arrival. They were housed two or three per cage with food and water provided ad libitum. All mice were synchronized with an alternation of 12 h of light and 12 h of darkness for 3 weeks in an autonomous chronobiologic animal facility (ESI-Flufrance, Arcueil, France) (Tampellini et al., 1995a). Times of drug injection and sampling were expressed in hours after light onset (HALO): 3, 7, and 11 HALO were located during the light span, when mice are usually at rest; 15, 19, and 23 HALO corresponded to the dark span, when mice are usually active.

Mortality was recorded daily, and body weight was recorded three times a week during 14 days for experiment 3, during 21 days for experiments 4 and 5, during 30 days for experiment 6, and during 60 days for experiments 7 and 8. Animals alive 60 days after tumor inoculation were considered as cured.

All procedures were performed in accordance with French guidelines for experimental animal care.

Tumor Model

P388 lymphocytic leukemia (P388) was obtained from Institut de Recherche Pierre Fabre (Castres, France). Tumors were maintained in DBA female mice (IFFA Credo, L'Arbresle, France) and passaged as weekly i.p. implants. A minimum of three passages was performed, and two to three donors were used for each experiment.

Drugs

Vinorelbine (solution for i.v. injection, concentration 10 mg/ml) was provided by Pierre Fabre Oncologie (Boulogne, France). It was diluted in 0.9% sodium chloride on each study day and injected i.v. (10 ml/kg of body weight) into the right retro-orbital venous sinus. The resulting drug concentration ranged from 2 mg/ml (20 mg/kg) to 3 mg/ml (30 mg/kg).

Study Design

Hematologic and Intestinal Toxicities. Two experiments were performed in a total of 279 female B2D6F₁ mice. Animals were injected with 26 mg/kg of vinorelbine at one of four different circadian times: 7, 11, 19, and 23 HALO. The dose and times of injection were selected on the basis of previously reported or unpublished data (Tampellini et al., 1995a; Filipski and Lévi, unpublished results). Seven and 19 HALO corresponded to the respective times of highest and lowest vinorelbine toxicity. Twenty-three HALO and 11 HALO, respectively, ranked as second to best and second to worst tolerability times. Control animals received 0.9% NaCl. For each administration time, two controls and six treated animals were sacrificed 1, 2, 4, 6, and 8 days after drug injection to measure leukocyte and neutrophil counts and to evaluate the extent of bone marrow and intestinal lesions. Blood samples (400 µl) were taken from the retro-orbital sinus vein. Circulating leukocytes were counted with Coulter Counter ZM (Coultronics, Margency, France), and neutrophil count was analyzed from blood smears. After being bled, mice were sacrificed by cervical dislocation, and samples of colon, small intestine, and bone marrow (femur) were fixed in Bouin Picroformol solution. Twenty-four h later, the samples were dehydrated and embedded into paraffin. Sections were made and stained with hematoxylin-eosin. Each slide was examined by the same histopathologist, and lesions were graded blind between 0 (normal) and 4 (extensive necrosis).

Antitumor Efficacy. Six experiments were performed in a total of 672 female B6D2F₁ mice (IFFA Credo, Arbresle, France). Overall, 552 mice were inoculated i.p. with P388 leukemia (10⁶ cells/mouse). Vinorelbine was injected i.v. to 398 tumor-bearing mice. Cure was defined as survival 60 days after tumor inoculation.

Experiment 3: possible role of P388 leukemia inoculation time upon survival. Groups of 10 mice each were inoculated with 10⁶ cells/mouse at one of six circadian times (3, 7, 11, 15, 19, or 23 HALO) and followed for survival.

Experiments 4 and 5: influence of circadian dosing time upon survival. Mice were randomized into two groups of 60; the first one received P388 leukemia alone, and the second one received P388, then vinorelbine 24 h later at a dose of 20 mg/kg. Overall, P388 leukemia or P388 and vinorelbine were administered to different subgroups of 10 mice at 3, 7, 11, 15, 19, or 23 HALO. The dose of 20 mg/kg was selected as being close to an LD10 in these mice (Tampellini et al., 1995a; Filipski and Lévi, unpublished data).

Experiment 6: single administration schedule-effect of dose and circadian time on survival. Mice were randomized to receive P388 leukemia cells alone (16 controls) or P388 then vinorelbine as a single injection 24 h later at 7, 11, 19, or 23 HALO (total of 128 mice). Subgroups of eight mice per time point received 20, 24, 26, or 30 mg/kg of vinorelbine.

Experiment 7: weekly schedule-effect of dose and circadian time on survival. One hundred and thirty-two mice were randomized to receive P388 leukemia alone (12 controls) or P388 then one of three vinorelbine doses (20, 24, or 26 mg/kg/inj) at one of four circadian times (7, 11, 19, or 23 HALO). Vinorelbine was injected 24 h after P388 inoculation, then 7 and 14 days later. This schedule produced the best vinorelbine efficacy in the P388 model (Cros et al., 1989).

Experiment 8: weekly schedule-effect of dose and circadian time on survival. Ninety-six mice were randomized to receive P388 leukemia alone (six controls) or P388 then one of three vinorelbine doses (24, 26, or 28 mg/kg/day) at one of two circadian times: 19 HALO (best) or 11 HALO (intermediate), selected on the basis of the results from experiment 7. Vinorelbine was injected 24 h after P388 inoculation, then 7 and 14 days later.

Endpoints and Statistics

Means and 1 S.E.M. were calculated for each variable. The statistical significance of differences observed between groups was validated by one- or two-way ANOVA. Differences in survival rates were analyzed by ² test. Survival curves were compared with the Log-Rank test. The dose-response relationship was tested with linear regression. Percentage of increase in life span (% ILS) was computed as follows:
where median survival time (MST) was the median day of death of treatment failures only. In experiments 4 to 7, a pooled MST from all the control mice regardless of the time of P388 inoculation was used for the % ILS calculation. This procedure was based upon the results from experiment 3.

All standard statistics were performed using SPSS for Windows software.

	Results

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Rhythm in Target Organ Toxicity

Leukocyte Count. Mean circulating leukocyte count of control mice (±S.E.M.) varied from 2494 cells/mm³ (±205) at 19 HALO to 5835 cells/mm³ (±485) at 7 HALO, as a result of the normal circadian rhythm in this variable (p from ANOVA < .001).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Mean relative change of leukocyte count (±S.E.M.) in female B6D2F1 mice receiving a single vinorelbine injection () (26 mg/kg i.v. on day 0). Statistically significant differences were found as a function of circadian time of injection (7, 11, 19, or 23 HALO) and interval since treatment administration (1, 2, 4, 6, or 8 days). Two-way ANOVA, Ftime = 26, p < .001; Finterval = 64, p < .0001.

Neutrophil Count. Mean neutrophil count of control mice (±S.E.M.) varied from 306 cells/mm³ (±38) at 19 HALO to 783 cells/mm³ (±242) at 7 HALO. Neutropenia was largest 4 days after vinorelbine administration (44 ± 16 cells/mm³). No significant difference was found as a function of injection time. The rate of recovery depended on drug dosing time; complete recovery was found 6 days after injection in mice treated at 19, 11, or 23 HALO but not in those receiving the drug at 7 HALO. The differences in extent of neutropenia were validated with ANOVA (p < .001).

Bone Marrow Toxicity. Bone marrow lesions ranged from moderate to extensive necrosis. Lesions were maximal 2 days after vinorelbine injection and had recovered completely 4 days later. On the day of maximal toxicity, extensive bone marrow necrosis (grade 4) was encountered in five of five mice treated at 7 HALO as compared with three of six mice injected with vinorelbine at 19 HALO. Recovery appeared to be the slowest in the mice treated at 7 HALO. Injection time-related differences in histologic grade were statistically validated with ANOVA (p = 0.004) (Table 1).

Intestinal Toxicity. No histological alteration was observed in small intestine or in colon.

Lack of Effect of P388 Leukemia Inoculation Time

Control mice died between the 9th and the 14th day after P388 leukemia cells inoculation; 52 of 60 animals died on day 9 or 10. No statistically significant difference in survival was observed as a function of the circadian time of tumor inoculation, whether the Log-Rank test was performed on pooled data from experiments 3 to 8 (p = 0.16) or on separate data sets from each study.

Circadian Time-Dependent Efficacy of Single-Dose Vinorelbine

Experiments 4 and 5. All untreated leukemic mice died from tumor progression within 12 days after inoculation; 57 of 60 animals died between days 8 and 10. A single vinorelbine injection of 20 mg/kg improved survival but failed to produce any cure. Vinorelbine dosing at 7 HALO resulted in 40% toxic deaths within 9 days after tumor inoculation, before the first death of an untreated tumor control. No toxic death was observed when vinorelbine was administered at 19 HALO. MST ranged from 14 days in the group of mice treated at 7 HALO to 17 days in groups treated at 15, 19, or 23 HALO (Table 2). The last survivor died 17 days after tumor inoculation in the group treated at 7 HALO, 18 days in the groups treated at 3 or 11 HALO, 19 days in those treated at 19 or 23 HALO, and 21 days in the group treated at 15 HALO. The survival curves differed significantly, vinorelbine being clearly most toxic and least active in mice treated at 7 HALO (Log-Rank = 16.7, p = .005).

Experiment 6. Untreated leukemic mice died within 10 days after inoculation. All four vinorelbine doses tested significantly prolonged the life span of P388- bearing mice as compared with untreated controls. Survival depended upon both dose and time of drug administration. All the mice treated with 20 or 24 mg/kg died within 21 days, whereas 7 of those treated with 26 or 30 mg/kg were still alive. Once more, lethal toxicity resulted from vinorelbine injection at 7 HALO, especially for the higher doses (24 mg/kg, one of eight; 26 mg/kg, two of eight; and 30 mg/kg, three of eight). Increasing vinorelbine dose at 7 HALO did not significantly improve MST (20 mg/kg, 16 days; 30 mg/kg, 16.5 days) (Fig. 2). A similar observation was made when vinorelbine was injected at 11 HALO (20 mg/kg, 16 days; and 30 mg/kg, 17.5 days). An increase in vinorelbine dose from 20 to 30 mg/kg at 19 or at 23 HALO resulted in an improvement in MST. It was significantly prolonged from 15.5 to 18.5 days at 19 HALO (p = .007) and from 16 to 18 days at 23 HALO (p = .01).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Relationship between vinorelbine dose (single injection) and median survival of P388-bearing B6D2F1 mice, as a function of circadian time of administration expressed in HALO. Median survival of vinorelbine-treated mice was increased as compared with that of untreated controls. Statistically significant correlation between dose and MST were found in the mice receiving vinorelbine at 19 or 23 HALO but not in those treated at 7 or at 11 HALO.

Circadian Time-Dependent Efficacy of Weekly Vinorelbine

Experiment 7. All untreated P388 leukemic mice died between the 8th and the 10th day after tumor inoculation. Weekly doses of vinorelbine prolonged survival as a function of the circadian time of administration (Fig. 3). Eleven mice clearly died from toxicity, 1 of 30 mice treated at 23 HALO, and 10 of 29 mice treated at 7 HALO. In the latter group, toxic lethality rate was dose dependent (20 mg/kg, 2 of 10; 24 mg/kg, 3 of 10; 26 mg/kg, 5 of 9) Furthermore, MST decreased as a function of dose from 29 days (20 mg/kg) to 17 days (24 mg/kg) and 6 days (26 mg/kg) in mice treated at 7 HALO, whereas it increased as a function of dose from 28.5 days (20 mg/kg) to 32 days (24 mg/kg) and 36 days (26 mg/kg) in the animals injected at 19 HALO. Although the lowest dose produced 1 of 40 cure (2.5%), 5 of 40 cures (12.5%) were obtained with 24 mg/kg and 6 of 38 (15.8%) with 26 mg/kg on day 60. Despite these limited numbers, the cure rate was 5-fold greater in the groups treated at 19 HALO (5 of 29, 17%) than in those injected at 7 HALO (1 of 29, 3%) (Table 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Survival curves of P388-bearing B6D2F1 female mice receiving three weekly vinorelbine injections () (26 mg/kg i.v. on days 1, 8, and 15). Results from experiment 7 (A), conducted in groups treated at one of four circadian times, expressed in HALO (p from Log-Rank < .05) and from experiment 8 (B), performed in groups treated at 11 or 19 HALO (p from Log-Rank < .0001).

Experiment 8. All untreated P388 leukemic mice died between the 9th and the 10th day after tumor inoculation. Vinorelbine treatment prolonged survival as compared with controls, yet the survival curves differed significantly according to drug dose and dosing time (Log-Rank 19.04, p < .0001).

	Discussion

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The circadian rhythm in vinorelbine toxicity was mostly related to hematological toxicity, as indicated by the lack of any histologic damage in jejunum or colon and by extensive bone marrow necrosis and profound leukoneutropenia. Bone marrow necrosis was largest 2 days after drug administration, preceding the nadir in leukocyte and neutrophil counts by 2 days. The extent of bone marrow necrosis and leukopenia were significantly enhanced in mice receiving the drug at 7 HALO. Furthermore, recovery from hematologic toxicity was slowest in those mice. The present results obtained in female B6D2F₁ mice were similar to those reported earlier in male mice of the same strain, using mortality and body weight loss as endpoints (Tampellini et al., 1995a). Least vinorelbine toxicity corresponded to an administration of this drug at 19 HALO, in the second half of the activity span, in B6D2F₁ mice of either sex. This time was similar to that earlier reported for vinblastine, a vinorelbine analog (Mormont et al., 1986).

The relationship between the circadian rhythm in drug tolerability and antitumor efficacy constitutes an essential issue for cancer chronotherapy. Using the transplantable P388 leukemia model, the present experiments demonstrated that vinorelbine lethal toxicity also followed a circadian rhythm in tumor-bearing female B6D2F₁ mice, receiving a single dose or weekly doses of this drug. The results showed that no toxic death resulted from vinorelbine administration at 19 HALO as compared with 7 HALO. The rhythm was similar to that found previously in male and in female B6D2F₁ mice (Tampellini et al., 1995a; Filipski and Lévi, unpublished results). These findings suggest that the rhythm in vinorelbine tolerance was influenced neither by sex nor by the presence of an early-stage tumor. It confirms similar findings with arabinosyl cytosine and docetaxel (Haus et al., 1972; Tampellini et al., 1998).

Median survival of leukemic mice increased as a function of the dose of a single vinorelbine administration; yet, the fact that vinorelbine toxicity varied as a function of circadian time allowed an increase in tolerable dose from 20 to 30 mg/kg at 19 or at 23 HALO but not at 7 or at 11 HALO. Thus, a positive dose-survival relationship could only be observed if a single vinorelbine dose was given in the second half of darkness.

The possibility to increase the dose at the time of the lowest toxicity of the drug was successfully exploited by administering three weekly vinorelbine injections. In this schedule, the longest median survival of mice treated at 7 HALO was 29 days and was obtained with the lowest dose (20 mg/kg/day), because higher doses were too toxic. The longest median survival of mice treated at 19 HALO was 36 days and was achieved with the highest dose (26 mg/kg/day). A single cure (3%) was obtained with vinorelbine dosing at 7 HALO, as compared with four cures (13%) at 11 HALO and five cures (17%) at 19 HALO. The superiority of a weekly administration of 26 mg/kg of vinorelbine at 19 HALO was further confirmed in a subsequent experiment with regard to median survival and cure rate. Furthermore, the cure rate achieved with administration of 26 mg/kg at 19 HALO was reproducible (33% in experiment 7 and 26% in experiment 8). Taken together, both experiments, performed at this optimal dose level, indicated that cure rate increased 3-fold between 11 HALO and 19 HALO. These dosing times, respectively, corresponded to the beginning and the second half of the activity span of mice.

A circadian regulation of normal cell cycle response to vinorelbine exposure might account for the circadian rhythm in vinorelbine tolerability. In a preliminary experiment, we found that in vivo vinorelbine administration blocked bone marrow cells in G₂-M phase then in G₁ phase. This G₁ blockage was accompanied by an increase in p53 expression, which depended upon drug dosing time. The fluorescence index of p53 expression was twice as high after vinorelbine treatment at 19 or 23 HALO as compared with 7 HALO. This suggested that the repair capacities of damaged bone marrow cells differed according to the time of vinorelbine exposure (Liu et al., 1998). Furthermore, p53 deficiencies in P388 cells might contribute to the synchrony of tolerability and efficacy rhythms in this model.

In conclusion, a 50% increase in tolerable vinorelbine dose was achievable by injecting this drug at 19 HALO as compared with 7 HALO. Vinorelbine delivery, at its maximum tolerated dose given at the least toxic circadian time, almost doubled median survival and more than tripled cure rate as compared with the best figure resulting from drug injection at 7 or at 11 HALO. We feel that these data support the investigation of clinical chronotherapy with vinorelbine in cancer patients.