Introduction

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A large number of physiological rhythms are controlled by the central nervous system, hormone secretion, and so on (Thomson et al., 1980; Kafka et al., 1981; Naber et al., 1981). Also, many drugs vary in potency and/or toxicity according to the time in the circadian cycle when they are administered (Walker and Owasayo, 1974; Frederickson et al., 1977; Ohdo et al., 1988, 1990, 1991, 1995b, 1996, 1997b, 1998).

Interferons, which belong to a group of cytokines, have been widely used as antiviral and antitumor agents in humans. However, interferons cause unavoidable adverse effects such as fever, fatigue, headache, rigors, and myalgias. One approach to increasing the efficiency of pharmacotherapy is the administration of drugs at times at which they are most effective and/or best tolerated. Certainly, use of a chronopharmacological strategy can improve the effects and reduce the toxicity of drugs. Administration of interferon- (IFN-) to cancer patients is better tolerated in the evening than in the morning, although the pharmacological effectiveness of IFN- has not been examined (Abrams et al., 1985; Iacobelli et al., 1995). There are significant dosing time-dependent differences reported in antitumor and myelosuppressive activity of IFN- in mice (Koren and Fleischmann, 1993; Koren et al., 1993). Also, the rhythmic changes of fever and antiviral activity induced by IFN- were examined in mice (Koyanagi et al., 1997; Ohdo et al., 1997a), but the mechanism was not investigated in detail.

IFN- elicits antitumor and antiviral activity by binding to a specific receptor on the cell surface (Alexander et al., 1986; Kumar and Korutla, 1995). Furthermore, IFN- elicits transcription of various genes through activation of interferon-stimulated gene factor (ISGF), via binding to specific receptor. IFN- is quickly eliminated from the body via several pathways. The main route of excretion is via the kidneys (Bino et al., 1982). IFN- is presumably filtered by glomeruli and taken up into tubules wherein it is metabolized. In this study, the dosing time-dependent change in antiviral activity induced by IFN- was examined in mice. The mechanisms underlying this phenomenon were investigated in terms of chronopharmacodynamics, the rhythmicity of receptor function, and chronopharmacokinetics, the rhythmicity of IFN- metabolism in kidney.

	Materials and Methods

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Animals and Chemicals. Male ICR mice (5 weeks old) were purchased from Charles River Japan Inc. (Kanagawa, Japan). Mice were housed 10 per cage in a light-controlled room (lights on from 7:00 AM to 7:00 PM) at a room temperature of 24 ± 1°C and a humidity of 60 ± 10% with food and water ad libitum. All mice adapted to their light/dark cycle for 2 weeks before the experiments. IFN- (Sumiferon) was supplied by Sumitomo Seiyaku Co. (Osaka, Japan). The drug supply was given within the time period from manufacture before expiration. Adequate stability studies had been conducted on the given lot. IFN- was diluted with sterilized saline to adjust the concentration to 2 MI.U./ml. The volume of injection was 0.05 ml/10 g of body weight. The drug solutions were used within 30 min after preparation to avoid decreasing their biological activity. The complete medium used in this study consisted of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Irvine Scientific, Santa Ana, CA), 0.5% penicillin, and 0.5% kanamycin.

2'-5'Oligoadenylate Synthetase (2'-5'OAS) Activity in Plasma. To examine the 2'-5'OAS activity induced by IFN-, groups of eight mice were i.v. injected with 10 MI.U./kg of IFN- or sterilized saline in the first half of the light phase (9:00 AM) or in the first half of the dark phase (9:00 PM). The dosage of IFN- was selected based on preliminary experiments in a previous study (Koyanagi et al., 1997). Blood samples were drawn by cardiac puncture at 24 h after IFN- injection and placed into microtubes containing 10 µl of EDTA (4%) solution. Plasma samples were obtained after centrifugation at 3000 rpm for 3 min and then stored at 20°C until assay. The plasma 2'-5'OAS activity was determined by radioimmunoassay (2-5A kit; Eiken, Tokyo, Japan).

IFN- Stimulating Effect on Lymphocytes. To study the IFN- stimulating effect on lymphocytes, blood from groups of six mice was taken with a 1-ml syringe at 9:00 AM or 9:00 PM, and placed into microtubes containing 30 µl of EDTA (4%). The blood lymphocytes were isolated by density gradient separation medium (Lympholyte-M; Cedarlane Laboratory Ltd., Ontario, Canada) and treated with hemolysis buffer (Tris-ammonium chloride buffer, 0.83% ammonium chloride/Tris buffer = 9:1, pH = 7.65) at 37°C for 5 min to get rid of the red cells. The remaining cells were counted by trypan blue dye exclusion and resuspended to 1 × 10⁵ viable cells/ml in complete medium. IFN- was diluted with sterilized saline consisting of 1% BSA (Sigma Chemical Co., St. Louis, MO) to adjust the concentration to 1 × 10⁴, 1 × 10⁵, and 1 × 10⁶ I.U./ml. Lymphocyte suspension (1 ml) was immediately transferred into microtubes. IFN- solution (final concentration: 0, 100, 1,000, 10,000 I.U./ml) and [³H]thymidine (10 µl, 25 µCi/ml) were added to each sample. The cell suspensions were incubated at 37°C for 24 h in humidified air containing 5% CO₂. After incubation, cells were harvested by centrifugation at 10,000 rpm, washed three times in complete medium, and transferred to scintillation vials containing 10 ml of aqueous counting scintillant (ACS II; Amersham Pharmacia Biotech Ltd., Little Chalfont, Buckinghamshire, UK). The uptake of [³H]thymidine by lymphocytes was quantitated by a liquid scintillation counter (LSC-1000; Aloka Co., Mitaka, Tokyo, Japan).

Iodination of IFN-. IFN- was iodinated using a solid-phase lactoperoxidase kit (ICN Pharmaceuticals, Inc., Irvine, CA). All operations were carried out at room temperature. HCl (0.01 N) was added to neutralize the 1 mCi of Na¹²⁵I (Amersham Pharmacia Biotech Ltd.) contained in the reaction vial, after which 10 µl of IFN- (1 µg/µl) (Pepro Tech EC Ltd., London, UK) was added. The lactoperoxidase was dissolved in 25 µl of distilled water, and added to the vial of radioiodine. Hydrogen peroxide (3%, 5 µl) was added to the vial and repeated four times every 10 min to initiate the reaction. Radioiodinated IFN- was separated from reactants with a PD-10 column (Amersham Pharmacia Biotech Ltd.), pre-equilibrated with 25 ml of phosphate buffer (pH 7.5) containing 0.1% BSA, using 0.05 M phosphate buffer (pH 7.5) containing 0.5% BSA, as an elution buffer. The radioactivity was determined by measuring the radioactivity of the precipitate that was not dissolved by 10% trichloroacetic acid (TCA). The concentration of ¹²⁵I-IFN- in the eluting fraction was determined by enzyme-linked immunosorbent assay (ELISA) (IFN- immunoassay kit; BioSource International Inc., Irvine, CA). The ¹²⁵I-IFN- was used within 1 month to avoid radiolysis.

IFN- Receptor Assay. To examine the IFN- bound to lymphocytes, blood was drawn from groups of six mice and rinsed with ice-cold PBS at 9:00 AM or 9:00 PM. The lymphocytes were isolated by the method described above. The cells were resuspended to 1 × 10⁶ cells/ml in complete medium. The binding experiments were performed at 4°C for 2 h with gentle shaking in a total volume of 200 µl of complete medium containing 0.2% BSA, various concentrations of ¹²⁵I-IFN-, and 1 × 10⁶ lymphocytes in the microtubes. Sodium azide (0.1%) was also added to prevent receptor internalization. Protamine sulfate (10 µg/ml; Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added to prevent nonspecific binding, and 2.5 mM CaCl₂ was also added to improve the binding. After incubation, cells were resuspended, transferred into 200 µl of FBS, and centrifuged at 10,000 rpm for 1 min. The medium and FBS were then aspirated, the tube tip containing bound ligand was cut, and the radioactivity was determined using a gamma counter (ARC-360; Aloka Co.). Nonspecific binding was determined in the presence of an at least 250-fold excess of unlabeled IFN-. Specific binding was defined as nonspecific binding subtracted from total binding. The data were plotted according to the method of Scatchard (1949). A molecular weight of 20,000 was assumed for the calculation of the receptor number per cell and the dissociation constant (K_d).

Western Blotting of ISGF. To examine the expression of ISGF in lymphocytes, blood was drawn from groups of three mice at 9:00 AM or 9:00 PM. The lymphocytes were isolated by the method described above. The cells were resuspended to 1 × 10⁵ viable cells/ml in complete medium. IFN- solution (final concentration: 1000 I.U./ml), 10 µl each, were added to each sample. The cell suspensions were incubated at 37°C for 24 h in humidified air containing 5% CO₂. After incubation, cells were harvested by centrifugation at 10,000 rpm. Cells were washed twice with buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8.0) and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 µM Na₂V₂O₅, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml) for 15 min on ice. The lysates were centrifuged at 4°C for 15 min. Cell lysates containing 30 µg of total protein were resolved by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose. The membrane was reacted with the anti-p91 monoclonal antibody (Stat1; N terminus, Transduction Laboratories, Lexington, KY). The antibody cross-reacts with Stat1 (91 kDa) expressing cells in human, dog, mouse, chick, and frog. For detection of the antigen-antibody complex on the membrane, an alkaline phosphatase-conjugated antibody was used as a secondary reagent, and visualized with 3,3',5,5'-tetramethylbenzidine substrate. The intensity was assessed by using the NIH Image program on a Macintosh computer. Plots of ISGF set the mean value of control at 9:00 AM at 100.

IFN- Concentration in Plasma. To study the plasma IFN- concentrations over time, groups of six mice were i.v. injected with 10 MI.U./kg of IFN- at 9:00 AM or 9:00 PM. Blood samples were collected by orbital sinus collection at 0.167, 0.5, 1, 2, 3, and 4 h after IFN- injection. Plasma samples were obtained after centrifugation at 3000 rpm for 3 min and stored at 20°C until assay. Plasma IFN- concentrations were determined by an ELISA kit (IFN- immunoassay kit; BioSource International Inc.). The titer was expressed in international units per milliliter, and the detection limit for the sample was 10 I.U./ml.

IFN- Metabolism in Renal Samples. To study IFN- renal metabolism, kidneys were removed from groups of six mice. Renal slices were cut and placed in 1 ml of 0.05% BSA-Krebs-Ringer solution (37°C, 95% O₂, 5% CO₂) at 9:00 AM or 9:00 PM. Renal slices were preincubated for 30 min, and 1000 I.U. of ¹²⁵I-IFN- was added to each tube. At 0, 20, 40, and 60 min after incubation, a 30-µl sample was transferred to 300 µl of PBS or 300 µl of 10% TCA. The PBS or TCA solutions were centrifuged at 10,000 rpm for 1 min, and 300 µl of supernatant was used for detection by a gamma counter. The radioactivity of TCA-soluble fraction was calculated as follows: % = ([the radioactivity of TCA-soluble fraction at each time  the radioactivity of TCA-soluble fraction at 0 min]/[the radioactivity of PBS solution at the corresponding time (total radioactivity)]) × 100. The residual activity of IFN- was assayed by ELISA.

Statistical Analysis. Pharmacokinetic parameters were calculated by moment analysis. ANOVA and Tukey's test were used for multiple comparisons. Student's t test was used for comparisons between two groups.

	Results

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Influence of Dosing Time on 2'-5'OAS Activity in Plasma. The influence of IFN- dosing time on 2'-5'OAS activity in plasma is shown in Fig. 1. 2'-5'OAS activity in plasma showed no significant difference between mice injected with saline at 9:00 AM and 9:00 PM. 2'-5'OAS activity in plasma at 24 h after IFN- (10 MI.U./kg, i.v.) injection was significantly higher for injections given at 9:00 AM than for those at 9:00 PM (P < .05). 2'-5'OAS activity in plasma after IFN- injection at 9:00 AM increased significantly compared with that after saline injection at 9:00 AM (P < .01).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Influence of dosing time on the plasma 2'-5'OAS activity at 24 h after IFN- (10 MI.U./kg, i.v.) injection at 9:00 AM or 9:00 PM. *P < .05; **P < .01 when compared with the saline group, or between the two dosing times. , saline; , IFN-.

Time Dependence of Lymphocyte Stimulating Effect. The stimulating effects of IFN- on lymphocytes are shown in Fig. 2. At the concentrations of 0, 100, 1,000, or 10,000 I.U./ml of IFN-, the uptake of [³H]thymidine by lymphocytes after 24-h incubation was significantly higher in the cells obtained at 9:00 AM than at 9:00 PM (P < .01). The uptake of [³H]thymidine by lymphocytes after 24-h incubation in the cells obtained at 9:00 AM was significantly higher in the IFN- (1,000 or 10,000 I.U./ml) group as compared with the saline group (P < .05).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Time-dependent difference in the stimulating effect of IFN- in lymphocytes prepared at 9:00 AM () or 9:00 PM (). The uptake of [3H]thymidine by lymphocytes after 24-h incubation with IFN- was used as an index of lymphocyte-stimulating effect of IFN-. *P < .05; **P < .01 when compared between the two groups.

Time Dependence of IFN- Receptor Function. The specific binding of ¹²⁵I-IFN- at concentrations of 5, 10, 20, or 30 ng/ml (1 ng = 300 I.U., expressed in nanograms per milliliter to estimate easily the parameters of IFN- receptor) was significantly higher in the cells obtained at 9:00 AM than in the cells obtained at 9:00 PM (P < .05, Fig. 3A). The specific binding of ¹²⁵I-IFN- at other concentrations showed no significant time-dependent difference. The specific binding data were replotted by the method of Scatchard as shown in Fig. 3B. The number of receptors per cell, calculated from the intercept of the Scatchard plot on the abscissa, was significantly larger in cells obtained at 9:00 AM than at 9:00 PM (P < .05, Table 1). The apparent K_d value did not differ significantly between cells obtained at 9:00 AM and 9:00 PM.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Time-dependent difference of IFN- receptor in lymphocytes prepared at 9:00 AM () or 9:00 PM (). A, binding isotherms. B, Scatchard analysis. *P < .05 when compared between the two groups.

View this table: [in this window] [in a new window]   TABLE 1 Time-dependent difference of IFN- receptor in lymphocytes prepared at 9:00 AM or 9:00 PM Values show the mean ± S.E. of six mice. Statistical significance is compared between the two groups.

Time-Dependent Expression of ISGF in Lymphocytes. The expression of ISGF in lymphocytes is shown in Fig. 4. The expression of ISGF in lymphocytes after 24-h incubation with saline showed no significant difference between the cells obtained at 9:00 AM and 9:00 PM. The expression of ISGF in lymphocytes after 24-h incubation with IFN- (1000 I.U./ml) was significantly higher in the cells obtained at 9:00 AM than at 9:00 PM (P < .05). The expression of ISGF in lymphocytes after 24-h incubation in the cells obtained at 9:00 AM was significantly higher in the IFN- group as compared with the saline group (P < .05).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Time-dependent difference in the expression of ISGF in lymphocytes stimulated for 24 h with IFN- (1000 I.U.) at 9:00 AM or 9:00 PM. The expression of ISGF was determined by Western blot analysis. *P < .05 when compared with the saline group or between the two dosing times. , saline; , IFN-.

Influence of Dosing Time on IFN- Concentration in Plasma. Plasma IFN- concentrations after IFN- (10 MI.U./kg, i.v.) injection decayed biphasically over time (Fig. 5). IFN- concentrations at 2, 3, and 4 h after IFN- injection were significantly higher for injections given at 9:00 AM than for those at 9:00 PM (P < .05). Clearance (CL) was significantly higher in mice injected with IFN- at 9:00 PM than in those injected at 9:00 AM (P < .05, Table 2). Area under the concentration-time curve, t_1/2, and mean residence time were significantly larger in mice injected with IFN- at 9:00 AM than at 9:00 PM (P < .05).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Influence of dosing time on plasma IFN- concentrations after IFN- (10 MI.U./kg, i.v.) injection at 9:00 AM () or 9:00 PM (). *P < .05 when compared between the two groups.

View this table: [in this window] [in a new window]   TABLE 2 Influence of IFN- dosing time on the pharmacokinetic parameters after IFN- (10 MI.U./kg, i.v.) injection at 9:00 AM or 9:00 PM Values show the mean ± S.E. of six mice. Statistical significance is compared between the two groups.

Time-Dependent Metabolism in Isolated Renal Slices. The metabolism of ¹²⁵I-IFN- (1000 I.U./ml) in renal slices is shown in Fig. 6. The radioactivity of TCA-soluble fraction at 60 min after incubation was significantly higher in renal slices obtained at 9:00 PM than at 9:00 AM (P < .05, Fig. 6A). The residual activity of IFN-, analyzed by ELISA, at 60 min after incubation was significantly higher in renal slices obtained at 9:00 AM than at 9:00 PM (P < .05, Fig. 6B).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Time-dependent difference of IFN- metabolism in renal slices taken at 9:00 AM () or 9:00 PM (). A, radioactivity of degraded IFN- in TCA-soluble fraction. B, residual activity of undegraded IFN- assayed by ELISA. *P < .05 when compared between the two groups.

	Discussion

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The antiviral activity of interferon is due, at least in part, to the 2'-5'OAS system (Baglioni, 1979). Plasma 2'-5'OAS activity is used as an index of the antiviral effect of interferon in patients with hepatitis. 2'-5'OAS activity in plasma at 24 h after IFN- injection was significantly higher for injections given at 9:00 AM than for injection at 9:00 PM, although it showed no significant time-dependent difference between mice injected with saline at 9:00 AM and 9:00 PM. This finding confirms previous observations with higher activity in the latter half of the dark phase and former half of the light phase, and lower activity in the latter half of the light phase and former half of the dark phase (Koyanagi et al., 1997). To clarify the mechanisms underlying the dosing time-dependent antiviral activity induced by IFN-, the amount of [³H]thymidine uptake by lymphocytes was used as an index of the stimulating effects of IFN- on lymphocytes. The uptake of [³H]thymidine by lymphocytes after 24-h incubation with IFN- was significantly higher in the cells obtained at 9:00 AM than in the cells obtained at 9:00 PM. This finding corresponded well to the dosing time-dependent antiviral activity of IFN-. A higher response of lymphocytes to IFN- was observed at 9:00 AM than at 9:00 PM. Because IFN- also modifies pituitary-adrenal function (Roosth et al., 1986), the secretion of corticosterone by IFN- may vary depending on dosing time.

IFN- elicits antiviral activity by binding to a specific receptor on cell surface (Aguet and Mogensen, 1983; Zoon and Arnheiter, 1984), and is partially lymphocyte-mediated (Alexander et al., 1986). The number of IFN- receptors per lymphocyte was significantly larger in cells obtained at 9:00 AM than at 9:00 PM. The next question is whether the increase in IFN- receptor number is functionally significant. IFN- exerts its biological effect presumably by inducing the expression of a series of IFN-induced genes. ISGF, a transcriptional activator, is considered a positive regulator in the biological action of IFN- (Kumar and Korutla, 1995). The expression of ISGF was significantly higher in cells obtained at 9:00 AM than at 9:00 PM. This result can partly explain the time-dependent antiviral activity of IFN- in terms of pharmacology. The number of IFN- receptor binding sites increases concomitantly with an increase in IFN-signaling, when the proportion of cells in the S (DNA synthesis) phase increases in leukemia (Tamura et al., 1997). This might be true in the case of this study. Certainly, the proportion of cells in the S phase was significantly higher in lymphocytes obtained at 9:00 AM than at 9:00 PM (9:00 AM, 6.7 ± 1.3%; 9:00 PM, 1.8 ± 0.3%, n = 5, mean ± S.E., P < .05). It should be clarified in the future why IFN- receptor expression varies with cell cycle distribution.

Plasma IFN- concentrations in the elimination phase after IFN- injection were significantly higher for injections given at 9:00 AM than for those at 9:00 PM. The dosing time-dependent difference of plasma IFN- concentrations corresponded well to that of 2'-5'OAS activity induced by IFN-. Therefore, the diurnal difference of 2'-5'OAS activity induced by IFN- can be explained in part by that of plasma IFN- concentration. A significant dosing time-dependent difference was also demonstrated for the pharmacokinetic parameters of IFN-, which showed higher CL for the injection at 9:00 PM than for that at 9:00 AM. The rhythmicity in CL seems to be closely related to that in plasma IFN- concentration. IFN- concentrations in plasma have been shown to decay biphasically after i.v. injection of IFN-, and the distribution phase lasted for 1 h after drug injection (Cantell and Pyhara, 1973). IFN- is quickly eliminated from the body via several pathways. The main route of excretion is via the kidneys (Bino et al., 1982). Renal tubular cells extract and break down many plasma proteins (Strober and Waldmann, 1974). IFN- is also internalized and catabolized intracellularly in the kidney via receptor-mediated endocytosis (Bocci et al., 1983). Both the renal elimination rate and liver metabolism rate increase during the active period in mice (Ohdo et al., 1995a). The rhythmicity can reflect not only the rhythmic activity of enzymes in the kidneys and liver, but also the rhythmic rate of blood flow (Labrecque et al., 1988). The rhythmicity of CL in our study corresponds well to that of blood flow. The blood flow in humans also increases during active periods (Lemmer and Nold, 1991). This data obtained in nocturnally active mice may correspond with the data in diurnally active humans, if referred to the species-specific rest-activity cycle. The renal metabolic activity in vitro was significantly higher in the renal slices prepared at 9:00 PM than in those prepared at 9:00 AM. The finding in vitro corresponded well to the dosing time-dependent difference of IFN- pharmacokinetics in vivo. Thus the circadian rhythm in IFN- pharmacokinetics may be caused by the diurnal rhythm of renal function. Although the circadian rhythm of receptor-mediated endocytosis has not yet been investigated, it should be clarified in the future.

These findings in this mouse model support the concept that choosing the most appropriate time of day for administration of IFN- associated with the rhythmicity of IFN- receptor function and renal function may increase the antiviral activity in experimental and clinical situations.