Decrease in the Time-Dependent Difference in Urinary Excretion of Furosemide with Age

The influence of age on the time-dependent difference in urinary excretion of furosemide, a loop diuretic agent, was examined in this longitudinal study. Male Wistar rats were maintained under conditions of light from 07:00 to 19:00 h and dark from 19:00 to 07:00 h. Furosemide (30 mg/kg) was given orally at 12:00 h (day trial) or 00:00 h (night trial) to rats at 3 months of age, and urine was collected for 8 h after dosage. Thereafter, the identical protocol was repeated using the same animals at 6, 9, 12, 15, 18, and 21 months of age. The urinary excretion of furosemide was significantly greater in the day than in the night trial at 3 months of age. Such a time-dependent difference was observed for up to 15 months, but disappeared at 18 and 21 months of age. The time-dependent difference in urinary excretion of furosemide (day trial — night trial) decreased gradually throughout the observation period of the study. These results suggest that the time-dependent difference in the urinary excretion of furosemide diminishes during the aging process and disappears by 18 months of age in male Wistar rats.     

Chronopharmacology of morphine in mice

Dosing-time-dependent changes in the effect and toxicity of morphine were examined in mice housed under alternating 12 h light (07:00 to 19:00 h) and dark (19:00 to 07:00 h) cycles. Morphine (0.5 mg/kg) was injected intraperitoneally (i.p.) in animals to assess its beneficial effect (i.e., protection against the kaolin-induced, bradykinin-mediated, writhing reaction) and its toxicity (i.e., alteration of the hepatic enzymes of aspartate aminotransferase [AST] alanine aminotransferase [ALT], and glutathione [GSH] in separate experiments). The magnitude of the analgesic effect of morphine depended on dosing time, with minimum effect at 02:00 h and maximum effect at 14:00 h. The serum hepatic enzyme levels of AST and ALT increased after dosing morphine (100 mg/kg) at 02:00 and 14:00 h. Time courses of these enzymes did not differ between the two trials. However, hepatic GSH, which is involved in the detoxification of chemical compounds, significantly decreased after i.p. morphine injection at 02:00 but not at 14:00 h. Overall, the results suggest that the analgesic effect of morphine is greater after dosing during the resting than during the activity phase of mice that have been induced with bradykinin-mediated pain. Drug-induced hepatic damage as inferred by GSH alteration, however, may be greater after dosing during the active phase.     

A blood plasma inhibitor is responsible for circadian changes in rat renal Na,K-ATPase activity

Rhythmic changes in activity following a circadian schedule have been described for several enzymes. The possibility of circadian changes in Na,K-ATPase activity was studied in homogenates of rat kidney cortex cells. Male Sprague-Dawley rats were kept on a schedule of 12h light (06:00-18:00 h) and 12 h darkness (18:00-06:00 h) for 2 weeks. At the end of the conditioning period, one rat was killed every 2 h, until completion of a 24 h cycle. Outermost kidney cortex slices were prepared, homogenized and assayed for Na,K-ATPase activity. The whole procedure was repeated six times. Na,K-ATPase activity shows an important oscillation (2 cycles/24 h). Peak activities were detected at 09:00 and 21:00 h, whereas the lowest activities were detected at 15:00 and 01:00-03:00 h. The highest activity was 40+/-3 nmoles Pi mg protein(-1)min(-1) (09:00 h), and the lowest was 79+/-3 nmoles Pi mg protein(-1)min(-1) (15:00 h). The amount of the Na+-stimulated phosphorylated intermediate is the same for the 09:00 h and 15:00 h homogenates. Preincubation of 09:00 h kidney cortex homogenates with blood plasma drawn from rats at either 03:00 h or 15:00 h, significantly inhibited their Na,K-ATPase activity. This inhibition was not seen when the preincubation was carried out with either 09:00 h or 21:00 h blood plasma. The striking oscillation (2 cycles/24 h) of the Na,K-ATPase activity of rat kidney cortex cells is ascribed to the presence of an endogenous inhibitor in blood plasma.     

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Dosing‐time–dependent changes in the effect and toxicity of morphine were examined in mice housed under alternating 12 h light (07:00 to 19:00 h) and dark (19:00 to 07:00 h) cycles. Morphine (0.5 mg/kg) was injected intraperitoneally (i.p.) in animals to assess its beneficial effect (i.e., protection against the kaolin‐induced, bradykinin‐mediated, writhing reaction) and its toxicity (i.e., alteration of the hepatic enzymes of aspartate aminotransferase [AST] alanine aminotransferase [ALT], and glutathione [GSH] in separate experiments). The magnitude of the analgesic effect of morphine depended on dosing time, with minimum effect at 02:00 h and maximum effect at 14:00 h. The serum hepatic enzyme levels of AST and ALT increased after dosing morphine (100 mg/kg) at 02:00 and 14:00 h. Time courses of these enzymes did not differ between the two trials. However, hepatic GSH, which is involved in the detoxification of chemical compounds, significantly decreased after i.p. morphine injection at 02:00 but not at 14:00 h. Overall, the results suggest that the analgesic effect of morphine is greater after dosing during the resting than during the activity phase of mice that have been induced with bradykinin‐mediated pain. Drug‐induced hepatic damage as inferred by GSH alteration, however, may be greater after dosing during the active phase.     

Day-night variation in the in vitro contractility of aorta and mesenteric and renal arteries in transgenic hypertensive rats

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

Administration-time differences in the pharmacokinetics of gentamicin intravenously delivered to human beings

Administration-time differences of gentamicin pharmacokinetics were studied by crossover design after a single intravenous administration of gentamicin (80 mg) to 10 human subjects at 09:00 (morning time) and 22:00 (nighttime). The profiles of serum gentamicin concentration showed a significant statistical difference between 09:00 and 22:00, suggesting circadian variations of pharmacokinetic behaviors. A significant circadian rhythm of pharmacokinetic parameters as a function of time of day was noted in human subjects, showing lower total body clearance Clt and higher serum area under the curve (AUC) when given at nighttime. The half-life t1/2 was shorter in the morning (2.82 h +/- 0.43 h) when compared to the nighttime (2.97 h +/- 0.36 h), but the difference was not statistically significant. The AUC was significantly greater in the morning (23.4 +/- 3.84 micrograms-h/mL) than that in the nighttime (26.3 +/- 5.79 micrograms-h/mL) (p < .05), most likely because the Clt was significantly higher when gentamicin was given in the morning (3.51 +/- 0.57 L/h) versus in the nighttime (3.18 +/- 0.65 L/h). Although the volume of distribution Vd decreased when given at nighttime, it was independent of the dosing time. From this study, there was an administration-time difference of gentamicin pharmacokinetics in human beings. The optimized dosing regimen of gentamicin can be decided by considering circadian rhythm and rest-activity routine so that minimized toxicity and effective therapy are established for patients. The current findings also can be applied to other drugs with circadian rhythms of pharmacokinetics and narrow therapeutic windows in clinical chronotherapeutics.     

Circadian variation of carrageenan-paw edema in the rat

The circadian variation of carrageenan (carr)-induced paw edema was studied in sham-operated and adrenalectomized rats. The edema was produced by carr injection into the plantar tissue at 03:00, 09:00, 15:00 and 20:00 hr. In sham-operated rats the rate of appearance of maximal edema was much faster in the evening than in the morning: at 20:00 hr, it was obtained 2 hours after carr injection while at 09:00 hr it took 4 hours. At 03:00 and 15:00 hr, maximal edema was found respectively 2.5 and 4 hours after carr. In adrenalectomized rats, maximal edema at 4 hours of investigation was always larger than in sham-operated animals but the rate of appearance of edema did not change throughout the day as it was obtained 3 hours after carr administration. At 09:00 and 20:00 hr injection of hydrocortisone (HC) to adrenalectomized rats produce the same dose-dependent effect on the rate of formation of edema. However, to reproduce in adrenalectomized animals the rates of formation of edema similar to those obtained in sham-operated rats, an injection of 18 mg/kg HC was required at 09:00 hr while less than 2 mg/kg was needed at 20:00 hr. The results suggest that the circadian rhythm of carr edema is related to circadian variation in the corticosteroid system.     

The effect of diets on different factors involved in the duration of resistance to low temperature in rats

Cold resistance of male Sprague Dawley rats (300 g) fed a laboratory chow (P) or a semi-purified diet (T4F or H) for 14 days was evaluated by the degree of hypothermia developed under unrestrained conditions at –18°C or under restraint at +6°C or +1°C. Cold tests were started either at 09:00 h, 14:00 h or 23:00 h. Rats fed a semi-purified diet were more resistant to cold than P-fed rats in winter and summer but not in spring or fall. Cold resistance followed a circadian cycle, being very high at 23:00 h, very low at 14:00 h and in between at 09:00 h. The higher resistance to cold of rats on semi-purified diets coincided with a higher liver glycogen reserve throughout the day and a higher production of corticosterone in stressful conditons than in rats on P diet. However, unrelated diurnal cycles of cold resistance and liver glycogen, absence of hypoglycemia and maintenance of a high level of blood corticosterone in hypothermic rats fed P or semi-purified diets indicate that cold resistance is not limited by glycogen availability in liver or glucose and corticosterone in blood respectively. The lower fecal lactobacilli content found in rats fed a semi-purified diet supports the hypothesis that heat producing organs of these animals may have to compete with a smaller bacterial population in their small intestine for essential nutrients than the P-fed rats which could be a factor in their greater degree of cold resistance.NRCC # 17311     

Regulation of hepatic synthesis of proteins by the chronology of protein ingestion

Circadian variations in liver protein synthesis were were assessed in control rats fed a mixed 10% protein diet and in rats fed proteins as a separate meal either at 09:00 (SF 09) or at 21:00 (SF 21) and provided with a protein-free diet ad libitum. Protein synthesis was measured by incorporation of labelled leucine over a short period of time (15 min) at time-points regularly spaced over 24 h. In controls, the circadian variations observed were of moderate amplitude (from 2.75 mg/h per g at 09:00 to 5.77 mg/h per g at 06:00) correlated with increased protein and RNA contents of the liver. In separately fed animals ingestion of the protein meal triggered a 300% increase in protein synthesis within 1 h while the feeding pattern was unaltered. In the SF 09 group, high synthetic activity was not followed by an increase of hepatic protein content while hepatic urea concentrations were sharply increased and glucogenic amino acid pools were greatly depleted. It is suggested that the high influx of amino acids consecutive to the absorption of the dietary proteins is the key factor stimulating protein synthesis, while synchronisation with the energetic metabolism controls the degree of degradation. The possible involvement of variations in the insulin to glucagon ratio is discussed.     

DAY-NIGHT VARIATION IN THE IN VITRO CONTRACTILITY OF AORTA AND MESENTERIC AND RENAL ARTERIES IN TRANSGENIC HYPERTENSIVE RATS*

TGR(mREN2)27 (TGR) rats develop severe hypertension and an inverted circadian blood pressure profile with peak blood pressure in the daytime rest phase. The present study investigated the in vitro responsiveness of different arteries of TGR rats during day and night. Twelve-week-old TGR rats and normotensive Sprague-Dawley (SPRD) controls, synchronized to 12h light, 12h dark (LD 12:12) (light 07:00 19:00), were killed at 09:00 (during rest) and 21:00 (during activity), and endothelium-dependent relaxation by acetylcholine and vascular contraction by angiotensin II were studied by measuring isometric force in ring segments of abdominal aorta and mesenteric and renal arteries. In SPRD rats, consistent day-night variation was found, with greater responses to angiotensin II during the daytime rest span. In TGR rats, biological time-dependent differences were found in the renal vasculature, but not in the aorta and mesenteric artery. Relaxation of SPRD rat aorta and mesenteric artery by acetylcholine was greater at 09:00, whereas in TGR rats, day-night variation was absent (mesenteric artery) or inverted (aorta). In conclusion, based on the study of two time points, daynight variation in vascular contractility of aorta and mesenteric artery is blunted in TGR rats, whereas renal artery segments showed an unchanged daynight pattern compared to SPRD controls. (Chronobiology International, 18(4), 665 681, 2001)     

Evaluation of chronopharmacodynamics of indomethacin by the kaolin-induced pain model in mice

We previously showed that the kaolin-induced writhe reaction exhibits 24h variation with a peak at the end of the resting period (14:00-18:00h) in mice maintained under light from 07:00 to 19:00h. In this study, we used this model to evaluate the administration-time-dependent (chronopharmacodynamic) effect of indomethacin. Indomethacin (0.5 mg/kg) was given orally to mice at 02:00, 08:00, 14:00, or 20:00h, and the suppressive effect on kaolin-induced writhing was determined after each timed dosing. After dosing at 08:00h, indomethacin remarkably reduced the number of writhes during the critical span of 14:00-18:00h—the time when writhing reaction was greatest during the 24h, while the suppressive effect of the medicine after dosing at the other clock times was relatively small. These data suggest the analgesic effect of indomethacin in mice with the kaolin-induced writhing is greater after dosing in the early resting period, which is similar to that reported in patients with nocturnal pain. The kaolin-induced pain mouse model seems to be useful for the chronopharmacodynamic evaluation of analgesic agents.     

Chronokinetics of Active Biliary Ampicillin Secretion in Rats

The existence of temporal variation in biliary excretion has been demonstrated for dibromosulfophthalein and ampicillin (AMP). This study was performed to investigate if the 24h rhythm of active AMP biliary secretion could be attributed to circadian rhythms in the capacity and/or binding affinity of the active secretion mechanism. In this study, 12 Sprague-Dawley rats, housed under a 12h light/12h dark environment, were used. Each rat four lh infusions of incremental doses of AMP during either the active (24: 00 group) or rest phase (12: 00 group) under pentobarbital anesthesia. High doses of AMP were administered to saturate the biliary secretion of AMP via the anion carrier system. Bile and plasma were collected at steady stale for each infusion and analyzed by a microbiological assay. The systemic clearance of AMP was increased approximately twofold during the active phase (24: 00 group) compared to the resting phase (12: 00 group). Plots of bile excretion rate versus plasma concentration indicated saturation of the anion carrier system. Analysis of the data using the Michaelis-Menten model revealed no significant difference in the binding affinity (1/Km) of the biliary anion carrier system between the 12: 00 and 24: 00 groups. However, the maximum AMP excretion rate attained in the bile (maximum transport or Vmax) showed a 50% increase during the active phase, thus implicating a day-night variation in transport capacity of the anionic pathway. Therefore, temporal variation in the capacity of the secretory mechanisms is a determinant contributor to the proposed circadian rhythm observed in the biliary elimination of AMP.     

Evaluation of Chronopharmacodynamics of Indomethacin by the Kaolin-Induced Pain Model in Mice

We previously showed that the kaolin-induced writhe reaction exhibits 24h variation with a peak at the end of the resting period (14:00–18:00h) in mice maintained under light from 07:00 to 19:00h. In this study, we used this model to evaluate the administration-time-dependent (chronopharmacodynamic) effect of indomethacin. Indomethacin (0.5 mg/kg) was given orally to mice at 02:00, 08:00, 14:00, or 20:00h, and the suppressive effect on kaolin-induced writhing was determined after each timed dosing. After dosing at 08:00h, indomethacin remarkably reduced the number of writhes during the critical span of 14:00–18:00h—the time when writhing reaction was greatest during the 24h, while the suppressive effect of the medicine after dosing at the other clock times was relatively small. These data suggest the analgesic effect of indomethacin in mice with the kaolin-induced writhing is greater after dosing in the early resting period, which is similar to that reported in patients with nocturnal pain. The kaolin-induced pain mouse model seems to be useful for the chronopharmacodynamic evaluation of analgesic agents.     

Melatonin excretion of man and rats: Effect of time of day,sleep, pinealectomy and food consumption

Pineal function was assessed in human subjects by measuring the excretion of melatonin. The hormone was extracted from the urine by adsorption on a nonionic polymeric resin and then elution with organic solvents; its concentration was determined with a bioassay based ontthe dermal melanophore response of larval anurans to melatonin in their bathing medium. Melatonin excretion among healthy, adult volunteers was 5- to 7-fold greater during the hours of sleep, darkness, and recumbancy (23:00–07:00 h) than during the active, waking hours (07:00–15:00 h or 15:00–23:00 h). When 2 subjects slept only during the daylight at odd, 8-hour intervals, their melatonin excretion was also greatest during their time of sleep. Three bedfast fracture patients (for whom sleep data were not available) failed to display melatonin rhythms. A new radioimmunoassay for melatonin was investigated in preliminary studies with experimental animals and was found to be expedient and sensitive; it was not as specific as the bioassay, however. Both analytical methods were used in studies on intact and pinealectomized rats, and the findings suggest that rhythmic melatonin excretion persists (although at a reduced amplitude) in the absence of the pineal. Fasting in the pinealectomized animal abolished the day/night variation in urinary melatonin, but did not have this effect in intact rats.     

Effects of hemodialysis on serum lipids and phospholipids of end-stage renal failure patients

There is a link between diabetes and oxidative stress. Hyperglycaemia leads to free radical generation and alterations of endogenous antioxidants. Our aim is to study the effect of orally administered L-tryptophan (TRP), the melatonin precursor, an endogenous antioxidant, on circulating levels of glycaemia, insulin and melatonin, and on the superoxide dismutase and catalase antioxidant systems in non-diabetic (ND) and type 2 diabetic (n5-STZ) male Wistar rats. At 19:30 every day for 15 days, TRP (125 mg/kg body weight) was administered orally. At 09:00 every two days the glycaemia was measured and every day the intake of food and water was recorded. At the beginning and end of treatment (at 09:00; 21:00; 02:00) plasma insulin and melatonin levels were measured, and (at 09:00) the enzymatic activities of catalase and superoxide dismutase (SOD) in erythrocytes were also measured. Glycaemia values were greater (p < 0.01) in n5-STZ rats than in ND rats, while insulin levels were lower (p < 0.05) at all times studied and these parameters were not altered by the TRP administration. Melatonin levels at 02:00 were lower in n5-STZ than in ND rats (p < 0.05). The TRP administration did not modify the circulating melatonin levels in ND rats, but raised (p < 0.01) the levels at 02:00 in the treated n5-STZ group. In ND rats after TRP administration there was a decline in catalase activity (p < 0.05), while in n5-STZ rats there was a rise (p < 0.01) at the end of treatment. However, there were no significant changes in SOD activity. There was increased food intake (g/day) in the treated n5-STZ group (p < 0.01). In conclusion, the oral administration of TRP did not modify glycaemia or insulinaemia levels, but raised melatonin levels in diabetic rats at 02:00, lowered catalase activity in ND rats but raised it in n5-STZ rats, and increased food intake in n5-STZ rats.     

Circadian variations of serotonin in plasma and different brain regions of rats

Most of the physiological processes that take place in the organism follow a circadian rhythm. Serotonin is one of the most important neurotransmitters in our nervous system, and has been strongly implicated in the regulation on the mammalian circadian clock, located in the suprachiasmatic nuclei (SCN). The present study analysed the levels of serotonin over a period of 24 h in the plasma and in different brain regions. The model used was of male Wistar rats, 14 +/- 2 weeks of age (n = 120), maintained under conditions of 12 h light and 12 h dark, and food and water ad libitum. The serotonin levels were measured by ELISA every hour at night (20:00-08:00 h) and every 4 h during the daytime (08:00-20:00 h). Ours results show that the maximum levels of serotonin in plasma were obtained at 09:00 and 22:00 and a minor peak at 01:00 h. In hypothalamus there was a significant peak at 22:00 and two minor peaks at 17:00 and 02:00 h; the same occurred in hippocampus with a significant peak at 21:00, and two secondary peaks at 24:00 and 05:00 h; in cerebellum there were two peaks at 21:00 and 02:00 h, while in striatum and pineal there were peaks at 21:00 h and 23:00, respectively. In conclusion, the higher levels of serotonin were during the phase of darkness, which varies depending on the region in which it is measured.     

Changes in inhibin activity, and progesterone, oestrogen and androstenedione concentrations, in rat follicular fluid throughout the oestrous cycle

Follicular fluid was aspirated from all visible surface follicles of rats at selected times of the oestrous cycle. Fluids from a pair of rat ovaries were pooled and assayed for inhibin activity by the rat anterior pituitary cell culture assay. Serum LH, FSH and progesterone as well as follicular fluid progesterone, total oestrogens and androstenedione were also measured. Follicular fluid inhibin activity was relatively constant throughout the oestrous cycle (30.7 +/- 3.4% inhibition of FSH per 0.1 microliter follicular fluid) except for a well defined surge at pro-oestrus (09:00-16:00 h, peak at 14:00 h = 84.0 +/- 7.2% inhibition of FSH per 0.1 microliter follicular fluid). The follicular fluid was not treated with charcoal before assay because a pilot experiment showed that such treatment did not alter the inhibin activity of follicular fluid. Steroids in follicular fluid were generally lowest on the afternoon of oestrus and the morning of dioestrus I and generally elevated during pro-oestrus.     

Effect of food intake on urinary excretions of histamine, N tau-methylhistamine, imidazole acetic acid and its conjugate(s) in humans and mice

The urinary excretions by young healthy men of histamine and its metabolites, N tau-methylhistamine, imidazole acetic acid, and imidazole acetic acid conjugate(s), increased 1-3 h after food intake. The increase was seen even after the intake of konnyaku (mannan) as a protein-deficient food, suggesting that physical stimulation of the gastric mucosa by food is the main cause of histamine release. This suggestion was confirmed by the following findings in patients and mice. In patients with stomach diseases, gastrectomy resulted in decreases in the excretion of histamine and its metabolites in the urine, and patients subjected to intravenous hyperalimentation excreted less histamine and its metabolites in the urine than normal subjects. In mice, a correlation of histamine excretion with food intake was demonstrated experimentally. Namely, mice fed only during the night (21:00-0:00) showed increased excretions of histamine and its metabolites at 23:00-3:00, whereas those fed in the morning (9:00-12:00) showed increased excretions of those compounds at 11:00-15:00. All these results are consistent with the idea that urinary histamine and its metabolites mainly originate from the stomach.     

Dosing time and sex-related differences in the pharmacokinetics of cefodizime and in the circadian cortisol rhythm

Two g cefodizime i.v. administration at 00:00, 06:00, 12:00 and 18:00 respectively, to 8 male and 8 female, young healthy volunteers, has shown: 1. a sex-related difference in both plasma (AUC) and total cumulative excretion of the agent with larger values in females than in males; 2. a dosing time-related difference in plasma (AUC) with the largest values for Rx at 00:00 and lowest for Rx at 18:00 in both males and females; 3. a dosing time-related difference in urinary concentration of cefodizime with largest values for Rx at 06:00 and lowest for Rx at 12:00. These urinary changes were highly correlated with changes in AUC for each dosing time; 4. no dosing time-related changes were observed for plasma T1/2 as well as cumulative urinary excretion of cefodizime; 5. curve patterns of plasma cortisol had similar aspects for both control and Rx at 00:00 with no sex-related differences. Curve patterns differed from control for other dosing times (p less than 0.005 to p less than 0.001 with ANOVA tests).     

Administration-Time Differences in the Pharmacokinetics of Gentamicin Intravenously Delivered to Human Beings

Administration-time differences of gentamicin pharmacokinetics were studied by crossover design after a single intravenous administration of gentamicin (80 mg) to 10 human subjects at 09:00 (morning time) and 22:00 (nighttime). The profiles of serum gentamicin concentration showed a significant statistical difference between 09:00 and 22:00, suggesting circadian variations of pharmacokinetic behaviors. A significant circadian rhythm of pharmacokinetic parameters as a function of time of day was noted in human subjects, showing lower total body clearance Cl_t and higher serum area under the curve (AUC) when given at nighttime. The half-life t_1/2 was shorter in the morning (2.82h ± 0.43h) when compared to the nighttime (2.97h ± 0.36h), but the difference was not statistically significant. The AUC was significantly greater in the morning (23.4 ± 3.84 μg-h/mL) than that in the nighttime (26.3 ± 5.79 μg-h/mL) (p<. 05), most likely because the Cl_t, was significantly higher when gentamicin was given in the morning (3.51 ± 0.57 L/h) versus in the nighttime (3.18 ± 0.65 L/h). Although the volume of distribution V_d decreased when given at nighttime, it was independent of the dosing time. From this study, there was an administration-time difference of gentamicin pharmacokinetics in human beings. The optimized dosing regimen of gentamicin can be decided by considering circadian rhythm and rest-activity routine so that minimized toxicity and effective therapy are established for patients. The current findings also can be applied to other drugs with circadian rhythms of pharmacokinetics and narrow therapeutic windows in clinical chronotherapeutics.