Anesth Analg 2005;101:1706-1708
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000184127.67866.2E
ANESTHETIC PHARMACOLOGY
The Dosing-Time Dependent Effects of Intravenous Hypnotics in Mice
Yuki Sato, MD, PhD*
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Norimasa Seo, MD, PhD*, and
Eiji Kobahashi, MD, PhD
*Department of Anesthesiology and Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan
Address correspondence and reprint requests to Eiji Kobayashi, MD, PhD, Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, 3311–1, Yakushiji, Minamikawachi, Kawachi, Tochigi 329–0498, Japan. Address e-mail to eijikoba{at}jichi.ac.jp.
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Abstract
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Chronobiology, which focuses on the biological rhythms that occur in the organization of living organisms, has been studied for several decades. Chronopharmacology, however, has received little attention until recently. We examined the hypnotic duration of intraperitoneally administered ketamine, pentobarbital, propofol, midazolam, and ethanol, to test whether they have obvious dosing-time dependent effects. Male C57BL/6 mice, which showed clear circadian rhythms of water-intake under a strict 12-h lighting cycle, were used. All tested drugs had significantly longer episodes of loss of righting reflex when administered at 22:00 (early active phase) than at 10:00 (early inactive phase). This dosing-time dependent hypnotic duration did not depend on the contents and activities of cytochrome P450 enzymes in the liver. These findings might be of clinical benefit in deciding the administration time and doses of anesthetics.
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Introduction
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Most physiological functions have a rhythm with a period of approximately 24 h (1). Drug efficacy also varies markedly within the 24-h period, depending on the time of administration (2). Anesthetic requirement is strongly correlated with the sleep-wake cycle, so that chronobiological aspects of anesthetics have been studied for several decades (3–5). However, the effect of dosing-time on the actions of common IV hypnotics has not yet been fully established (6).
Gamma-aminobutyric acid type A (GABAA) receptors and N-methyl-d-aspartate (NMDA) receptors are now viewed as important sites for general anesthetic action. Circadian variation in the number and activity of GABAA and NMDA receptors has recently been demonstrated (6). We (7) have previously reported a clear chronopharmacological effect of ketamine, a noncompetitive NMDA receptor antagonist. Ketamine was most effective when administered at 22:00 (early active phase) and least effective at 10:00 (early inactive phase). In the present study, we tested other IV hypnotics (pentobarbital, propofol, midazolam, and ethanol), which act mainly through GABAA receptors (8), to investigate whether these drugs also have increased hypnotic duration in the early inactive phase.
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Methods
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All animal procedures and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of Jichi Medical School. Male C57BL/6 mice aged 10 to 12 wk were used. They were maintained in a pathogen-free room in a controlled environment (22°C ± 2°C room temperature, 12-h light/dark cycle, light on at 07:00). To measure daily variations in locomotor activities, their water intake was measured every 6 h as described previously (n = 6) (8). Briefly, drinking water was provided in 15-mL test tubes (DM-G1; O’Hara, Tokyo, Japan) after the animals had become familiar with their environment over 7 days. For 3 days the tubes were weighed 4 times daily, at 01:00, 07:00, 13:00, and 19:00.
To study the chronopharmacological effect of IV hypnotics, 200 mg/kg of ketamine (Sankyo CO, Tokyo, Japan), 50 mg/kg of pentobarbital (Dainippon CO, Tokyo, Japan), 100 mg/kg of propofol (AstraZeneca CO, Osaka, Japan), or 4 g/kg of 20% (volume/volume) ethanol (Wako Pure Chemical Industries, Osaka, Japan) solution in saline was injected intraperitoneally at 10:00 or 22:00 (n = 10 in each group). Each mouse was served as its own control by being given a single drug at the two different times on different days. Hypnosis was evaluated as the duration of loss of righting reflex (9), as described previously (8).
To measure hepatic metabolism at different times of a day, we further examined the total content of cytochrome P450 enzymes (CYPs) and activity levels of isoenzymes in the liver at 10:00 or 22:00 by high-performance liquid chromatography (HPLC) (n = 4 in each group), as described previously (8,10). The total content of P450 was measured spectrally by the original method using carbon monoxide difference (11). The interassay and the intra- assay coefficients of variation for the total content of P450 were 8% and 5%, respectively. To measure the activity levels of CYP1A1/2, 7-ethoxyresorufin deethylase activities were determined using 0.5 µM 7-ethoxyresorufin (Sigma Chemical CO, St. Louis, MO) as a substrate. The reaction, which contained 0.2 mg/mL microsomal protein, was initiated by adding of the NADPH-generating system, which contained 2.5 mM NADP+ (Oriental Yeast CO, Tokyo, Japan), 25 mM glucose 6-phosphate (Oriental Yeast CO), 10 mM MgCl, and 2U glucose-6-phosphate dehydrogenase (Oriental Yeast CO). After incubating for 10 min at 37°C the reaction was terminated by adding 0.1 mL ethanol (Wako Pure Chemical Industries). HPLC analysis was performed using HPLC Gulliver 1500 analysis (Nihonbunnko, Tokyo, Japan) with a reversed phase analytical column. The excitation and emission wavelengths were set at 575 and 595 nm. The quantification was achieved by using a resorufin (Sigma Chemical CO) as a standard. To measure the activity levels of CYP2C8/9, tolbutamide hydroxylase activities were determined using 400 mM tolubutamide (Sigma Chemical CO) as a substrate. The reaction, which contained 0.5 mg/mL microsomes, was initiated by adding of the NADPH-generating system. After 60 min incubation the reaction was terminated by adding 25 mL hydrochloric acid (4N, Wako Pure Chemical Industries) and 4 mL ethylacetate (Wako Pure Chemical Industries) and then chlorpropamide (Sigma Chemical CO) was added as an internal standard. HPLC analysis was performed with a reversed phase analytical column and the elute was monitored at 230 nm. The quantification was achieved by using a hydroxytolbutamide (Sumika Chemical Analysis Service. Osaka, Japan) as a standard. To measure activity levels of CYP3A, testosterone-6b-hydroxylase activities were determined using 150 mM testosterone (Wako Pure Chemical Industries) as a substrate. The reaction, which contained 0.5 mg/mL microsomes, was initiated by adding of the NADPH-generating system. After 10 min incubation the reaction was terminated by adding 2 mL ethylacetate (Wako Pure Chemical) and then 17a-methyltestosterone (Wako Pure Chemical) was added as an internal standard. HPLC analysis was performed with a reversed phase analytical column and the elute was monitored at 245 nm. The quantification was achieved by using a 6b-hydroxytestosterone (Sigma Chemical CO) as a standard. The interassay and the intra- assay coefficients of variation for the activity levels of isoenzymes (A1/2, 2C8/9, and 3A) were 5% and 3%, respectively.
Statistical analysis was performed using the paired Student’s t-test. Data are expressed as mean ± sd. A value of P < 0.05 was taken as significant.
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Results and Discussion
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Circadian locomotor activities of the experimental animals were measured as the volume of water consumed every 6 h for 3 consecutive days (n = 6). Figure 1 shows that water consumption follows a circadian pattern. It peaked between 19:00 and 01:00 in each dark phase and was least between 07:00 and 19:00 in each light phase. This indicates that these nocturnal animals were most active between 19:00 to 01:00, in the dark phase, and least active between 7:00 and 19:00, in the light phase.
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Figure 1. Daily variation of water intake in male C57BL/6 mice. Water intake was measured every 6 h (01:00–07:00, 07:00–13:00, 13:00–19:00, and 19:00–01:00) for 3 days. The average amount of water intake in each interval was plotted at the mid-time of each interval. Water consumption displayed a circadian pattern, implying a daily locomotor rhythm of the experimental animals. Error bar, sd (n = 6).
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Recent molecular and gene targeting observations suggest that propofol and pentobarbital enhance GABAA receptor b1 or b3 subunit and that benzodiazepine enhances b3 subunit to achieve their anesthetic effects (7). Ethanol mediates transmembrane (TM) domain 2 and TM3 of GABAA receptor a1, a2, and a3 subunits (12,13). All tested drugs induced loss of righting reflex at both 22:00 and 10:00. We found that, as well as ketamine (P = 0.001), the hypnotic duration of these GABAergic drugs (pentobarbital, propofol, midazolam, and ethanol) had significantly longer episodes of loss of righting reflex when administered at 22:00 (early active phase) than at 10:00 (early inactive phase) (n = 10 for each) (P = 0.001, P = 0.03, P = 0.01, and P = 0.02, respectively) (Table 1). To investigate whether the differences in anesthetic effect with dosing time are influenced by hepatic metabolism, we examined the total content of CYPs and activities of isoenzymes (A1/2, 2C8/9, and 3A) in the liver at 10:00 and 22:00. Although the hepatic flow increases in the active phase, total content CYPs (mmol/mg) and activities of isoenzymes (pmol/mg/min) showed no marked differences between the two different dosing times (n = 4 for each) (Table 2). These results were also supported by our previous findings that no significant difference was observed in the plasma blood concentration of ketamine at different dosing times (at 10:00 and 22:00), suggesting that the effects of dosing time are attributable to differences in sensitivity of the central nervous system (CNS) rather than pharmacokinetics.
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Table 2. Total Content of Cytochrome P450 enzymes (CYPs) and their Isoenzyme Activities in Male C57BL/6 Mice at 2 Different Times of Day (10:00 and 22:00)
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Anesthetic drugs possess dosing time dependent effects (3,5). In recent clinical studies, chronopharmacology of intrathecal sufentanil and ropivacaine for labor analgesia has been reported (14,15). In the present study, we found that a NMDA receptor antagonist (ketamine) and GABAA receptor agonists, such as pentobarbital, propofol, midazolam, and ethanol, showed increased hypnotic duration in the early active phase (at 22:00) in C57/BL6 mice. Although the mechanisms of these dosing-time dependent effects have not been determined, several reports show that postsynaptic GABAA receptor activity is increased during nocturnal hours (16) and that there are also circadian effects in the expression of NMDA receptors in the brain (17). These receptor activity levels over a day might therefore be related to the efficacy of hypnotic drugs, both NMDA receptor antagonists and GABAA receptor agonists. Also, the lack of statistically significant differences in the total content of CYPs and the activity levels found for CYPs in the liver indicate that these dosing time dependent hypnotic durations might be explained by differing sensitivity of the CNS (pharmacodynamics) rather than pharmacokinetics.
In conclusion, we have demonstrated the larger hypnotic duration of various IV hypnotics in the early active phase in C57BL/6 mice.
The authors thank Dr. Satoshi Suzuki (HAB Biomedical Research Institute, Chiba, Japan) for measurement of the content of CYPs and their isoenzymes activities.
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Footnotes
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Supported, in part, by "21st Century Center of Excellence (COE)" program of Japan’s Ministry of Education (Tokyo, Japan) and by the Research Award to Jichi Medical School Graduate Student (Tochigi, Japan) to Y.S.
Presented, in part, at the 5th Annual Eurosiva Congress, April 5–6, 2001, Nice, France.
Accepted for publication June 29, 2005.
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Abstract
Introduction
