Chronopharmacological study of KE-SI-TO in mice

Life Sciences, Vol. 60, No. 23, pp. 2091~2O!B,1997 Copyright e 1997 E!kevier Science Inc. Printed in the USA. All rights reserved cm-3205197 517.co + .oo

ELSEVIER

PII SOO24-3205(97)00196-3

CHRONOPHARMACOLOGICAL STUDY OF KE-St-TO IN MICE Nobuya Ogawa ‘, Shigehiro Ohdo *, Jian Guo Song 3 and Shun Higuchi ’ ‘Department of Pharmacology, Ehime University School of MedicineShigenobu-Cho, Onsen-Gun, Ehime 791-02, Japan, Division of Pharmacokinetics, *Department of Clinical Pharmaceutical Science, Kyushu University, 3-l -1, Maidashi, Higashi-Ku, Fukuoka, 812 Japan and 3Department of Pharmacology, Wannan Medical College, Wuhu, Anhui Province, China 241001 (Received in final form February 27,1997)

Summary Influence of dosing time on pharmacological effects and toxicity of KE-SI-TO (KST), analgesic and antipyretic drug, was investigated in ICR male mice under LD (12:12) cycle. Significant circadian rhythms were demonstrated for analgesic and hypothermal effects of KST (1 g/kg, i.p.) with higher values in the dark and lower ones in the light ( p
!&o-Japanese

medicine, cluonopharmacology,

chronotoxicity

KE-SI-TO(KST), a classical traditional Sino-Japanese medicine, is used in the clinical Corresponding Author: Dr. Shigehiro Ohdo, Ph.D., Department of Clinical Pharmacokinetics, Division of Pharmaceutical Science, Kyushu University, 3-l -1, Maidashi, Higashi-Ku, Fukuoka 812 Japan;Tel: 092-641-l 151 (ex.6198) Fax: 092-651-7483

2m2

Chronophamacdogy of KESI-TO

Vol. 60,No. 23,1!?37

practice to treat disorders, such as influenza. The analgesic and antipyretic activities of the drug are well established and recognized (1). KST has a marked therapeutic potentiality in the management of fever, headache, nerve pain, muscle and joint stiff. KST (7.5 g/day, oral dose for human) contains five crude drugs: Cinnamomi Cortex, Paeoniae Radix, Zingiberis Rhizoma, Glycyrrhizae Radix and Zizyphi Fructus (4:4:1.5:2:4). Cinnamomi Cortex and Paeoniae Radix are principal ingredients and others are co-ingredients. Paeoniflorin (terpenoid) extracted from Paeoniae Radix (2), shogaol and gingerol (phenyl ketone) extracted from Zingiberis Rhizoma (1) have analgesic effects. Cinnamaldehyde (essential oil) extracted from Cinnamomi Cortex, shogaol and gingerol show antipyretic and hypothermal effects (3). These chemical substances together with other ingredients in KST, such as liquiritin (flavonoids) extracted from Glycyrrhizae Radix (2), contribute to the analgesic and antipyretic effects of the drug. lt is of interest that, according to the theory of traditional Chinese medicine and the observations in clinical practice (4,5), KST is more effective when administered during the active span than during the rest span. Both painful sensitivity to the thermal stimulus and body temperature also show circadian stage-dependent changes (6-8). These findings suggest that KST may show a circadian stage-dependent effect. Biological responses to various drugs follow circadian rhythms in experimental animals as well as human beings (9-13). The findings indicate that it is important to state what time a drug is studied in terms of biological time. However, the exact information on the chronopharmacological effect of Sino-Japanese medicine has not been demonstrated up to date. The present study was undertaken in order to examine the existence of circadian rhythm in the pharmacological effects, analgesic and hypothermal effects, and the toxicity of KST in mice. To elucidate the mechanism underlying the rhythms of analgesic and hypothermal effects, the rhythms were compared with those in mice injected with saline. Method Animals and treatments ; Male ICR mice (Clea Inc. Osaka, Japan), 6 weeks old with body weight from 28 to 32 g, were housed 10 per cage in a light-controlled room (lights on from 0700 to 1900) at a room temperature of 24 -t 1 “c and a humidity of 60 + 10 % with food and water ad libitum. In the study observing the circadian rhythm in analgesic and hypothermal effects, groups of 10 mice each were injected intraperitoneally ( i.p.) a single dose of KST (1 g/kg) (Kanebo Co., Ltd., Osaka, Japan) or saline at once of six times: 0900, 1300, 1700,2100, 0100 and 0500 hr. The drug was dissolved in saline to yield appropriate concentration of lg/lOml. The volume of injection was 0.1 ml/log body weight. The KST-induced analgesic and hypothermal effects were determined at 0.5 hr after KST injection. In order to study the time course of analgesic effect of KST, groups of 10 mice each were given a single i.p. dose of KST (1 S/kg) or saline on two occasions: in the early half of the light (0900) or in the early half of the dark (2100). The

Vol. 60, No. 23,1997

Chronophmacology

latency time to either hind foot-lifting and 4 hr after the drug groups

or jumping

described

The dosage above.

2m3

were recorded before and at 0.51 ,152

For the time course of hypothermal

of 10 mice each were given

determined. single

injection.

of KE-SI-TO

effect of KST,

KST (1 g/kg )and the rectal temperatures

and the procedure

In the toxicological

were

were the same as the analgesic

study, groups

effect

of 10 mice each were injected

i.p. dose of KST(6 g/kg) at once of six times as described

above.

a

After KST

injection, the mice were returned to the cages and KST-induced

mortality was observed

for 7 days. Dead animals

The dosages

experiment

were removed

were determined

Determination

of analgesic

at each observation.

by preliminary

dose-response

effect ; A thermal technique

for each

trials. (Hot plate for analgesia

test,

KN-205D type, Natume, Tokyo, Japan) was used to evaluate analgesic effect(l4). The animals were placed on a hot plate (55-tO.5 ‘C) after an injection of KST or saline. Then

the time to either

likelihood

of habituation

hind

foot-lifting

or tolerance

or jumping

were

recorded.

To avoid

the

of mice to the hot plate, the animals were not used

repetitively. Determination by measuring lubricated

of hypothermal effect ; the rectal temperature

thermocouple

digital thermometer

was inserted

The KST-induced hypothermia was determined (FIT) after an injection of KST or saline. A 2 cm into the rectal of mice. RT was read on a

(‘Takara thermistor, Digimulti

D611, Takara, Tokyo, Japan)(l5).

Statistical analysis ; Analysis of variance (ANOVA) and Tukey’s test were applied for the multiple comparison. x ’ -test was used for the mortality data. A probability level of eO.05 was considered to be significant. Results The mice given

saline

showed

a significant

circadian

rhythm

in the latency

of their

response to the thermal stimulus, spending significantly longer latency during the dark than during the light (~~0.01, Fig.1). The latency was shorter at 0900 and increased during the light. Then it was significantly

longer

at 1700 (~~0.01) and peaked at 0100

(peO.01). The mice given KST also showed a significant circadian rhythm in the time spent on the hot plate with the shortest latency at 0900 and the longest one at 0100 (~~0.01). The time spent on the hot plate after KST injection during 24 hr cycle when compared pattern of KST-induced analgesia

was significantly

longer

with that after saline injection (p-zO.01). The rhythmic resembled overall the rhythm occuring after saline

injection. The time course of the time spent on the hot plate after saline injection showed circadian stage-dependent changes with longer latency at 2100 and shorter one at 0900 (pcO.01, Fig.2). 2100 was significantly respectively).

The time spent on the hot plate after KST injection at 0900 or longer when compared with that after saline injection (p~O.01,

The mice given

KST at 2100 showed

significantly

longer

latency

as

Chronopharmacology

2094

II

I

’

I

Vol. 60, No. 23.1997

of KE-SI-TO

r

’

I

I

’

I

0900 $300 1700 2100 0100 0500 Time of drug Injection (clock hourr)

Fig.1. Circadian rhythm of latency of mice to hot plate at 0.5 hr after KST(l g/kg) injection (0) or saline injection (0). Each point represents the mean +SE of 10 mice.

0

2

1 time

after

3

4

drug InjectIon

5

(hr)

Fig.2.

The time course of KST-induced anafgesiceffect in mice. Each point represents the mean?SE of 10 mice. 0:safine injection at 0900, l :KST (1 g/kg) injection at 0900, 0 :saline injection at 2100, m:KST (1 g/kg) injection at 2100.

?! 3 5 :: 2E

36 37 36

0900

1300

1700

2100

Time of drug InJectIon

0100 (clock

0500 hours)

Fig. 3.

Circadian rhythm of rectal temperature of mice at 0.5 hr after KST (1 g/kg) injection (@) or saline injection (0). Each point represents the mean +SE of 10 mice.

Chronopharmacology

Vol. 60, No. 23,1997

-l

I

.

I

-

t

*

Time after

I

.

I

3

2

1

0

of KE-S-TO

-

4

drug in)ection

5

(hr)

Fig.4. The

time course

of KST-induced

injection at 0900,

hypothermal

effect

in mice. Each

point

of 10 mice. O:saline injection at 0900, l :KST (1 g/kg)

represents the mean *SE

q:saline

m:KST (1 g/kg) injection at 2100.

injection at 2100,

100 g

90

2

a0

;

70

5

60

E

50

= z :: ; p

40 30 20 1Y-b 10 0.

, 0900

.

1 1300

.

, 1700

.

, 2100

Time of drug Injection

, 0100 (clock

.

, 1 0500

hours)

Fig.5. Circadian rhythm of mortality after KST (6 g/kg) injection. Each point represents the data from 10 mice. 0:

mortality within 1 day, 0: mortality within 7 days.

compared with those given KST at 0900 (p
course

of RT after saline

injection

showed

circadian

stage

dependent

changes (Fig.4). For mice given saline at 0900, RT gradually decreased and was significantly lower at 4 hr after saline injection (p
20%

Chronopharmacology of KE-U-TO

Vol. 60, No. 23,1!3!?7

at 2100, FIT gradually increased and was significantly higher at 4 hr after saline injection (p
Vol. 60, No. 23,1997

A significant injection. showed

circadian

rhythm was demonstrated

All of the dead mice died within the inhibitory

behavioral showed

Chronopharmacdogy of KE-SI-TO

inhibition

respiratory

inhibitory

effects of central

inhibition

cause chronotoxicity and

chronotoxicity

nervous

The animals

system (such as sedative

and decrease

(23). Circadian

of body temperature

affect

respiration

symptoms,

rhythms are shown for respiratory

thermoregulatory

Several

of the drugs that act on the autonomic

and/or

system

those factors may contribute

inhibitory

before death. Central

and body temperature

(7,8,24).

(25). Therefore, has central

for the mortality within 24 hr after KST

3 days after KST injection.

and loss of righting reflex) before falling to sleep. The animals also

drugs such as barbiturates

temperature

2097

amines central

to the chronotoxicity

effect and hypothermal

,

function,

body

influence nervous

the

system

of KST, since

effect, and also influences

and

KST

electrolyte

(1,2,3,26,27). Rhythmic changes,

including

circadian

and circannual

ones, have been reported in the

pharmacokinetics of drugs (9,28). The chronopharmacokinetics with the rhythms in physiological and/or biochemical functions

of drugs are associated such as gastric function

(29), plasma protein (30), blood flow (31), microsomal enzyme (32,33) and urinary acidity (34,35). The pharmacokinetics of KST may also vary depending on dosing time. The present

study suggests

that the injection

shows clearly higher analgesic biological

rhythm

should

of KST during the active period of mice

effect and lower toxicity. The principles

be included

effects and toxicity of KST, Sino-Japanese

in the consideration

and concepts of

of the pharmacological

medicine.

Acknowledgements Kanebo them.

Co. Ltd. (Osaka, Japan) generously

supplied

the KE-SI-TO. We are grateful to

References 1. M. YAMADA and S. TEI, Handbook

of Crude Drugs, Juntendou,

2. K TAKAGI and M. HARADA, J. Pharmaceu.

Tokyo (1985).

Sot. Japan 89-879-886

(1969).

3. M. HARADA and Y OZAKI, J. Pharmaceu. Sot. Japan 92_ 135-l 40 (1972). 4. YC. ZHANG, Shantong Traditional Chinese Medicine 3 6. (1988). 5. J.B. HU, Chronomedication of Traditional Chinese Medicine 123-124, Anhui Scientific

Press, Hefei (1989).

6. C.B. BOURDALLE-BADIE,

B. BRUGUEROLLE,

G. LABRECQUE,

ERNY, Annu. Rev. Chronopharmacol. & 155-l 81 (1990). 7. R. REFINElTI and M. MENAKER, Physiol. Behav. 51613-637

S. ROBERT and P

(1992).

8. A. REINBERG and M. SMOLENSKY, Thermoresulation: Physiology and Biochemistry, E. Schonbaum and P Lomax (Eds), 61-100, Pergamon Press, New York (1990). 9. E. HAUS, F. HALBERG, J.F.W. KUHL and D.J. LAKATUA, Chronobiological Aspects of Endocrinology,

J. Aschoff, F Ceresa and F. Halberg (Eds), 269-304,

Schattauer-

2098

Chronopharmacologyof KE-SI-TO

Vol. 60, No. 23,1!??7

Verlag , Stuttgart and New York (1974). 10. A. REINBERG, Chronobioloqical and F. Halberg

(Eds), 305337,

Aspects of Endocrinology, Schattauer-Verlag,

J. Aschoff, F. Ceresa

Stuttgart and New York (1974).

11. S. OHDO, S. NAKANO and N. OGAWA, Japan, J. Pharmacol.

g

12. S. OHDO, N. OGAWA and J.G. SONG, Eur. J. Pharmacol. 293

11-l 9 (1988). 151-l 57 (1995).

13. S. OHDO, N. OGAWA, S. NAKANO and S. HIGUCHI, J. Pharmacol. 74-81 (1996). 14. M. KAVALIERS and M. HIRST, Brain Research 279 387-393 15. A. CABANAC

and E. BRIESE, Physiol. Behav. 5195-98

16. R.C.A. FREDERICKSON,

Exp. Ther. 278

(1983).

(1991).

V. BURGIS and J.D. EDWARDS, Science -756-758

(1977). 17. D.L. WESCHE and R.C.A. FREDERICKSON, Life Sci. 29-2199-2205 (1981). 18. D. NABER, A. WIRZ-JUSTICE and MS. KAFKA, Neurosci. Lett. 2145-50 (1981). 19. R. PRZEWLOCKI, W. LASON, A.M. KONECKA, C. GRAMSCH, REID, Science 219 71-73 (1983). 20. F. HALBERG, H.A. ZANDER, M.W. HOUGLUM Physiol. 177

361-366

A. HERZ and L.D.

and H.R. MUHLEMANN,

(1954).

21. R. REFINElTI, H. MA and E. SATINOFF, Exp. Gerontol. 25533-543 22. W. FELDBERG and R.D. MYERS, J. Physiol. 173226-237 (1964). 23. L.E. SCHEVING,

Am. J.

(1990).

D.F. VEDRAL and J.E. PAULY, Anat. Rec. 160 741-750

(1968).

24. M.H. SMOLENSKY and G.E. D’ALONZO, Trends in Chronopharmacology, W.Th.J.M. Hekkens, G.A. Kerkhof and W.J. Rietveld (Eds), 243-260, Pergamon

Press,

Oxford and New York (1988). 25. C.A. WALKER and J.O. OWASOYO, Int. J. Chronobiol. 2 125-130 (1974). 26. D.M. ANDERSON and W.G. SMITH, J. Pharm. Pharmacol, 13396-404 (1961). 27. K. TAKAGI and M. HARADA, J. Pharmaceu. 28. A. REINBERG and M.H. SMOLENSKY, 29. K.J. VENER

Sot. Japan 89 893-898

Clin. Pharmacokinet.

and J.G. MOORE, Annu. Rev. Chronopharmacol.

(1969).

z 401-420

30. S. NAKANO, H. WATANABE, K. NAGAI and N. OGAWA, Clin. Pharmacol. 271-277

(1982).

4 257-281

(1988). Ther. 36

(1984).

31. G. IABRECQUE, P.M. BELANGER, F. DORE and M. IALANDE, Annu. Rev. Chronopharmacol. 2445-448 (1988). 32. J.A. GURR, R.S. ELVEHJEM and V.R. POTTER, Life Sci.3 1301-l 308 (1978). 33. M.A. MILLER, J.M. PARKER and A.E. COLAS, Life Sci. 23 217-222

(1978).

34. A.H. BECKETT and M. ROWLAND, Nature 204 1203-l 204 (1964). 35. A. REINBERG, Z. ZAGULA-MALLY, J. GHATA and F. HALBERG, Proc. Sot. Exp. Biol. Med. 124 826-832 (1967).