Pentazocine-induced biphasic analgesia in mice

Life Sciences, Vol. 48, pp. 1827-1835 Printed in the U.S.A.

PENTAZOCINE-INDUCED

Tsutomu

Suzuki 1

F

Minoru

Pergamon Press

BIPHASIC

Narita 1 f

ANALGESIA

IN M I C E

Miwa M i s a w a 1 a n d H i r o s h i

Nagase 2

iDepartment 2Basic

of A p p l i e d Pharmacology, School of Pharmacy, Hoshi University, Shinagawa-ku, Tokyo 142, Japan R e s e a r c h Laboratories, Toray Industries, Inc., K a m a k u r a 248, Japan (Received in final form March i, 1991)

P e n t a z o c i n e (PZ) is well known to act as an opioid mixed a g o n i s t - a n t a g o n i s t analgesic. In the present study, we selected the mouse w a r m plate test condition of 51 ± 0.5"C instead of 55 ± 0.5°C to determine the analgesic action of PZ. As a result, i.c.v. PZ p r o d u c e d a biphasic antinociceptive response, while U-50,488H (U-50) and morphine (MRP) showed a m o n o p h a s i c response. P r e t r e a t m e n t with i.c.v. 8-FNA (mu antagonist) a n t a g o n i z e d the initial response, whereas the delayed one was a n t a g o n i z e d by pretreatment with nor-BNI (kappa antagonist). In addition, pretreatment with NTI (delta antagonist) s i g n i f i c a n t l y a t t e n u a t e d the initial response but not the d e l a y e d one. These results suggest that the initial and delayed responses may be m e d i a t e d mainly by m u / d e l t a and kappa receptors, respectively. With regards to the interaction between MRP and PZ, a low dose of PZ a n t a g o n i z e d the analgesic action of MRP, while a high dose PZ plus MRP showed the additive effect. Furthermore, tolerance d e v e l o p e d almost e q u a l l y to both initial and delayed responses, indicating that tolerance to the kappa compoment of PZ may be d e v e l o p e d as well as the mu c o m p o n e n t of action of PZ.

G e n e r a l l y three receptor types (mu, delta and kappa) are c o n s i d e r e d to p a r t i c i p a t e in the analgesic actions of many opioid analgesic agents. Pentazocine, one of these analgesics, is well known to act as an opioid a g o n i s t - a n t a g o n i s t . A l t h o u g h Levine et al. (i) r e c e n t l y reported that the analgesic action p r o d u c e d by p e n t a z o c i n e may be p r e d o m i n a n t l y m e d i a t e d by kappa receptors, it is not yet clear which receptors mediate p e n t a z o c i n e - i n d u c e d analgesia. A change in the reaction time of mice exposed to a thermal noxious stimulus has been widely used in determining the analgesic activity. Of the various methods used for e v a l u a t i o n of 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

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analgesic agents, the 55°C hot plate is standard in this field. We r e c e n t l y d e t e r m i n e d that the analgesic p o t e n c y of p e n t a z o c i n e is w e a k e r than m o r p h i n e (mu-agonist), DPDPE (delta-agonist) and U-50,488H (kappa-agonist) on the 55°C hot plate (2). These results led us to examine a simple and effective m e t h o d to detect weak analgesics, such as pentazocine, other mixed agonista n t a g o n i s t s and kappa agonists. In the process of e x a m i n i n g the hot plate m e t h o d to detect weak analgesic action like that of pentazocine, we found that i.c.v, p e n t a z o c i n e p r o d u c e d a biphasic a n t i n o c i c e p t i v e response c o n s i s t i n g of an initial and a d e l a y e d response on the warm plate test (51 • 0.5°C instead of 55 ± 0.5°C). We used selective opioid antagonists: 8-funaltrexamine (8-FNA) for mu, n a l t r i n d o l e (NTI) for delta and n o r - b i n a l t o r p h i m i n e (nor-BNI) for kappa (3, 4, 5), to study the b i p h a s i c response to p e n t a z o c i n e and to determine which receptor types are involved in the response. In the p r e s e n t study, we report the results of these attempts and also describe the tolerance to the initial and d e l a y e d r e s p o n s e s induced by pentazocine.

Methods

Materials

and animals.

Male ddY mice o b t a i n e d from Tokyo Animal L a b o r a t o r y Inc., Tokyo, Japan w e i g h i n g 18-25 g (6 weeks) at the b e g i n n i n g of e x p e r i m e n t s were used. Subjects were housed 7-10 per cage (20 x 30 x 15 cm) in which food and water were c o n t i n u o u s l y available. The h o u s i n g rooms were m a i n t a i n e d under a 12 hr light-dark cycle, with a t e m p e r a t u r e of 22 ± Ioc, and a relative humidity of 55 ~ 5 %. The mice were allowed to adapt to this e n v i r o n m e n t for a p e r i o d of 1 week before experiments. Procedure. A n a l g e s i c responses were d e t e r m i n e d using a 51°C w a r m - p l a t e as the n o c i c e p t i v e stimulus with the latency to fore-paw lick or escape by jumping taken as the endpoint. The test was r e p e a t e d every i0 min after t r e a t m e n t with agonists. A cut-off time of 60 sec was employed. The p e r c e n t effect at each time point was c a l c u l a t e d a c c o r d i n g to the following formula: % analgesic effect = 100 X (test latency - control latency) / (cut-off time control latency) (6). All the compounds to be tested were given into the lateral cerebral ventricle of u n a n e s t h e t i z e d mice in a volume of i0 ~i using a m o d i f i c a t i o n of the m e t h o d of Haley and M c C o r m i c k (7) as p r e v i o u s l y r e p o r t e d (8). In order to determine the receptor types involved in mediating the pentazocine-induced analgesia, various opioid antagonists were used. 8-FNA (i ~g), an irreversible mu antagonist, was injected i n t r a c e r e b r o v e n t r i c a l l y (i.c.v.) 24 hr before p e n t a z o c i n e injection. NTI (5 ~g), a highly selective delta antagonist, and nor-BNI (5 ~g), a highly selective kappa antagonist, were a d m i n i s t e r e d i0 min before testing. In this schedule, treatments with the antagonists had no effect on control latencies in naive mice (data not shown). In the study of

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Pentazocine-lnduced Biphasic Analgesia

--A---[3--

1829

Pentazocine 50,ug U- 50,Z.88H 30~g

60

Peak 1

Peak 2

t~

~

~0

o~

c

2O

0

I

I

I

I

1

I

1

I

I

tO

20

60

80

90

100

120

150

180

Time

after Fig.

injection

(rain)

1.

Time course changes in % analgesia induced by p e n t a z o c i n e (50 ~g, i.c.v.) and U - 5 0 , 4 8 8 H (30 lag, i.c.v.) in the mouse 51°C warm plate test. Each point represents the mean ± S.E.M. of 20 animals. 100

80

--O--

Morphine(5)

-- - -

Pentazocine(50) Pentazocine(50) ÷

60

~

Morphine(5)

4o

20

o

"6 I

I

0

10

I

I

i

30 60 90 Time a f t e r i n j e c t i o n ( m i n )

1

120

Fig. 2. Time course changes in % analgesia (the mouse 51°C warm plate) induced by p e n t a z o c i n e (50 ~g, i.c.v.), m o r p h i n e (5 ~g, i.c.v.) and p e n t a z o c i n e (50 ~g, i.c.v.) in c o m b i n a t i o n with m o r p h i n e (5 ~g, i.c.v.). Each point r e p r e s e n t s the mean ± S.E.M. of 20 animals.

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interaction between morphine and pentazocine, morphine (5 ~g/body, i.c.v.) was coinjected with either 5 ~g or 50 ~g of pentazocine. The analgesic effect of morphine plus pentazocine was tested i0 min after the coinjection. We also examined the development of tolerance to pentazocine-induced analgesia. Mice were injected with pentazocine (50 ~g/body, i.c.v.) every 24 hr for 5 days. Four hr after the last injection, the analgesic effect was tested to assess tolerance. Drugs. The drugs used were morphine hydrochloride (Sankyo Co., Tokyo, Japan), pentazocine hydrochloride (Yamanouchi Pharmaceutical Co., Tokyo, Japan) and 8-FNA (8-funaltrexamine hydrochloride; Research Biochemicals, Inc., Natick, MA, USA). NTI (naltrindole hydrochloride), nor-BNI (nor-binaltorphimine hydrochloride) and U-50,488H (trans-3,4-dichloro-N-(2-(lpyrrodinyl)-cyclohexyl)benzenacetamide) methane sulfonate hydrate) were synthesized by Dr. Nagase. All doses refer to the salt forms of the drugs. All the drugs were dissolved in 0.9% saline. Statistical

analyses.

Data are expressed as the mean ± S.E.M. The Student's t-test and Dunnett's test were used for statistical analyses. EDKo value of pentazocine analgesia and its 95% confidence limi~ were determined using the analysis of variance and linear regression techniques (9). Results

The effects of i.c.v, administrations of pentazocine (50 ~g), U-50,488H (30 ~g) and morphine (5 ~g) are shown in Figures 1 and 2. Pentazocine produced a biphasic antinociceptive response, while U-50,488H and morphine showed a monophasic one which peaked at i0 min. The initial response to pentazocine peaked at i0 min and the delayed peaked at 90 min. In addition, higher doses of U-50,488H (50 ~g) and morphine (i0 ~g) only produced the initial phase. The initial but not the delayed response of pentazocine was antagonized by pretreatment with i.c.v. 8-FNA (Table i), which significantly antagonized morphine analgesia but not DPDPE and U-50,488H. On the other hand, the delayed but not the initial response was significantly antagonized by pretreatment with nor-BNI, which significantly antagonized U-50,488H-induced supraspinal analgesia but not DPDPE and morphine. In addition, the delayed but not theinitial response induced by 150 ~g pentazocine was greatly antagonized by pretreatment with nor-BNI (61.5 % inhibition). Table 2 shows that NTI significantly antagonized the initial peak but not the delayed one. Additionally, pretreatment with NTI greatly antagonized the initial but not delayed response induced by 150 ~g pentazocine (31.1 % inhibition). This dose of NTI significantly antagonized the analgesic activity induced by selective delta agonists (e.g., i.c.v. DPDPE and DADLE) without

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affecting the analgesic potency of morphine and U-50,488H.

TABLE

Antagonistic effect of p e n t a z o c i n e in the mouse

Pretreatment

None

8-FNA a

Peak time (min)

1

8-FNA on antinociceptive 51"C warm plate.

EDen of p e n t a z o c i n e ( ~ / b o d y , i.c.v.)

i0 (peak i)

76.3

(49.1-118.65)

90 (peak 2)

224.4

(122.1-412.4)

activity

Relative p o t e n c y (none=l)

10 (peak i) 1678.4"(184.0-15313.5)

0.045

90 (peak 2)

0.76

296.6

(55.2-1593.4)

of

aS-FNA (i ~g, i.c.v.) was administered 24 hr before the p e n t a z o c i n e injection. To calculate ED. 0 values, at least 4 drug doses were used and 7-20 mice were D used for each dose. Numbers in p a r e n t h e s i s indicate the 95 % confidence limits. *P<0.05 vs. n o n - p r e t r e a t m e n t group.

TABLE

2

% Change in analgesia induced by p e n t a z o c i n e (50 ~g, i.c.v.) after each specific antagonist treatment in the mouse 51°C warm plate.

Pretreatment

Initial peak

D e l a y e d peak

none

i00.0 ± 13.5

i00.0 ± 24.0

(control)

8-FNA NTI

(i ~g, i.c.v.)

(5 ~g, i.c.v.)

nor-BNI

(5 ~g, i.c.v.)

35.2 ± 14.2"

85.2 ± 14.4

58.4 ±

9.6*

80.6 ± 13.5

72.5 ±

8.8

8.9 ± 12.9"*

8-FNA, NTI and nor-BNI were a d m i n i s t e r e d i.c.v. 24 hr, i0 min and i0 min before testing, respectively. Each value represents the mean p e r c e n t a g e of changes from control (± S.E.M.) for i0 mice. Statistical significance was tested using the Dunnet's test. *p< 0.05, **p<0.01 vs. n o n - p r e t r e a t m e n t (none).

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When we investigated the interaction between morphine (i.c.v.) and p e n t a z o c i n e (i.c.v.), low dose of pentazocine (5 ~g) which exerted little analgesic effect by itself inhibited the analgesic activity of morphine, while high dose of pentazocine (50 ug) which alone exerted a significant analgesic effect e n h a n c e d the m o r p h i n e - i n d u c e d analgesia (Fig. 3). In addition, 1 100

%

ID

o,

50

I

\\\\\,l

C

i~

O"

Mor(5)

Pz(5)

Pz(5)+Mor(5) Mor(5)

•i

Pz(50) Pz(50)+Mor(5)

Fig. 3. A n a l g e s i c effects of low (5 ~g, i.c.v.) and high (50 ~g, i.c.v.) doses of p e n t a z o c i n e (PZ) and their combinations with m o r p h i n e (MRP; 5 ug, i.c.v.) in the mouse 51°C w a r m plate test. The analgesic activity was m e a s u r e d 10 min after i.c.v, treatment. Each column represents the mean ± S.E.M. of 10 mice. *p<0.05 vs. morphine alone.

~g morphine alone showed 38.7 ± 8.6 (% analgesia) i0 min after morphine injection, while 1 ~g morphine plus 50 ~g p e n t a z o c i n e (43.5 ± 5.9 %) showed 77.7 ± 8.7 (% analgesia), indicating that the analgesic effect of m o r p h i n e plus high dose p e n t a z o c i n e was additive. Since both mu and kappa agonists have been shown to produce tolerance to their analgesic effect, we examined the development of tolerance to both the initial and delayed responses induced by pentazocine. As a result, tolerance developed almost equally to both responses (Fig. 4). Discussion Several researchers showed that reducing the intensity of the heat noxious stimulus greatly enhances the sensitivity of antinociceptive tests to the a g o n i s t - a n t a g o n i s t compounds. Shaw

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et al. (10) r e p o r t e d that EDen values of a g o n i s t - a n t a g o n i s t s were capable of being e s t i m a t e d ~ the abdominal c o n s t r i c t i o n test, but not by the 55°C hot plate test, although the a n t a g o n i s t i c effects of these agents were detected. P e n t a z o c i n e shows a potent analgesic action against chemical and p r e s s u r e - i n d u c e d pain in the mouse and rat (ii). Luttinger (12) has reported that p e n t a z o c i n e and nalorphine, which were not active in the 55°C tail immersion test, p r o d u c e d large increments in the reaction time to a 45"C stimulus. On the basis of these experiments, we selected the warm plate test condition to determine p e n t a z o c i n e effect on the heat noxious stimuli. The warm plate can be useful for detecting the analgesic effects of the a g o n i s t - a n t a g o n i s t compounds and kappa receptor agonists (13, 14).

i

100

T

T

~n o n - t o l e r a n t mice

~\~\.:~ t o l e r a n t

mice

C

C

5(;

C 0 o

Peak 1(10 rain )

Peak 2(90rain)

Fig. 4. T o l e r a n c e to the initial (Peak i) and delayed (Peak 2) a n a l g e s i a induced by p e n t a z o c i n e (50 ~g, i.c.v.). Mice were injected i.c.v, with p e n t a z o c i n e (50 ~g) every 24 hr for 5 days. Four hr after the last injection, the analgesic effect was tested for the assessment of tolerance. Each column represents the mean ± S.E.M. of 10 mice. *p<0.05 vs. acute treatment (non-tolerant).

The i.c.v, a d m i n i s t r a t i o n of p e n t a z o c i n e p r o d u c e d a biphasic a n t i n o c i c e p t i v e response. P r e t r e a t m e n t s with i.c.v. 8-FNA and nor-BNI significantly antagonized the initial and delayed responses, respectively. P r e t r e a t m e n t s with B-FNA and nor-BNI did not affect U - 5 0 , 4 8 8 H and morphine analgesia, respectively. In addition, DPDPE-induced analgesia was u n a f f e c t e d by both 8-FNA

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and nor-BNI. Furthermore, NTI, which did not affect both morphineand U-50,488H-induced analgesia, significantly attenuated the initial but not the delayed response. These results suggest that the initial and delayed responses induced by pentazocine may be mainly mediated by mu/delta and kappa receptors, respectively. Jhamandas et al. (15) r e p o r t e d that intrathecally injected dynorphin (1-8) and dynorphin (1-13) produced a similar biphasic antinociceptive response c o n s i s t i n g of both the initial and delayed responses in the rat tail-flick test. The reason why the position of delayed response to dynorphin differs from that to U - 5 0 , 4 8 8 H which shows only a m o n o p h a s i c response is due to the existence of interaction between mu and kappa receptors in the former. In the present study, the biphasic antinociceptive response of i.c.v, pentazocine also might be based on the interaction between mu and kappa receptors. Pentazocine has been known to act as an a g o n i s t - a n t a g o n i s t analgesic. Its analgesic activity is mainly m e d i a t e d by kappa receptor and p a r t i a l l y by mu and delta receptors in the mouse supraspinal site (2). Additionally, it has been known that the antagonistic activity of p e n t a z o c i n e against m o r p h i n e - i n d u c e d p h a r m a c o l o g i c a l actions was p r o d u c e d by a n t a g o n i s m on mu receptor. On the other hand, Ramarao et al. have reported that some of kappa agonists (bremazocine and U-50,488H) s i g n i f i c a n t l y suppressed m o r p h i n e - i n d u c e d analgesia in rats (16). Thus, the dissociation of mu/delta and kappa components of i.c.v, pentazocine-induced analgesic action p r o v i d e d a better basis for u n d e r s t a n d i n g the m e c h a n i s m of its antagonistic action. In the present study, we investigated the interaction between morphine and p e n t a z o c i n e in the supraspinal analgesia in mice. Low dose of p e n t a z o c i n e antagonized the analgesic action of morphine at I0 min after morphine treatment, while the high dose of p e n t a z o c i n e p r o d u c e d an additive analgesic effect with morphine. In addition, i.c.v, morphine produced an additive analgesic effect with i.c.v, p e n t a z o c i n e at 90 min after 50 ~g pentazocine treatment (data not shown). Pentazocine p o s s e s s e s affinity for mu, delta and kappa receptors. The rank order ~f the ~ffinity for each receptor is mu (naloxone) >> delta (D-AIa -D-Leu -enkephalin) > kappa (ethylketocyclazocine) sites (17). These results suggest that low dose of i.c.v, pentazocine may p r e d o m i n a n t l y act as a partial mu and/or delta antagonist when p e n t a z o c i n e was a d m i n i s t e r e d i.c.v, by combinations with m o r p h i n e in mice. However, high dose of i.c.v, p e n t a z o c i n e may act as a mu, delta and kappa agonist and may produce an additive analgesic effect with morphine which is m e d i a t e d through kappa receptor as well as mu and delta receptors in the mild thermal noxious stimulus test. Moreover, this additive effect may be m e d i a t e d through the interaction of m u - d e l t a complex on the initial response. Furthermore, we also found that tolerance to pentazocine analgesia d e v e l o p e d almost equally to both initial and delayed responses. These results suggest that tolerance to p e n t a z o c i n e analgesia may develop to both the kappa and m u / d e l t a components of pentazocine.

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Acknowled@ments We w i s h to t h a n k Dr. A.E. T a k e m o r i a n d Dr. D.B. T u a z o n t h e i r h e l p f u l c o m m e n t s and Ms. Y. T a k a h a s h i and Mr. M. K i z a w a technical assistance.

for for

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