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Studies on the effect of systemic PD134308 (CAM 958) in spinal reflex and pain models with special reference to interaction with morphine and intrathecal galanin
Neuropeptides (1991) 19, (Suppl.) 79-84 0 Longman Group UK Ltd 1991
Studies on the Effect of Systemic PD134308 (CAM 958) in Spinal Reflex and Pain Models with Special Reference to Interaction with Morphine and Intrathecal Galanin 2. WIESENFELD-HALLIN”,
X-J. XU*, J. HUGHESt,
D. C. HORWELLt
and T. HOKFELTS
Karolinska Institute, *Department of Clinical Physiology, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86, Huddinge, Sweden, tParke-Davis Research Unit, Cambridge, UK and #Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden (Reprint requests to ZW-HI
Abstract-The effect of intravenous (i.v.1 PD134308, which is a CCK-B antagonist, morphine and intrathecal (i.t.) galanin (GAL) on the excitability of the spinal nociceptive flexor reflex and in the hot plate test was examined in rats. PD134308 (1 mg/kg, i.v.) caused a weak, naloxone-reversible depression of the flexor reflex and moderate antinociception in the hot plate test. PD134308 significantly potentiated the antinociceptive effect of morphone, as well as its depressive effect on the flexor reflex. PD134308 and i.t. GAL synergistically depressed the flexor reflex, which was reversed by naloxone. Finally, the magnitude and duration of the depression of the flexor reflex by morphine was synergistically increased by coadministering i.v. PD134308 and i.t. GAL. The results demonstrate that a CCK antagonist directed to the central CCK-B receptor potentiates the analgesic effects of opioid and non-opioid drugs at spinal level in the rat, thus supporting the notion that CCK in the CNS may be an endogenous, physiological opioid antagonist.
areas of the spinal cord which are believed to have a role in nociception. Studies on the effects of exogenously applied CCK and related peptides on pain modulation in rodents have given conflicting results. CCK has been reported to cause analgesia after various routes of administration, whereas there are many reports that CCK reduced the analgesic effect of morphine and endogenous opioids (see 5 for review).
Introduction Cholecystokinin (CCK) is present in many areas of the central nervous system (CNS) (1). This peptide fulfils many of the criteria for a neurotransmitter and may have a role in various CNS functions (2). In the spinal cord genuine CCK-like immunoreactivity has been described in local neurons in the dorsal horn and in neurons in lamina X (3, 4), 79
80
Evidence has been presented that there might be multiple receptors for CCK and that the most abundant receptor type for CCK in the periphery differs from that in the CNS (6, 7), designated as CCK-A and CCK-B, respectively (7). More recently a number of CCK antagonists have become available, opening up possibilities to resolve the role of CCK in nociception. Thus, the non-selective, weak CCK receptor antagonists proglumide and benzotript potentiated the analgesic effect of morphine and other opioids (8). Studies with more recently developed specific CCK-A (Devazepide, formerly MK 329 and L365, 031) and CCK-B (L365, 260) antagonists further supported a role for CCK as a physiological opioid antagonist, as these drugs enhanced the analgesic effect of morphine and abolished morphine tolerance (9, 10). The rank order of potency of these drugs for enhancing morphine analgesia was L365,260>MK329>L365,031, which correlated with their potency in blocking the central CCK-B receptor in rats, indicating that the site of CCW opioid interaction is in the CNS and is mediated by CCK-B receptors (10). Another highly selective and potent CCK-B PD134308 (formerly receptor antagonist, CAM958), has recently been developed (11). We have now examined the effect of PD134308 on nociception by using two experimental models, the spinal nociceptive flexor reflex in decerebrated, spinalised, unanesthetised rats and a non-reflexive behavioral test of nociception, the hot plate test (12). It was shown that PD134308 enhances the analgesic effect of morphine and of morphine combined with galanin (GAL), a neuropeptide which may have endogenous antinociceptive effect at spinal level and potentiates the spinal analgesic effect of morphine (13).
NEUROPEPTIDES
animals were briefly anesthetised by methohexytal (70mg/kg, i.p.), decerebrated by aspiration of the fore- and midbrain and then artificially ventilated. The spinal cord was sectioned at Th8-9. In some rats, an i.t. catheter (PElO) was implanted caudally to the transection with its tip at the lumbar enlargement (L4-5). The reflex was continuously elicited by test stimuli of single shocks (l/min) to the sural nerve (1 ms, 5 mA) to activate C-fibers. The number of action potentials elicited during the reflex was integrated over 2s. A stable reflex magnitude was established for at least 20min prior to drug injection. Behavioral tests were carried out on male rats. The antinociceptive effect of the drug was assessed with a hot plate (IITC, Woodland Hills, CA, USA) at 54 + 0.2”C. The latency to licking a hind paw was determined to an accuracy of 0.1 s. Before any drugs were administered, the rats were habituated by testing on the hot plate for 5 days in order to obtain a stable response value. The average baseline response latency was 4.2 + 0.2s on the day before drug administration and there was no significant difference between groups. Morphine hydrochloride (Apoteksbolaget , Stockholm, Sweden) and PD134308 (ParkeDavis, Cambridge, UK) and naloxone (DuPont, Delaware, USA) were injected in a volume of 0.2ml i.v. in the physiological studies and i.p. in the behavioral experiments. Porcine GAL and Switzerland) Bubendorff, (Bachem, sulphated CCK 26-33 (Cambridge Research Biochemicals, Cambridge, UK) were dissolved in 0.9% saline and injected i.t. in a lOl.~l volume followed by 10 ul saline to flush the catheter. Results Potentiation of the analgesic effect of morphine by PDI34308
Materials and Methods Physiological studies were carried out on SpragueDawley rats of both sexes (b. wt. 250-300g) as described previously (14). Briefly, the magnitude of the hamstring flexor reflex in response to activation of high threshold afferents was examined in decerebrate, spinalised, unanesthetised rats by recording the e.m.g. from the posterior bicep femoris/semitendinosus muscles. The
PD134308
(lmg/kg) injected i.v. caused a weak depression of the flexor reflex by about 25% below control reflex magnitude for about 30min (Table, Fig. 1). The effect was reversed by 1 mg/kg naloxine i.v. Pre-treatment with this dose of PD134308 i.v. also antagonised the facilitatory effect of the flexor reflex by 1OOng and lpg i.t. CCK. PD134308 (1 mg/kg) and morphine (1 mg/kg) interacted synergistically to depress the flexor
STUDIES
ON THE EFFECT
PO134306
1
1
PD134308
(CAM 958) IN SPINAL
Morphine
Ok-----150
OF SYSTEMIC
Time (hz
PD134306+Yorphine
6
7 Time (hr)
Fig. 1 (Upper) PD134308 (lmg/kg) or morphine (lmg/kg) moderately depressed the flexor reflex for 30-60 min. (Lower, continuation of abscissa from Upper). The magnitude and duration of depression were greatly enhanced by coadministration of the two drugs. (Reproduced with permission from Proc. Natl. Acad. Sci. USA)
Table
The magnitude
and duration
of the depressive
AND
81
PAIN MODELS
reflex (Table, Fig. 1). After coadministration the reflex was depressed about twice as much as to each drug alone and the duration of depression lasted about three times as long as after morphine alone (Table). PD134308 at low doses (0.1,0.3mg/kg) had no effect on lick latency in the hot plate test, whereas larger doses had a fairly weak, but significant, analgesic effect for 20min (1 mg/kg) and 40min (3 mg/kg). Morphine (1 mg/kg) caused moderate analgesia for about 50min and PD134308 potentiated the analgesic effect of morphine in a dose-dependent fashion. With the three higher doses of PD134308 the analgesic effect of morphine was significantly potentiated at all intervals examined, starting 1Omin after injection. The duration of analgesia was increased from 5090min with 0.3mg/kg and to 130min with 1 and 3mg/kg PD134308.
2
5
REFLEX
Interaction of i. t. GAL and systemic PO134308 and morphine
One hundred ng GAL briefly facilitated the flexor reflex without causing reflex depression and 1 kg
effect of PD134308, morphine and GAL and the combination
of these drugs on the flexor reflex Drugs
N
% reflex depression
Duration of depression (min)
PD134308 1 mg/kg i.v.
6
24.5 ?C5.9
28.6k7.6
Morphine 1 mg/kg i.v.
8
38.1+ 10.7
60.5 + 27.2
PD134308 lmg/kg i.v. + Morphine lmg/kg i.v.
6
62.5 + 11.2”.’
195.3 f 24.2a,c
15
0
0
GAL 100 ng i.t. + Morphine 1 t.rg i.t.
5
58.5 f 15.1b
80.1 f 15.2
GAL 100 ng i.t. + PD134308 lmg/kg i.v.
4
66.0f 15.9”*’
152.2 + 38.9b,C
GAL 100 ng i.t. + Morphine 1 mg/kg i.v. + PD134308 1mgIkg i.v.
4
85.6 f 13.7d,e
250.2 f 14.1d,‘,s
GAL 100 ng i.t.
Statistics were performed by two-way ANOVA followed by Scheffe F-test, a = compared to morphine p
82
NEUROPEPTIDES
0
1
2
3
4
Tilz
mr,
Fig. 2 Effect of GAL at 100 ng i.t., PD134308 at 1 mg/kg i.v., and the combination of the two drugs on the excitability of the flexor reflex. (Reproduced with permission from Proc. Natl. Acad. Sci. USA)
morphine caused a weak facilitation followed by reflex depression for about 1 h. When GAL and morphine were coadministered i.t. at these doses the reflex depression became significantly stronger and its analgesic duration was also increased (Table). The effect of 1OOng i.t. GAL and lmg/kg i.v. PD134308, administered within a 5min interval, are summarised in the Table and illustrated in Figure2. Both the magnitude and duration of the reflex depression by lmg/kg i.v. PD134308 were significantly potentiated by 100ng i.t. GAL. In fact, the reflex depression by PD134308 plus GAL was significantly stronger and longer lasting than after 1 mg/kg morphine. Furthermore, the depressive effect of GAL plus PD134308 was naloxone reversible. An extremely strong potentiation of the effect of i.v. PD134308 plus i.v. morphine was observed after i.t. administration of GAL (Table, Fig. 3). The reflex was depressed by about 85% below control level, which was significantly greater than the effect of GAL plus morphine or PD134308 plus morphine. The duration of the reflex depression was about 250min, which was significantly longer than the effect of GAL plus morphine, PD134308 plus morphine or PD134308 plus GAL (Table). Discussion
In the present report we have shown that systemic PD134308, a specific CCK-B receptor antagonist,
dose-dependently potentiates the analgesic effect of morphine both in the hot plate test and in the flexor reflex model in spinalised rats, supporting the hypothesis that CCK-8 is an endogenous opioid antagonist. Since PD134308 has only very low affinity to the CCK-A receptor, the results strongly indicate that the interaction between CCK and opioids occurs through CCK-B receptors in the CNS. The potentiating effect of PD134308 on morphine-induced analgesia is robust, apparently stronger than the effect reported with another CCK-B antagonist, L365,260 (10). Also PD134308 tested over a 30-fold dose range did not reveal a bell-shaped distribution of dose effectiveness, which was seen with L365,260 (10). It is, however, difficult to make a direct comparison between the efficacy of these two drugs in potentiating the analgesic effect of morphine, since different animal models were employed. Systemic PD134308 by itself at high doses had a weak analgesic effect and depressed the flexor reflex in a naloxone-reversible fashion. It seems unlikely that this effect was due to a direct interaction of PD134308 with opioid receptors, since PD134308 has been shown to be inactive or
‘y
Galanin+ PDl34308+Morphina
I
Fig. 3 Effect of GAL at 100 ng i. t., PD134308 at 1 mg/kg i.v., morphine at 1 mg/kg i.v. (Upper), and combination of all three drugs (Lower, continuation of abscissa) on the flexor excitability in a single experiment. (Reproduced with permission from Proc. Natl. Acad., Sci. USA)
STUDIES ON THE EFFECT OF SYSTEMIC PD134308 (CAM 958) IN SPINAL REFLEX AND PAIN MODELS
only weakly active in the displacement of the binding of radioligands to kappa, mu or delta opioid receptors (11). Analgesia has not been reported with other CCK receptor antagonists. It is possible that the antagonism by CCK on opioid analgesia operates to some extent tonically through the CCK-B receptor at spinal level and that blockade by PD134308 results in removal of this antagonism, leading to increased opioid peptide release. We have previously demonstrated that GAL given i.t. potentiated the effect of morphine on flexor reflex excitability and a behavioral test of nociception in a naloxone-reversible fashion (13). If the analgesic effect of PD134308 involves the endogenous opioid system one would predict that it should be potentiated by GAL as well. Indeed, the depressive effect of systemic PD134308, when combined with i.t. GAL, was reversed by naloxone and was stronger than that after lmg/kg morphine. Since we have previously shown that naloxone could not reverse the reflex depression by high doses of GAL (14), it appears as though the GAL receptor is independent of both the opioid and CCK-B receptors, but can interact with them to cause a synergistic opioid-mediated analgesia. The synergistic interaction between GAL, PD134308 and morphine further strengthens this suggestion. A major site of the analgesic effect of PD134308 appears to be within the spinal cord, since we observed a similar effect in intact and in spinalised rats. Furthermore, the interaction between PD134308 and morphine was also similar in intact and spinalised animals. Finally, the strongest argument for a direct spinal effect of systemic PD134308 is that it effectively antagonised the facilitatory effect of i.t. CCK and interacted with i.t. GAL to cause synergistic reflex depression. Previous experiments have documented a spinal site of interaction between CCK and opioids (see 5). Moreover, extensive systems containing genuine CCK peptide and CCK mRNA are present in the rat dorsal horn (3,4,15). However, CCK-B receptors also exist in many brain regions, including the periaqueductal grey (6,7), known to be involved in opioid analgesia, and the interaction between CCK, its antagonist and opioids has been reported after intracerebral or intracere-
83
broventricular injection (5). We thus cannot rule out the possibility that the potentiation of morphine-induced analgesia by PD134308 on the hot plate test may be partly mediated by CCK-B receptors in the brain. In conclusion, the present results demonstrate that a newly developed CCK-B antagonist in two experimental models can enhance the analgesic effect of morphine, especially when combined with the neuropeptide GAL, indicating new approaches to pain treatment based on combinations of drugs. Acknowledgements Thisstudy was supported by the Bank of Sweden Tercentenary Foundation, the Swedish MRC (project no. 07913,04X-2887), NIMH (MH43230-02), Marianne och Marcus Wallenberg Stiftelse, Konung Gustav V och Drottning Victorias Stiftelse, Fredrik och Ingrid Thurings Stiftelse and research funds of the Korolinska Institute. X.-J. Xu was supported by the WennerGren Center Foundation.
References 1. Hokfelt,
2.
3.
4.
5.
T., Skirboll, L., Everitt, B. J.. Meister, B., Brownstein, M., Jacobs, T., Faden, A., Kuga, S., Goldstein, M., Markstein, R., Dockray, G. and Rehfeld, J. (1985) Distribution of cholecystokinin-like immunoreactivity in the nervous system with special references to coexistence with classical neurotransmitters and other neuropeptides. In: Vanderhaeghen, J. J. and Crawley J. (eds) Neuronal Cholecystokinin. Annals of the New York Academy of Sciences, Vol. 448, New York, p. 255-274. Vanderhaeghen, J. J. and Crawley, J. (eds) (1985) Neuronal Cholecystokinin. Annals of the New York Academy of Sciences, Vol. 448, New York. Hokfelt, T., Herrera-Marschitz, M., Seroogy, K., Ju, G., Staines, W. A., Holets, V., Schalling, M., Ungerstedt, U., Post, C., Rehfeld, J. F., Frey, P., Fischer, J., Dockray, G., Hamaoka, T., Walsh, J. H. and Goldstein, M. (1988) Immunohistochemical studies on cholecystokinin (CCK)immunoreactive neurons in the rat using sequence specific antisera and with specific reference to the caudate nucleus and primary sensory neurons. Journal of Chemical Neuroanatomy, 1: 11-52. Williams, R. G., Dimaline, R., Varro, A., Isetta, A. M., Trizio, D. and Dockray, G.J. (1987) Cholecystokinin octapeptide in rat central nervous system: immunocytochemical studies using a monoclonal antibody that does not react with GCRP. Neurochemistry International 4: 433442. Baber, N. S., Dourish, C. T. and Hill, D. R. (1989) The role of CCK, caerulein, and CCK antagonists in nociception. Pam 39: 307-328.
84 6. Innis, R. B. and Snyder, S. H. (1980) Distinct cholecystokinin receptors in brain and pancreas. Proceedings of the National Academy of Sciences USA, 77: 6917-6921. 7. Moran, T. H., Robinson, P. H., Goldrich, M. S. and McHugh, P. R. (1986) Two brain cholecystokinin receptors: implications for behavioral actions. Brain Research 362: 175-179. 8. Watkins, L. R., Kinscheck, I. B. and Mayer, D. J. (1984) Potentiation of opiate analgesia and apparent reversal of morphine tolerance by proglumide. Science 224: 395-396. 9. Dourish, C. T., Hawley, D. and Iversen, S. D. (1988) Enhancement of morphine analgesia and prevention of morphine tolerance in the rat by the cholecystokinin antagonist L-364,718. European Journal of Pharmacology 147: 469-472. 10. Dourish, C. T., O’Neill, M. F., Coughlan, J., Kitchener, S. J., Hawley, D. and Iversen, S. D. (1990) The selective CCK-B receptor antagonist L365,260 enhances morphine analgesia and prevents morphine tolerance in the rat. European Journal of Pharmacology 176: 35-44. 11. Hughes, J., Boden, P., Costall, B., Domeney, A., Kelly, E., Horwell, D. C., Hunter, J. C., Pinnock, R. D. and
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12.
13.
14.
15.
Woodruff, G. N. (1990) Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity. Proceedings of the National Academy of Sciences USA, 87: 6728-6732 Wiesenfeld-Hallin, Z., Xu, X.-J., Hughes, J., Horwell, D. C. and Hakfelt, T. (1990) PD134308, a selective antagonist of cholecystokinin type B receptor, enhances the analgesic effect of morphine and synergistically interacts with galanin to depress spinal nociceptive reflexes. Proceedings of the National Academy of Sciences USA, 87: 71057109 Wiesenfeld-Hallin, Z., Xu, X.-J., Villar, M. J. and Hokfelt, T. (1990) Intrathecal galanin potentiates the spinal analgesic effect of morphine: electrophysiological and behavioral studies. Neuroscience Letters 109: 217-221. Wiesenfeld-Hallin, Villar, M. J. and Hiikfelt, T. (1989) Effects of intrathecal galanin and C-fiber stimulation on the stimulation on the flexor reflex in the rat. Brain Research 486: 205-213. Cortts, R., Arvidsson, U., Schalling, M., Ceccatelli, S. and Hokfelt, T. (1990) Journal of Chemical Anatomy, in press.
Studies on the Effect of Systemic PD134308 (CAM 958) in Spinal Reflex and Pain Models with Special Reference to Interaction with Morphine and Intrathecal Galanin 2. WIESENFELD-HALLIN”,
X-J. XU*, J. HUGHESt,
D. C. HORWELLt
and T. HOKFELTS
Karolinska Institute, *Department of Clinical Physiology, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86, Huddinge, Sweden, tParke-Davis Research Unit, Cambridge, UK and #Department of Histology and Neurobiology, Karolinska Institute, Stockholm, Sweden (Reprint requests to ZW-HI
Abstract-The effect of intravenous (i.v.1 PD134308, which is a CCK-B antagonist, morphine and intrathecal (i.t.) galanin (GAL) on the excitability of the spinal nociceptive flexor reflex and in the hot plate test was examined in rats. PD134308 (1 mg/kg, i.v.) caused a weak, naloxone-reversible depression of the flexor reflex and moderate antinociception in the hot plate test. PD134308 significantly potentiated the antinociceptive effect of morphone, as well as its depressive effect on the flexor reflex. PD134308 and i.t. GAL synergistically depressed the flexor reflex, which was reversed by naloxone. Finally, the magnitude and duration of the depression of the flexor reflex by morphine was synergistically increased by coadministering i.v. PD134308 and i.t. GAL. The results demonstrate that a CCK antagonist directed to the central CCK-B receptor potentiates the analgesic effects of opioid and non-opioid drugs at spinal level in the rat, thus supporting the notion that CCK in the CNS may be an endogenous, physiological opioid antagonist.
areas of the spinal cord which are believed to have a role in nociception. Studies on the effects of exogenously applied CCK and related peptides on pain modulation in rodents have given conflicting results. CCK has been reported to cause analgesia after various routes of administration, whereas there are many reports that CCK reduced the analgesic effect of morphine and endogenous opioids (see 5 for review).
Introduction Cholecystokinin (CCK) is present in many areas of the central nervous system (CNS) (1). This peptide fulfils many of the criteria for a neurotransmitter and may have a role in various CNS functions (2). In the spinal cord genuine CCK-like immunoreactivity has been described in local neurons in the dorsal horn and in neurons in lamina X (3, 4), 79
80
Evidence has been presented that there might be multiple receptors for CCK and that the most abundant receptor type for CCK in the periphery differs from that in the CNS (6, 7), designated as CCK-A and CCK-B, respectively (7). More recently a number of CCK antagonists have become available, opening up possibilities to resolve the role of CCK in nociception. Thus, the non-selective, weak CCK receptor antagonists proglumide and benzotript potentiated the analgesic effect of morphine and other opioids (8). Studies with more recently developed specific CCK-A (Devazepide, formerly MK 329 and L365, 031) and CCK-B (L365, 260) antagonists further supported a role for CCK as a physiological opioid antagonist, as these drugs enhanced the analgesic effect of morphine and abolished morphine tolerance (9, 10). The rank order of potency of these drugs for enhancing morphine analgesia was L365,260>MK329>L365,031, which correlated with their potency in blocking the central CCK-B receptor in rats, indicating that the site of CCW opioid interaction is in the CNS and is mediated by CCK-B receptors (10). Another highly selective and potent CCK-B PD134308 (formerly receptor antagonist, CAM958), has recently been developed (11). We have now examined the effect of PD134308 on nociception by using two experimental models, the spinal nociceptive flexor reflex in decerebrated, spinalised, unanesthetised rats and a non-reflexive behavioral test of nociception, the hot plate test (12). It was shown that PD134308 enhances the analgesic effect of morphine and of morphine combined with galanin (GAL), a neuropeptide which may have endogenous antinociceptive effect at spinal level and potentiates the spinal analgesic effect of morphine (13).
NEUROPEPTIDES
animals were briefly anesthetised by methohexytal (70mg/kg, i.p.), decerebrated by aspiration of the fore- and midbrain and then artificially ventilated. The spinal cord was sectioned at Th8-9. In some rats, an i.t. catheter (PElO) was implanted caudally to the transection with its tip at the lumbar enlargement (L4-5). The reflex was continuously elicited by test stimuli of single shocks (l/min) to the sural nerve (1 ms, 5 mA) to activate C-fibers. The number of action potentials elicited during the reflex was integrated over 2s. A stable reflex magnitude was established for at least 20min prior to drug injection. Behavioral tests were carried out on male rats. The antinociceptive effect of the drug was assessed with a hot plate (IITC, Woodland Hills, CA, USA) at 54 + 0.2”C. The latency to licking a hind paw was determined to an accuracy of 0.1 s. Before any drugs were administered, the rats were habituated by testing on the hot plate for 5 days in order to obtain a stable response value. The average baseline response latency was 4.2 + 0.2s on the day before drug administration and there was no significant difference between groups. Morphine hydrochloride (Apoteksbolaget , Stockholm, Sweden) and PD134308 (ParkeDavis, Cambridge, UK) and naloxone (DuPont, Delaware, USA) were injected in a volume of 0.2ml i.v. in the physiological studies and i.p. in the behavioral experiments. Porcine GAL and Switzerland) Bubendorff, (Bachem, sulphated CCK 26-33 (Cambridge Research Biochemicals, Cambridge, UK) were dissolved in 0.9% saline and injected i.t. in a lOl.~l volume followed by 10 ul saline to flush the catheter. Results Potentiation of the analgesic effect of morphine by PDI34308
Materials and Methods Physiological studies were carried out on SpragueDawley rats of both sexes (b. wt. 250-300g) as described previously (14). Briefly, the magnitude of the hamstring flexor reflex in response to activation of high threshold afferents was examined in decerebrate, spinalised, unanesthetised rats by recording the e.m.g. from the posterior bicep femoris/semitendinosus muscles. The
PD134308
(lmg/kg) injected i.v. caused a weak depression of the flexor reflex by about 25% below control reflex magnitude for about 30min (Table, Fig. 1). The effect was reversed by 1 mg/kg naloxine i.v. Pre-treatment with this dose of PD134308 i.v. also antagonised the facilitatory effect of the flexor reflex by 1OOng and lpg i.t. CCK. PD134308 (1 mg/kg) and morphine (1 mg/kg) interacted synergistically to depress the flexor
STUDIES
ON THE EFFECT
PO134306
1
1
PD134308
(CAM 958) IN SPINAL
Morphine
Ok-----150
OF SYSTEMIC
Time (hz
PD134306+Yorphine
6
7 Time (hr)
Fig. 1 (Upper) PD134308 (lmg/kg) or morphine (lmg/kg) moderately depressed the flexor reflex for 30-60 min. (Lower, continuation of abscissa from Upper). The magnitude and duration of depression were greatly enhanced by coadministration of the two drugs. (Reproduced with permission from Proc. Natl. Acad. Sci. USA)
Table
The magnitude
and duration
of the depressive
AND
81
PAIN MODELS
reflex (Table, Fig. 1). After coadministration the reflex was depressed about twice as much as to each drug alone and the duration of depression lasted about three times as long as after morphine alone (Table). PD134308 at low doses (0.1,0.3mg/kg) had no effect on lick latency in the hot plate test, whereas larger doses had a fairly weak, but significant, analgesic effect for 20min (1 mg/kg) and 40min (3 mg/kg). Morphine (1 mg/kg) caused moderate analgesia for about 50min and PD134308 potentiated the analgesic effect of morphine in a dose-dependent fashion. With the three higher doses of PD134308 the analgesic effect of morphine was significantly potentiated at all intervals examined, starting 1Omin after injection. The duration of analgesia was increased from 5090min with 0.3mg/kg and to 130min with 1 and 3mg/kg PD134308.
2
5
REFLEX
Interaction of i. t. GAL and systemic PO134308 and morphine
One hundred ng GAL briefly facilitated the flexor reflex without causing reflex depression and 1 kg
effect of PD134308, morphine and GAL and the combination
of these drugs on the flexor reflex Drugs
N
% reflex depression
Duration of depression (min)
PD134308 1 mg/kg i.v.
6
24.5 ?C5.9
28.6k7.6
Morphine 1 mg/kg i.v.
8
38.1+ 10.7
60.5 + 27.2
PD134308 lmg/kg i.v. + Morphine lmg/kg i.v.
6
62.5 + 11.2”.’
195.3 f 24.2a,c
15
0
0
GAL 100 ng i.t. + Morphine 1 t.rg i.t.
5
58.5 f 15.1b
80.1 f 15.2
GAL 100 ng i.t. + PD134308 lmg/kg i.v.
4
66.0f 15.9”*’
152.2 + 38.9b,C
GAL 100 ng i.t. + Morphine 1 mg/kg i.v. + PD134308 1mgIkg i.v.
4
85.6 f 13.7d,e
250.2 f 14.1d,‘,s
GAL 100 ng i.t.
Statistics were performed by two-way ANOVA followed by Scheffe F-test, a = compared to morphine p
82
NEUROPEPTIDES
0
1
2
3
4
Tilz
mr,
Fig. 2 Effect of GAL at 100 ng i.t., PD134308 at 1 mg/kg i.v., and the combination of the two drugs on the excitability of the flexor reflex. (Reproduced with permission from Proc. Natl. Acad. Sci. USA)
morphine caused a weak facilitation followed by reflex depression for about 1 h. When GAL and morphine were coadministered i.t. at these doses the reflex depression became significantly stronger and its analgesic duration was also increased (Table). The effect of 1OOng i.t. GAL and lmg/kg i.v. PD134308, administered within a 5min interval, are summarised in the Table and illustrated in Figure2. Both the magnitude and duration of the reflex depression by lmg/kg i.v. PD134308 were significantly potentiated by 100ng i.t. GAL. In fact, the reflex depression by PD134308 plus GAL was significantly stronger and longer lasting than after 1 mg/kg morphine. Furthermore, the depressive effect of GAL plus PD134308 was naloxone reversible. An extremely strong potentiation of the effect of i.v. PD134308 plus i.v. morphine was observed after i.t. administration of GAL (Table, Fig. 3). The reflex was depressed by about 85% below control level, which was significantly greater than the effect of GAL plus morphine or PD134308 plus morphine. The duration of the reflex depression was about 250min, which was significantly longer than the effect of GAL plus morphine, PD134308 plus morphine or PD134308 plus GAL (Table). Discussion
In the present report we have shown that systemic PD134308, a specific CCK-B receptor antagonist,
dose-dependently potentiates the analgesic effect of morphine both in the hot plate test and in the flexor reflex model in spinalised rats, supporting the hypothesis that CCK-8 is an endogenous opioid antagonist. Since PD134308 has only very low affinity to the CCK-A receptor, the results strongly indicate that the interaction between CCK and opioids occurs through CCK-B receptors in the CNS. The potentiating effect of PD134308 on morphine-induced analgesia is robust, apparently stronger than the effect reported with another CCK-B antagonist, L365,260 (10). Also PD134308 tested over a 30-fold dose range did not reveal a bell-shaped distribution of dose effectiveness, which was seen with L365,260 (10). It is, however, difficult to make a direct comparison between the efficacy of these two drugs in potentiating the analgesic effect of morphine, since different animal models were employed. Systemic PD134308 by itself at high doses had a weak analgesic effect and depressed the flexor reflex in a naloxone-reversible fashion. It seems unlikely that this effect was due to a direct interaction of PD134308 with opioid receptors, since PD134308 has been shown to be inactive or
‘y
Galanin+ PDl34308+Morphina
I
Fig. 3 Effect of GAL at 100 ng i. t., PD134308 at 1 mg/kg i.v., morphine at 1 mg/kg i.v. (Upper), and combination of all three drugs (Lower, continuation of abscissa) on the flexor excitability in a single experiment. (Reproduced with permission from Proc. Natl. Acad., Sci. USA)
STUDIES ON THE EFFECT OF SYSTEMIC PD134308 (CAM 958) IN SPINAL REFLEX AND PAIN MODELS
only weakly active in the displacement of the binding of radioligands to kappa, mu or delta opioid receptors (11). Analgesia has not been reported with other CCK receptor antagonists. It is possible that the antagonism by CCK on opioid analgesia operates to some extent tonically through the CCK-B receptor at spinal level and that blockade by PD134308 results in removal of this antagonism, leading to increased opioid peptide release. We have previously demonstrated that GAL given i.t. potentiated the effect of morphine on flexor reflex excitability and a behavioral test of nociception in a naloxone-reversible fashion (13). If the analgesic effect of PD134308 involves the endogenous opioid system one would predict that it should be potentiated by GAL as well. Indeed, the depressive effect of systemic PD134308, when combined with i.t. GAL, was reversed by naloxone and was stronger than that after lmg/kg morphine. Since we have previously shown that naloxone could not reverse the reflex depression by high doses of GAL (14), it appears as though the GAL receptor is independent of both the opioid and CCK-B receptors, but can interact with them to cause a synergistic opioid-mediated analgesia. The synergistic interaction between GAL, PD134308 and morphine further strengthens this suggestion. A major site of the analgesic effect of PD134308 appears to be within the spinal cord, since we observed a similar effect in intact and in spinalised rats. Furthermore, the interaction between PD134308 and morphine was also similar in intact and spinalised animals. Finally, the strongest argument for a direct spinal effect of systemic PD134308 is that it effectively antagonised the facilitatory effect of i.t. CCK and interacted with i.t. GAL to cause synergistic reflex depression. Previous experiments have documented a spinal site of interaction between CCK and opioids (see 5). Moreover, extensive systems containing genuine CCK peptide and CCK mRNA are present in the rat dorsal horn (3,4,15). However, CCK-B receptors also exist in many brain regions, including the periaqueductal grey (6,7), known to be involved in opioid analgesia, and the interaction between CCK, its antagonist and opioids has been reported after intracerebral or intracere-
83
broventricular injection (5). We thus cannot rule out the possibility that the potentiation of morphine-induced analgesia by PD134308 on the hot plate test may be partly mediated by CCK-B receptors in the brain. In conclusion, the present results demonstrate that a newly developed CCK-B antagonist in two experimental models can enhance the analgesic effect of morphine, especially when combined with the neuropeptide GAL, indicating new approaches to pain treatment based on combinations of drugs. Acknowledgements Thisstudy was supported by the Bank of Sweden Tercentenary Foundation, the Swedish MRC (project no. 07913,04X-2887), NIMH (MH43230-02), Marianne och Marcus Wallenberg Stiftelse, Konung Gustav V och Drottning Victorias Stiftelse, Fredrik och Ingrid Thurings Stiftelse and research funds of the Korolinska Institute. X.-J. Xu was supported by the WennerGren Center Foundation.
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