Pain, 49 (1992) 137-144 0 1992 Elsevier Science
137 Publishers
B.V. All rights reserved
0304-3959/92/$05.00
PAIN 02024
Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists Christine Miaskowski a, Kimberly A. Sutters a, Yetunde 0. Taiwo ‘,* and Jon D. Levine b,c @Schools of Nursing, ’ Medicine and ’ Dentistry, University of California at San Francisco, San Francisco, CA 94143 (USA) (Received
22 January
1991, revision
received
16 July 1991, accepted
11 October
1991)
Interactions between selective opioid agonists acting at spinal p-, a-, and K-opioid receptors were Summary evaluated by co-administering a low-antinociceptive dose of the selective S-agonist, DPDPE, or the selective K-agonist, U50,488H, with sequentially increasing doses of the selective p-agonist, DAMGO, intrathecally. Antinociceptive synergy (i.e., a more than additive antinociceptive effect) was observed with both combinations of opioid agonists tested. The demonstration of antinociceptive synergy suggests that the subtypes of spinal opioid receptors can act, at least in part, through a common neural circuit. Since our measure of antinociception, the Randall-Selitto paw-withdrawal test, is dependent on a normally functioning motor system, we also evaluated the effects of these same combinations of opioid peptides on motor coordination using a rotarod treadmill. A low-antinociceptive dose of DPDPE or U50,488H co-administered intrathecally, with sequentially increasing doses of DAMGO, did not worsen the decrement in rotarod performance observed with the same doses of DAMGO administered as a single agent. In fact, the low-antinociceptive dose of DPDPE significantly attenuated the decrease in rotarod performance produced when the same dose of DAMGO was administered as a single agent. The results of this study suggest that intrathecal combinations of selective CL-with both 6- or K-selective opioid agonists can produce antinociceptive synergy without producing an increase in motor side effects. In addition, the results of a secondary analysis, that compared the antinociceptive data from this study with data from our previous studies of intrathecal combinations of sequentially increasing doses of U50,488H with low doses of DPDPE or DAMGO and sequentially increasing doses of DPDPE with low doses of DAMGO or U50,488H, demonstrate distinct differences in the magnitude of the antinociceptive interactions. The most prominent finding from the secondary analysis was that administration of the p-opioid agonist, DAMGO, as a component of any combination regimen, resulted in the largest enhancement in antinociceptive effects. Key words: Antinociceptive
synergy; p-Opioid;
&Opioid; K-Opioid; Motor coordination;
Introduction
While it is well known that opioid peptides administered intrathecally (i.t.> act at p-, S-, and K-OpiOid receptors to produce antinociceptive effects (Yaksh et
* Present address: The Procter OH 45239-8707. USA.
and
Gamble
Company,
Cincinnati,
Correspondence too: Jon D. Levine, M.D., Ph.D., Division of Rheumatology, U426/Box 0724, University of California, San Francisco, CA 94143-0724, USA.
Spinal intrathecal
al. 1978; Yaksh 1983; Porreca et al. 1984, 1987; Hylden et al. 1986), the potential interactions between selective opioid agonists acting on different receptor subtypes have not been investigated in detail. A few studies have shown that i.t. combinations of various opiates and opioid peptides with different receptor specificity produce enhanced or even synergistic interactions (Vaught and Takemori 1979; Larson et al. 1980; Lee et al. 1980; Levine et al. 1982; Roerig and Fujimoto 1989). In order to determine if there are interactions between the various subtypes of receptors in spinal antinociceptive circuitry, we have systematically investigated the antinociceptive effects produced by combina-
13X
tions of selective p-, S-, and Ic-opioid receptor agonists. In previous studies, we have demonstrated that i.t. co-administration of a low-antinociceptive dose of a selective S- or K-agonist with a range of doses of the other agonist (Miaskowski et al. 1990) or that i.t. co-administration of a low-antino~iceptiv~ dose of a selective p-agonist with a range of doses of either a selective 6or a selective K-agonist @utters et al. 1990) produced antinociceptive synergy (i.e., the combination regimens produced greater than additive antinociceptive effects). We concluded that these synergistic antinociceptive effects occurred because the various subtypes of spinal opioid receptors can act, at least in part, through common neural circuits. We now report on the synergistic effects produced by Lt. co-administration of a low-antinociceptive dose of the selective &agonist, DPDPE (Mosberg et al. 1983) or the seiective K-agonist, U50,488H (Von Voigtlander et al. 19831, with a range of doses (0.5 ng to 5 pg) of the selective p-agonist, DAMGO (Handa et al. 1981). In addition, since we have recently shown a marked decrease in motor coordination associated with i.t. administration of DAMGO (Miaskowski et al. 1991), this study also evaluated the effects of the same two combination regimens on motor coordination. Finally, a secondary analysis of the data from our present and previous antinociceptive synergy studies (Miaskowski et al. 1990; Sutters et al. 1990) was performed to evaluate differences in the magnitude of the antinociceptive effects produced by the various i.t. combinations of selective opioid agonists (see Appendix).
tmean+S,E.M., N = 40). The effects of each of the opioid agonists on nociceptive threshold were tested 15, 20, and 25 min after i.t. administration, at the time of peak effect of these agents (Heyman et al. 1986; Porreca et al. 1987; Millan 1989). The change in nociceptive threshold, produced by the opioid agonists, was calculated as the average percentage change from baseline threshold of these 3 consecutive measurements. A cut-off of SO0 g was used for the pawwithdrawal test and a value of 500 g assigned if the animal reached cut-off.
Drug administration protocol A dose-response curve for i.t. administration of the selective p-opioid agonist, DAMGO (Peninsula Laboratories. Belmont, CA), was determined by administering sequentially increasing doses of the agonist, in a volume of IO PI at 30 min intervals. To test for synergistic interactions between the antinociceptive effects of the selective p-agonist with the selective 6- and K-agonists the following combinations were tested: (If a low-antinociceptive dose (i.e., a dose which produced approximately a 20% increase in nociceptive threshold; in this case 0.5 ng) of the selective &agonist, [D-Pen2,5~nkephalin (DPDPE, Peninsula Laboratories. Belmont, CA), was co-administered with each sequentially increasing dose of DAMGO and (2) a low-antinociceptive dose (5 ng) of the selective K-agonist, U50,488H (a generous gift from Dr. Robert Lahti, Upjohn, Kalamazoo, MI), was co-administered with each sequentialiy increasing dose of DAMGO. All drugs were dissolved in saline. Since the cumulative dosing regimen might have an effect on nociceptive thresholds, we determined whether i.t. administration of a single high dose (i.e., 5 pg) of an opioid produced an increase in nociceptive threshold of similar magnitude as when the same dose was administered as the last dose in an ascending series, as performed in these experiments. We found no differences in the antin~iceptive effects produced by the highest dose of opioid agonist administered alone or as the last dose in an ascending series (unpublished results). Therefore, neither the lower doses preceding the highest dose nor the time in the apparatus, nor the time of exposure to elevated paw pressures appear to contribute to the antinociceptive effects produced in these experiments by the highest dose of the opioid agonist.
Materials and methods
Motor coordination testing The experiments were performed using 240-300 g, male Sprague-Dawley rats (Bantin and Kingman. Fremont, CA). One week prior to the experiments, under pentobarbital anesthesia (65 mg/kgf. i.t. catheters were inserted through the atianto-occipital membrane and passed caudally 8.5 cm to a site just rostra1 to the lumbosacral enlargement (Yaksh and Rudy 1976). Rats that exhibited neurological deficits following the surgical procedure were eliminated from the study.
Aniinoc~ceptj~e testing The Randall-Selitto paw-withdrawal test (Randall and Selitto 1957) was used to measure the mechanical nociceptive threshold. The mechanical stimulus was applied with a Basile analgesymeter (Stoelting Co., Chicago, IL) which generates a linearly increasing mechanical force, applied by a conical piece of plastic with a domeshaped tip, to the dorsal surface of the rat’s hind paw. The nociceptive threshold was defined as the force in grams at which the animal withdraws its paw. The rats were trained in the test procedure for 3 h daily for 5 days prior to data collection (Taiwo et al. 1989). On the day of the experiment, rats were first tested at 5 min intervals for 2 h. Baseline thresholds were defined as the mean of the last 6 measurements prior to drug administration. The average baseline nociceptive threshold for these rats was lOl.lO+ 2.30 g
In separate groups of rats, motor coordination was tested using an accelerating rotarod treadmill (Rota-Rod, Ugo Basile, Stoelting Co., Chicago, IL) (Dunham and Miya 19S7; Watzman et al. 1967). The rotarod was set in motion at a constant speed and the rats were placed into individual sections of the apparatus. Once all the rats were in position. the timers were set to zero and the rotarod was switched to the accelerating mode. The rotarod accelerated from a rate of 3.7 to 37.5 r.p.m.s in a period of 5 min. The animals’ performance time in seconds was recorded when the rat, unable to stay on the rotarod, tripped a plate and stopped the timer. A minimum of 2 training sessions of 3 h duration were performed to condition the animals to the treadmill and establish consistent baseline performance scores. On the day of the experiment, the rats were tested on the rotarod at 5 min intervals for 2 h. A baseline rotarod performance score was defined as the mean of the last 6 measurements prior to drug administration. The average rotarod performance score for these rats
was 97.48i6.37 set (meanrtS.E.M., N = 29). The effect of the opioid agonists alone and in combination on rotarod performance scores (i.e., length of time on the rotarod) was tested using the same drug administration paradigm used in the antinociceptive mance scores
testing. The effect was also calculated
baseline performance score.
of the drugs on rotarod perforas a percentage change from
139
Statistical analyses Statistical analysis of the dose-dependent effects of DAMGO, as a single agent, on nociceptive threshold and rotarod performance time was performed using l-way analysis of variance (ANOVA). While several methods of analysis are currently available to analyze for interactions between biologically active agents (Berenbaum 1989), the method used in this study was a 2-factor, repeated measures ANOVA (Cohen and Cohen 1983; Melton and Tsokos 1983; Sokal and Rohlf 1987; Marascuilo and Serlin 1988). ANOVA
A.
ADDITIVE
INTERACTION
O-O
Drug
.-•Drug
A A + low-dose
80 --
60.-
40 -.------O/O
200-------
which has been used to identify interactions between biologically active agents in research studies in cardiovascular physiology (Caplan and Su 1986; Katahira et al. 1989; Kopia et al. 1989; Thompson and Epstein 19911, immunology (Sat0 et al. 1989; Oksenberg et al. 19901, oncology (Shaikh et al. 1989), and toxicology (Harvey and Klaassen 1983; Brennan and Jastreboff 1989) studies was chosen because this method of analysis allowed us to evaluate for a statistically significant interaction between 2 groups (in this case, a single drug compared to a combination regimen) across a series of measures (in this case, cumulative dosing across 5 orders of magnitude). A statistically significant interaction term, in a 2-factor repeated measures ANOVA, demonstrates that the differences between 2 groups over a repeated measure (in this case, increasing doses of the opioid agonist) deviate from parallelism and, therefore, are more than additive that is synergistic (Fig. 1). Differences were considered statistically significant at the P < 0.05 level. The number of rats in each group is indicated in the figure legends. The results of a secondary analysis that compared the differences in the magnitude of the antinociceptive effects produced by the various combinations of opioid agonists used in this study with data from our previous Lt. studies of different combinations of opioid agonists (Miaskowski et al. 1990; Sutters et al. 1990) are presented in the Appendix.
OH 0
2
1
0
1 3
Results 6.
SYNERGISTIC
INTERACTION
Intrathecal administration of sequentially increasing doses of DAMGO produced statistically significant, dose-dependent increases in nociceptive thresholds (F (5, 45) = 315.47, P < 0.001; Fig. 2A) and statistically significant, dose-dependent decreases in rotarod performance scores (F (5, 35) = 13.41, P < 0.001; Fig. 2B) demonstrated by l-way ANOVA.
Dose
of Drug
1. A: hypothetical dose-dependent antinociceptive effects of increasing doses of drug A and a combination of a low-antinociceptive dose of drug B with increasing doses of drug A. The zero dose for the single agent is saline and the zero dose for the combination regimen is the low dose of drug B. The dose-response curve for the combination regimen is displaced by an equal amount across the entire dose range tested (i.e., there is a parallel displacement of the curves), indicating that the combination of a low-antinociceptive dose of drug B with increasing doses of drug A produced only additive antinociceptive effects. B: hypothetical dose-dependent antinociceptive effects of increasing doses of drug A and a combination of a low-antinociceptive dose of drug B with increasing doses of drug A. The zero dose for the single agent is saline and the zero dose for the combination regimen is the low dose of drug B. The dose-response curve for the combination regimen is displaced by an increasing amount that is more than the sum of the antinociceptive effects of the two drugs given as single agents, across the entire dose range tested (i.e., the curves are divergent, deviating from parallelism). In this case, a significant interaction term in a 2-factor repeated measures ANOVA would indicate that the combination of a low-antinociceptive dose of drug B with increasing doses of drug A produced a synergistic interaction.
6 /p Agonist effects A comparison of the antinociceptive effects of i.t. co-administration of a low-antinociceptive dose (0.5 ng) of DPDPE with each sequentially increasing dose of DAMGO to the antinociceptive effects produced by DAMGO, as a single agent (Fig. 2A), using a 2-factor repeated measures ANOVA, demonstrated significant main effects of group and dose (F (1, 18) = 13.96, P < 0.002 and F (4, 72) = 146.61, P < 0.001, respectively) as well as a significant group x dose interaction (F (4, 72) = 7.19, P < 0.001). The statistically significant interaction term indicates that the combination regimen produced antinociceptive synergy. A comparison of the effects of the same combination regimen on rotarod performance, with the effects produced by DAMGO as a single agent (Fig. 2B), demonstrated significant main effects of group and dose (F (l,ll)= 8.73, P < 0.02 and F (5, 55) = 22.99, P < 0.001, respectively); however, the group X dose interaction was not significant (F (5, 55) = 1.55, P > 0.05). The combination regimen improved rotarod performance compared to the single agent as indicated by the significant main effect of group.
A.
A.
PAW-WITHDRAWAL 500
-
400
--
300
--
200
--
PAW-WITHDRAWAL
0
, O-O
DAMGO
n -m
DAMGO (IT) + 0.5 ng DPDF’E (IT)
(IT)
B. B
o-ODAMGO
(IT)
.-•DAMGD
(IT) + 5 “9 “50.488H
(IT)
ROTAROD
ROTAROD 6040 -20 --
I
-60
--
-40 -60
---
-80
-
-1001 1001
0 5
“9
5
“9
50
“9
500
“9
5 w
Fig. 2. A: dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of DAMGO (N = 10) and a low-antinociceptive dose (0.5 ng) of DPDPE co-administered with each sequentially increasing dose of DAMGO (N = 10) on paw-withdrawal nociceptive thresholds. The 5 pg dose of DAMGO was not tested with a low-antinociceptive dose of DPDPE because 60% of the animals were already at cut-off for the paw-withdrawal test at the 500 ng dose of DAMGO. B: dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of DAMGO (N = 8) and a low-antinociceptive dose (0.5 ng) of DPDPE co-administered with each sequentially increasing dose of DAMGO (N = 5) on rotarod performance scores (i.e., length of time on the rotarod). All responses are graphed as percentage change from baseline paw-withdrawal threshold (A) or rotarod score (B) after drug administration. Each point in the figures represents the mean +S.E.M. Some error bars are contained within the symbols.
K / p Agonist effects effects of i.t. A comparison of the antinociceptive dose (5 ng> co-administration of a low-antinociceptive of U50,488H with each sequentially increasing dose of DAMGO to the antinociceptive effects produced by DAMGO as a single agent (Fig. 3A) demonstrated significant main effects of group and dose (F (1, 18) = 22.65 and F (4, 72) = 265.30, respectively, both P < 0.001) as well as a significant group X dose interaction (F (4, 72) = 7.87, P < 0.001). Again, the statistically significant interaction term indicates that the combination regimen produced antinociceptive synergy. A comparison of the effects of the same combination regimen on rotarod performance, with the effects
t ‘\i \ I 0.5 “9
5 “9
50 ng
500 “9
5 u9
Fig. 3. A: dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of DAMGO (N = 10) and a low-antinociceptive dose (5 ng) of U50,488H co-administered with each sequentially increasing dose of DAMGO (N = 10) on paw-withdrawal nociceptive thresholds. Again, the 5 fig dose of DAMGO was not tested with a low-antinociceptive dose of U50,488H because 20% of the animals were already at cut-off for the pawwithdrawal test at the 500 ng dose of DAMGO. B: dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of DAMGO (N = 8) and a low-antinociceptive dose (5 ng) of U50,488H co-administered with each sequentially increasing dose of DAMGO (N = 8) on rotarod performance scores (i.e., length of time on the rotarod). All responses are graphed as percentage change from baseline paw-withdrawal threshold (A) or rotarod score (B) after drug administration. Each point in the figures represents the mean+ S.E.M. Some error bars are contained within the symbols.
produced by DAMGO as a single agent (Fig. 3B) demonstrated significant main effects of dose (F (5, 70) = 12.40, P < 0.001) but not group (F (1, 14) = 1.67, P > 0.05), and the group X dose interaction was not significant (F (5, 70) = 0.65, P > 0.05). The combination regimen did not significantly affect rotarod performance scores as indicated by the insignificant group effect.
Discussion The results of this study demonstrate that i.t. co-administration of low-antinociceptive doses of DPDPE or U50,488H with sequentially increasing doses of
141
DAMGO produced antinociceptive synergy. In addition, a low-antinociceptive dose of DPDPE or U50,488H co-administered with sequentially increasing doses of DAMGO did not worsen the decrement in rotarod performance observed with the same dose of DAMGO administered as a single agent. In fact, coadministration of a low-antinociceptive dose of DPDPE attenuated the decrease in rotarod performance produced by DAMGO. The data from this study demonstrate that the antinociceptive synergy produced by the combination regimens was not caused by decrements in motor coordination. The synergistic interactions between low-dose 6and p- opioid agonists and between low-dose K- and p-opioid agonists, observed in this study, as well as those interactions seen in previous i.t. studies (Miaskowski et al. 1990; Sutters et al. 1990) using different combinations of low and varying doses of selective p-, 6-, and K-opioid agonists, demonstrate that all pairs of spinal opioid receptors can interact to produce marked enhancements in antinociceptive effects. However, there are differences in the magnitude of the interactions produced by activation of different combinations of opioid receptors. To evaluate these differences, a secondary statistical analysis (see Appendix) was performed that compared the differences in the magnitude of the antinociceptive effects produced by the various combinations of opioid agonists in this and our previous i.t. studies (Miaskowski et al. 1990; Sutters et al. 1990). Results of the secondary analysis (see Appendix) demonstrated that a low-antinociceptive dose of DAMGO markedly enhanced the antinociceptive effects of DPDPE and U50,488H. In contrast, when a low dose of DPDPE was given with U50,488H or a low dose of U50,488H was given with DPDPE, although antinociceptive synergy was demonstrated, it was significantly less than that produced when a low dose of DAMGO was co-administered with DPDPE or U50,488H. These data demonstrate that activation of p-opioid receptors produces the most marked enhancement in antinociceptive effects. Further studies are needed, including those using receptor-selective opioid antagonists, to confirm the important role of the CL-opioid receptor in spinal antinociceptive circuitry as well as to elucidate further the mechanisms underlying the interactions between spinal w-, a-, and K-opioid receptors observed in this study. While the exact mechanisms underlying antinociceptive synergy are unknown, there are several lines of evidence that support the possibility of interactions between spinal opioid receptors. For example, Fields et al. (1980) demonstrated the presence of both p- and S-opioid receptors on primary afferent fibers and on dorsal horn neurons in the rat. In addition, positive cooperativity for interactions between p- and a-bind-
ing sites in vitro (Rothman and Westfall 1982) and for CL- and a-mediated analgesia in vivo (Larson et al. 1980; Lee et al. 1980, Vaught et al. 1982) have been shown to occur. Finally, Werz et al. (1987) characterized IL, 6, and K receptors that are coupled to calcium or potassium ion channels on single dorsal root ganglion cells, in vitro. In order to demonstrate that the increases in nociceptive thresholds observed with the low-dose 6 with p and the low-dose K with p combinations were not the result of decreases in motor coordination, the effects of these combinations of opioid agonists on rotarod performance scores were determined. The rotarod performance data suggest that the enhanced antinociceptive effects produced by these combinations of opioids is not secondary to enhanced motor side effects. In conclusion, the data from this and our previous i.t. studies of combinations of opioid agonists (Miaskowski et al. 1990; Sutters et al. 1990) demonstrate that all three subtypes of opioid receptors (i.e., p, 6, and K) can interact to produce antinociceptive synergy. However, the magnitude of the interactions vary markedly depending on which combinations of selective opioid receptor agonists are administered. The data from these studies demonstrate that co-activation of the p-opioid receptor, with either 6- or K-Opioid receptors, results in the largest enhancement in antinociceptive effects. Importantly, these marked enhancements in antinociception are not attributable to increases in motor deficits.
Acknowledgements
We would like to thank Dr. Steven Paul for statistical consultation. This work was supported by NIH grant DE08973 and a grant from Sigma Theta Tau International, Inc.
Appendix Secondary analysis comparing the magnitude of the antinociceptive effects of intrathecal combinations of p,-, 6-, and K-opioid agonists
A secondary analysis of the data from our present and previous antinociceptive synergy studies (Miaskowski et al. 1990; Sutters et al. 1990) was performed to evaluate differences in the magnitude of the antinociceptive effects produced by the various i.t. combinations of selective opioid agonists. This analysis was done, using a 2-factor repeated measures ANOVA, that compared the dose-response effects among the control group and two groups receiving combination regimens. Post hoc contrasts, using the Scheffe test, were performed to determine statistically significant
142 O-0
U50,4BBH
(IT)
0-0
DPDPE
(IT)
n---o
U50.488H
(IT)
+ 0.5
ng DPDPE
(IT)
0-n
DPDPE
+ 0.5
“g DAMGO
A-A
U50.488H
(IT)
+ 0.5
ng DAMGO
(IT)
A-A
DPDPE
+ 5.0
ng U50.488H
(IT) (IT)
T
300 -250 -200 --
I 5 “9
50
ng
500
“g
5 ug
50
ug
0.5
“g
50
5 ng
500
“g
“g
5 ug
Fig. 4. Dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of U50,488H (N = 12). a low-antinociceptive dose (0.5 ng) of DPDPE co-administered with sequentially increasing doses of U50,488H (N = lo), and a low-antinociceptive dose (0.5 ng) of DAMGO co-administered with each sequentially increasing dose of U50,488H (N = 10) on paw-withdrawal nociceptive thresholds. All responses are graphed as percentage change from baseline paw-withdrawal threshold after drug administration. Each point in the figures represents the mean + S.E.M. Some error bars are contained within the symbols.
Fig. 5. Dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of DPDPE (N = 24), a low-antinociceptive dose (0.5 ng) of DAMGO co-administered with sequentially increasing doses of DPDPE (N = lo), and a low-antinociceptive dose (5 ng) of U50,488H co-administered with each sequentially increasing dose of DPDPE (N = 10) on paw-withdrawal nociceptive thresholds. All responses are graphed as percentage change from baseline paw-withdrawal threshold after drug administration. Each point in the figures represents the mean f S.E.M. Some error bars are contained within the symbols.
differences in group means averaged across dose (Cohen and Cohen 1983; Melton and Tsokos 1983; Sokal and Rohlf 1987; Marascuilo and Serlin 1988). The first question addressed in the secondary analysis was whether the low-antinociceptive i.t. dose of DAMGO or DPDPE produced the larger increase in nociceptive thresholds when given in combination with sequentially increasing doses of i.t. U50,488H (Fig. 4). A comparison of the antinociceptive effects of sequentially increasing doses of U50,488H with the effects produced by combination regimens of a low-antinociceptive dose of DAMGO with U50,488H and a low-antinociceptive dose of DPDPE with U50,488H, using a 2-factor repeated measures ANOVA, demonstrated a significant main effect for all three groups averaged across dose (F (2, 29) = 105.62, P < 0.001). Post hoc contrasts demonstrated that there was a statistically significant difference in the group mean averaged across dose for the low-dose p and K combination compared with the low-dose 6 and K combination (P < 0.01). These results indicate that a low dose of DAMGO produced a significantly larger increase in the antinociceptive effects of U50,488H than did a low dose of DPDPE administered i.t. The second question addressed in the secondary analysis was whether the low-antinociceptive i.t. dose of DAMGO or U50,488H produced the larger increase in nociceptive thresholds, when given in combination with sequentially increasing doses of DPDPE (Fig. 5). A comparison of the antinociceptive effects of sequentially increasing doses of DPDPE with the ef-
fects produced by combination regimens of a low-antinociceptive dose of DAMGO with DPDPE and a lowantinociceptive dose of U50,488H with DPDPE, using a 2-factor repeated measures ANOVA, demonstrated a significant main effect for all three groups averaged across dose (F (2, 41) = 117.56, P < 0.001). Post hoc O-0
DAMGO
(IT)
0-0
DAMGO
(IT)
+ 0.5
“g
DPDPE
A-A
DAMGD
(IT)
+ 5.0
“g
U50.4BBH
0.5
ng
5 ng
50
“g
(IT)
500
(IT)
ng
5 ug
Fig. 6. Dose-dependence relationship for the effects of intrathecal administration of sequentially increasing doses of DAMGO (N = lo), a low-antinociceptive dose (0.5 ng) of DPDPE co-administered with sequentially increasing doses of DAMGO (N = lo), and a low-antinociceptive dose (5 ng) of U50,488H co-administered with each sequentially increasing dose of DAMGO (N = 10) on paw-withdrawal nociceptive thresholds. All responses are graphed as percentage change from baseline paw-withdrawal threshold after drug administration. Each point in the figures represents the mean i_ S.E.M. Some error bars are contained within the symbols.
143
contrasts demonstrated that there was a statistically significant difference in the group mean averaged across dose for the low-dose w and 6 combination compared with the low-dose K and 6 combination (P < 0.001). These results indicate that a low dose of DAMGO produced a significantly larger increase in the antinociceptive effects of DPDPE than did a low dose of U50,488H, administered i.t. The final question addressed in the secondary analysis was whether the low-antinociceptive i.t. dose of DPDPE or U50,488H produced the larger increases in nociceptive thresholds when given in combination with sequentially increasing doses of i.t. DAMGO (Fig. 6). A comparison of the antinociceptive effects of sequentially increasing doses of DAMGO with the effects produced by the combination regimens of a low-antinociceptive dose of DPDPE with DAMGO and a lowantinociceptive dose of U50,488H with DAMGO, using a 2-factor, repeated measures ANOVA, demonstrated a significant main effect for all three groups averaged across dose (F (2, 27) = 9.38, P < 0.001). Post hoc contrasts demonstrated that there was no statistically significant difference in the group mean averaged across dose for the low-dose S and p combination compared with the low-dose K and p combination. These results indicate that there is no difference in the increases in nociceptive threshold produced by a low dose of DPDPE or a low dose of U50,488H when they were co-administered i.t. with sequentially increasing doses of DAMGO.
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