- Email: [email protected]
κ-opioid agonist combinations in Planaria
BR A IN RE S E A RCH 1 1 14 ( 20 0 6 ) 3 1 –3 5
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Subadditive withdrawal from cocaine/κ-opioid agonist combinations in Planaria Robert B. Raffa⁎, Gregory W. Stagliano, Ronald J. Tallarida Department of Pharmaceutical Sciences, Temple University School of Pharmacy (RBR, GWS) and Department of Pharmacology, Temple University Medical School (RJT), Philadelphia, PA 19140, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
We have previously developed and extensively characterized a convenient and sensitive
Accepted 13 July 2006
metric for the quantification of withdrawal responses using Planaria. Planaria are
Available online 17 August 2006
particularly valuable for these studies because of their permeable exteriors and their relevant neurotransmitter systems (e.g., dopaminergic, opioid, and serotonergic). In the
Keywords:
present study, we used this metric and mathematically rigorous joint-action analysis to
Withdrawal
investigate poly-drug withdrawal from fixed-ratio cocaine/κ-opioid agonist combinations.
Cocaine
The D50 (concentration producing half-maximal effect) for cocaine and U-50,488H was 10.3
κ-opioid
and 1.02 μg, respectively. The D50 for 19:1 or 1:19 combinations did not differ significantly
U-50,488H
(p > 0.05) from expected additive values (11.6 ± 3.0 vs. 9.9 ± 1.4 and 1.1 ± 0.2 vs. 1.5 ± 0.1,
Planaria
respectively), but the 3:1, 1:1, and 1:3 ratios did (34.5 ± 6.9 vs. 7.7 ± 1.1; 55.1 ± 10.0 vs. 5.7 ± 0.7; and 40.8 ± 8.9 vs. 3.3 ± 0.4, respectively), indicating subadditive interaction at these ratios. The finding of subadditivity in this model suggests that abstinence-induced withdrawal from the combination is less intense than that predicted from the individual drug potencies. The concept that certain combinations of drugs leads to attenuated withdrawal might generalize to humans. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Planarians have a centralized nervous system (cephalic ganglia and spinal processes) and mammalian-like neurotransmitter systems. They respond to dopaminergic agonists, antagonists, or neuronal uptake inhibitors (e.g., Welsh and Williams, 1970; Carolei et al., 1975; Algeri et al., 1983; Venturini et al., 1989; Palladini et al., 1996). Changes in 2nd-messenger levels induced by dopamine agonists (Palladini et al., 1996) suggest that the effects are mediated via dopamine receptors. The presence of an enkephalinergic system in Planaria was identified by means of radioimmunological and immunocytochemical techniques (Venturini et al., 1983) and a dopamine-opioid behavioral interaction has been described
(Passarelli et al., 1999). In a previous work, we developed a behavioral model that quantifies physical dependence and withdrawal in Planaria (Raffa and Valdez, 2001; Raffa et al., 2001, 2003). In brief, following exposure to drug, planarians display a dose-related decrease in spontaneous locomotor velocity (pLMV) when placed into drug-free water, but not drug-containing, water (i.e., abstinence-induced withdrawal) and receptor-selective antagonists decrease pLMV of drugexperienced planarians in a dose-related and enantiomericspecific manner (i.e., precipitated withdrawal) (Raffa et al., 2001). Specific withdrawal signs are noted at higher doses (Raffa and Desai, 2005). The phenomenon is not due to adverse effects of the drugs on locomotor activity or to changes in pH, temperature, or osmolarity (Umeda et al., 2004).
⁎ Corresponding author. Fax: +1 215 707 5228. E-mail address: [email protected] (R.B. Raffa). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.07.037
32
BR A IN RE S EA RCH 1 1 14 ( 20 0 6 ) 3 1 –35
The individual mechanisms of action of cocaine and opioids are relatively well known, involving modulation of biochemical processes within the central nervous system that can lead to abuse, craving, and relapse (e.g., Koob and Le Moal, 1997; Self and Nestler, 1998; Ahmed and Koob, 1998). However, little is known about possible interactions between these drugs. In the few studies that have been conducted on combinations, interactions have been reported between dopamine and opioid (rats) (e.g., Turchan et al., 1998; Woolfolk and Holtzman, 1996; Collins et al., 2001), dopamine and serotonin (rats) (e.g., Sasaki-Adams and Kelley, 2001; Filip et al., 2001), and cocaine with opioid (rats and rhesus monkeys) (Sharpe et al., 2000; Yuferov et al., 2001; Wang et al., 2001). We report a quantitative assessment of planarian withdrawal from fixed-ratio combinations of cocaine and the κopioid agonist U-50,488H (trans-(±)-3,4-Dichloro-N-methyl-N(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide). We used U-50,488H instead of morphine or heroin because planarians are more sensitive to this opioid (Passarelli et al., 1999). We applied ‘joint-action’ analysis to characterize the nature of the interaction between the two compounds (Tallarida, 2000).
2.
Results
2.1.
Cocaine
Consistent with previous reports (Raffa et al., 2001), cocainenaïve planarians displayed a characteristically nearly con-
Fig. 2 – Withdrawal (expressed as the 5-min cumulative mean ± SD decreased pLMV) when planarians (N = 10 each point) are placed into water following exposure to the doses indicated of cocaine alone or the opioid U-50,488H alone.
stant locomotor velocity (pLMV) of approximately 15–16 gridlines/min when tested in cocaine-free water and planarians that were exposed for 1 h to cocaine then tested in water containing the same concentration of cocaine displayed pLMV which was not significantly different from cocaine-naïve planarians tested in cocaine-free water. Hence, exposure to cocaine did not alter pLMV under these conditions. However, planarians exposed to cocaine for 1 h then placed into cocaine-free water displayed a significantly reduced pLMV. Notably, the pLMV remained constant over the 5-min observation period, even during withdrawal from the highest concentration of cocaine, suggesting a physiologic, not toxic, or local anesthetic, effect of cocaine on the planarians. The reduction in pLMV was dose-related to prior exposure to 10− 7– 10mg/ml (10− 8–10− 3 M) cocaine (Fig. 1). The D50 value (dose producing half-maximal effect) for cocaine (MW = 339.81) was 10.3 μg. The duration of withdrawal signs was previously shown to persist greater than 1 h (Raffa and Desai, 2005) followed by recovery of the animals.
Table 1 – The predicted additive and experimentally determined mean D50 ± SD (μg/ml) for each fixed-dose ratio of cocaine:U-50,488H combinations Ratio a Additive (total) D50 Experimental (total) D50 Fig. 1 – Solid lines: pLMV (expressed as the mean ± SD of the cumulative number of gridlines crossed per minute) of planarians exposed to cocaine (concentration in M indicated) for 1 h and then tested in cocaine-free water. Dotted line: cocaine-naïve animals tested in water. Dashed line: cocaine-exposed animals tested in the same concentration (1 × 10− 5 M) of cocaine. N = 10 for all groups.
1:19 1:3 1:1 3:1 19:1 a
1.5 ± 0.2 3.3 ± 0.4 5.7 ± 0.8 7.7 ± 1.1 9.9 ± 1.4
1.1 ± 0.2 40.8 ± 8.9 55.1 ± 10.0 34.5 ± 6.9 11.6 ± 3.0
Proportion of each drug's D50 value (cocaine:U-50,488H).
P n.s. <0.05 <0.05 <0.05 n.s.
BR A IN RE S E A RCH 1 1 14 ( 20 0 6 ) 3 1 –3 5
2.2.
U-50,488H
Consistent with previous reports (Raffa et al., 2003), U-50,488Hnaïve planarians displayed a characteristically nearly constant locomotor velocity of approximately 15–16 gridlines/min when tested in drug-free water and planarians that were exposed for 1h to U-50,488H then tested in water containing the same concentration of U-50,488H displayed pLMV which was not significantly different from cocaine-naïve planarians tested in drug-free water. However, planarians exposed to U-50,488H for 1h then placed into drug-free water displayed a significantly reduced pLMV. The pLMV remained constant over the 5-min observation period, even during withdrawal from the highest concentration of U-50,488H, suggesting a physiologic, not toxic, effect of U-50,488H on the planarians. The reduction in pLMV was dose-related to prior exposure to 10− 7–10mg/ml U-50,488H (Fig. 2). The D50 value for U-50,488H (MW = 465.43) was 1.02 μg.
2.3.
Cocaine/U-50,488H combinations
Planarians that were exposed to combinations of cocaine plus U-50,488H for 1 h and then placed into drug-free water displayed dose-related reduction in pLMV. The D50 value for each cocaine:U-50,488H combination is given in Table 1 and graphed as an isobologram (Fig. 3). The 19:1 and 1:19 cocaine: U-50,488H ratios yielded D50 values that were not significantly different (p > 0.05) from the expected additive values (11.6 ± 3.0 vs. 9.9 ± 1.4 and 1.1 ± 0.2 vs. 1.5 ± 0.1, respectively), indicating that there was no interaction between the two compounds at these ratios. However, the 3:1, 1:1, and 1:3 cocaine:U-50,488H
Fig. 3 – Isobologram of D50 values of cocaine and U-50,488H. All points on the line are dose pairs (additive isoboles) that give the half-maximal effect if joint action is in accord with their individual potencies. The thick diagonal line is the locus of theoretical points representing doses which in combination are additive. The actual combination ratios of 3:1, 1:1, and 1:3 produced subadditive withdrawal.
33
ratios yielded D50 values that were each significantly different (p > 0.05) from the expected additive values for these ratios (34.5 ± 6.9 vs. 7.7 ± 1.1; 55.1 ± 10.0 vs. 5.7 ± 0.7; and 40.8 ± 8.9 vs. 3.3 ± 0.4, respectively). These results indicate that there is an interaction between the two compounds on withdrawal at these ratios and that the withdrawal from these combinations is less than the expected additive amount.
3.
Discussion
Many drug abusers engage in ‘polydrug’ abuse, the co-incident use of more than one controlled substance. Of particular interest is the selection of specific combinations of substances. It is assumed that these combinations are preferred because they maximize the euphoric effect. An alternative, or additional, view is that the combination might result in less intense withdrawal during periods of abstinence. The model selected for this study involves the use of Planaria. While mammals would clearly be more clinically relevant with regard to understanding the complexities of human drug abuse, Planaria provide an advantageous model for examining questions about drug interactions. Because planarians absorb low molecular weight chemicals from their surroundings, their exposure to drugs can be expressed as a concentration, rather than a dose (a factor desirable for the mathematical analysis of drug combinations) and there are fewer sites for possible confounding pharmacokinetic drug– drug interactions. The metric used in the present study has previously been reported to demonstrate a withdrawal phenomenon in planarians (e.g., Raffa and Valdez, 2001; Raffa et al., 2001, 2003). The observed withdrawal signs (Raffa and Desai, 2005) are reversible, not due to changes in the temperature, pH, or osmolarity of the solutions, and in the case of U-50,488H are enantiomer-selective and antagonized by naloxone and the more κ-opioid receptor antagonist norBNI (nor-Binaltorphimine) (Raffa et al., 2003). ‘Joint-action’ analysis is a mathematical method used to quantify interactions between drugs. Bliss (1939) and Finney (1942) were among the first to study interactions between two drugs/toxins used in combination. Analysis of dose–response relations for each separate agent and for the combination provides information to help determine the expected response and, thereby, the nature of any interaction. The mathematical basis of joint-action analysis, development of appropriate statistical evaluations, and applications to pharmacologic endpoints have been extensively published by Tallarida (Tallarida, 1992, 2000; Tallarida et al., 1997a,b, 1999). In the present study, cocaine:U-50,488H ratios of 19:1 or 1:19 demonstrated no deviation from additivity, that is, the withdrawal was not significantly different from the expected additive effect of the two drugs. In contrast, the 3:1, 1:1, and 1:3 ratios displayed subadditive interaction at these ratios. Subadditivity in this model suggests that abstinence-induced withdrawal is less intense than predicted from the individual drug potencies. Therefore, these findings suggest that coexposure to cocaine and a κ-opioid attenuates development of physical dependence and subsequent abstinence-induced withdrawal.
34
BR A IN RE S EA RCH 1 1 14 ( 20 0 6 ) 3 1 –35
The mechanism of the observed subadditive interaction between cocaine and U-50,488H for abstinence-induced withdrawal cannot be determined from the present study. However, others have reported opioid–dopamine interaction on at least one other endpoint in Planaria (Passarelli et al., 1999) and a cocaine/opioid connection for physical dependence is supported by our previous finding that cocaine withdrawal is attenuated by co-exposure to the opioid antagonist naloxone and by more receptor subtype-selective (μ, δ, and κ) opioid antagonists (manuscript in preparation). Humans are unlikely to abuse combinations of cocaine plus a κ-opioid agonist. However, it will be important to ask if the concept suggested by our findings – i.e., that certain combinations of drugs leads to attenuated withdrawal – generalizes to humans.
4.
Experimental procedures
4.1.
Materials
Planarians (Dugesia dorotocephala) were purchased from Carolina Biological Supply Co. (Burlington, NC), maintained and acclimated to temperature-controlled (21 °C) room conditions in the jars in which they were shipped, and tested within 72 h (average weight about 2 mg). Each planarian was used only once. Cocaine and U-50,488H were purchased from Sigma Chemical Co. (St. Louis, MO).
4.2.
Behavioral measurement
To measure pLMV, planarians were placed individually into a clear plastic petri dish (14 cm diameter) containing roomtemperature tap water (treated with AmQuel® water conditioner). The dish was located over graph paper composed of gridlines spaced 0.5 cm apart. pLMV was quantified by an unbiased observer as the mean ± SD number of gridlines planarians crossed or re-crossed each minute over a 5-min observation period. Prior to measurement of pLMV, each planarian was placed into individual 0.5 ml vials containing room-temperature water or test compound(s) for 1 h. Each planarian was exposed individually for 1 h to one of the following treatments: water, U-50,488H, cocaine, or fixed-ratio mixtures (19:1, 3:1, 1:1, 1:3, 1:19) of cocaine and U-50,488H.
4.3.
Joint-action analysis
Bliss (1939) and Finney (1942) were among the first to study interactions between two drugs/toxins used in combination. The analysis has been extended by Tallarida (Tallarida, 1992, 2000; Tallarida et al., 1997a,b, 1999). The cocaine/U-50,488H combination meets a requirement of joint-action analysis for similar and independent action, since abstinence-induced withdrawal is observed for each tested individually. For a constant potency ratio R the quantity of drug B equivalent to a dose a of drug A is a / R. Hence, combination (a, b) is equivalent to a / R + b of drug B. Therefore, the a,b combination equivalent to Bx (effect level x) is such that b + a / R = Bx, or a / Ax + b / Bx = 1, which is a straight line with intercepts Ax and Bx, called the isobole of additivity. The dose
pair of the combination that experimentally produces the same effect (e.g., 50% of maximum) may plot as a point that is located either below (superadditivity) or above (subadditivity) the line of additivity. The graph containing the additive line and the plotted experimental points is an ‘isobologram’. A non-additive joint action is demonstrated when the difference between the actual effect produced by a fixed-ratio combination is significantly different from the predicted additive effect (see Tallarida, 2000).
Acknowledgments The authors thank Timothy Shickley, Ph.D. for the suggestion of Planaria as a test model. This work was supported by Grant R01-DA15378 from the NIH, NIDA (to R.B.R.) and by Grant R01DA09793 from the NIH, NIDA (to R.J.T.).
REFERENCES
Ahmed, S.H., Koob, G.F., 1998. Transition from moderate to excessive drug intake: change in hedonic set point. Science 282, 298–300. Algeri, S., Carolei, A., Ferretti, P., Gallone, C., Palladini, G., Venturini, G., 1983. Effects of dopaminergic agents on monoamine levels and motor behaviour in Planaria. Comp. Biochem. Physiol. 74C, 27–29. Bliss, C.I., 1939. The toxicity of poisons applied jointly. Ann. Appl. Biol. 26, 585–615. Carolei, A., Margotta, V., Palladini, G., 1975. Proposal of a new model with dopaminergic–cholinergic interactions for neuropharmacological investigations. Neuropsychobiology 1, 355–364. Collins, S.L., D'Addario, C., Izenwasser, S., 2001. Effects of kappa-opioid receptor agonists on long-term cocaine use and dopamine neurotransmission. Eur. J. Pharmacol. 426, 25–34. Filip, M., Nowak, E., Papla, I., Przegaliski, E., 2001. Role of 5-hydroxytryptamine1B receptors and 5-hydroxytryptamine uptake inhibition in the cocaine-evoked discriminative stimulus effects in rats. J. Physiol. Pharmacol. 52, 249–263. Finney, D.J., 1942. The analysis of toxicity tests on mixtures of poisons. Ann. Appl. Biol. 29, 82–94. Koob, G.F., Le Moal, M., 1997. Drug abuse: hedonic homeostatic dysregulation. Science 278, 52–58. Palladini, G., Ruggieri, S., Stocchi, F., De Pandis, M.F., Venturini, G., Margotta, V., 1996. A pharmacological study of cocaine activity in planaria. Comp. Biochem. Physiol., C 115, 41–45. Passarelli, F., Merante, A., Pontieri, F.E., Margotta, V., Venturini, G., Palladini, G., 1999. Opioid–dopamine interaction in planaria: a behavioral study. Comp. Biochem. Physiol., C 124, 51–55. Raffa, R.B., Desai, P., 2005. Description and quantification of cocaine withdrawal signs in Planaria. Brain Res. 1032, 200–202. Raffa, R.B., Valdez, J.M., 2001. Cocaine withdrawal in Planaria. Eur. J. Pharmacol. 430, 143–145. Raffa, R.B., Holland, L.J., Schulingkamp, R.J., 2001. Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint. J. Pharmacol. Toxicol. Methods 45, 223–226. Raffa, R.B., Stagliano, G.W., Umeda, S., 2003. κ-Opioid withdrawal in Planaria. Neurosci. Lett. 349, 139–142. Sasaki-Adams, D.M., Kelley, A.E., 2001. Serotonin–dopamine interactions in the control of conditioned reinforcement and motor behavior. Neuropsychopharmacology 25, 440–452.
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Self, D.W., Nestler, E.J., 1998. Relapse to drug-seeking: neural and molecular mechanisms. Drug Alcohol Depend. 51, 49–60. Sharpe, L.G., Pilotte, N.S., Shippenberg, T.S., Goodman, C.B., London, E.D., 2000. Autoradiographic evidence that prolonged withdrawal from intermittent cocaine reduces mu-opioid receptor expression in limbic regions of the rat brain. Synapse 37, 292–297. Tallarida, R.J., 1992. Statistical analysis of drug combinations for synergism. Pain 49, 93–97. Tallarida, R.J., 2000. Drug Synergism and Dose–Effect Data. Chapman and Hall/CRC Press, Boca Raton, FL. Tallarida, R.J., Stone Jr., D.J., Raffa, R.B., 1997a. Efficient designs for studying synergistic drug combinations. Life Sci. 61, PL417–PL425. Tallarida, R.J., Kimmel, H.L., Holtzman, S.G., 1997b. Theory and statistics of detecting synergism between two active drugs: cocaine and buprenorphine. Psychopharmacology 133, 378–382. Tallarida, R.J., Stone Jr., D.J., McCary, J.D., Raffa, R.B., 1999. A response surface analysis of synergism between morphine and clonidine. J. Pharmacol. Exp. Ther. 289, 8–13. Turchan, J., Przewlocka, B., Lason, W., Przewlocki, R., 1998. Effects of repeated psychostimulant administration on the prodynorphin system activity and kappa opioid receptor density in the rat brain. Neuroscience 85, 1051–1059. Umeda, S., Stagliano, G.W., Raffa, R.B., 2004. Cocaine and κ-opioid
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withdrawal in Planaria blocked by D-, but not L-glucose. Brain Res. 1018, 181–185. Venturini, G., Carolei, A., Palladini, G., Margotta, V., Lauro, M.G., 1983. Radioimmunological and immunocytochemical demonstration of Met-enkephalin in planaria. Comp. Biochem. Physiol. 74, 23–25. Venturini, G., Stocchi, F., Margotta, V., Ruggieri, S., Bravi, D., Bellantuono, P., Palladini, G., 1989. A pharmacological study of dopaminergic receptor in Planaria. Neuropharmacology 28, 1377–1382. Wang, N.S., Brown, V.L., Grabowski, J., Meisch, R.A., 2001. Reinforcement by orally delivered methadone, cocaine, and methadone–cocaine combinations in rhesus monkeys: are the combinations better reinforcers? Psychopharmacology 156, 63–72. Welsh, J.H., Williams, L.D., 1970. Monoamine containing neurons in Planaria. J. Comp. Neurol. 138, 103–116. Woolfolk, D.R., Holtzman, S.G., 1996. The effects of opioid receptor antagonism on the discriminative stimulus effects of cocaine and D-amphetamine in the rat. Behav. Pharmacol. 7, 779–787. Yuferov, V., Zhou, Y., LaForge, K.S., Spangler, R., Ho, A., Kreek, M.J., 2001. Elevation of guinea pig brain preprodynorphin mRNA expression and hypothalamic–pituitary–adrenal axis activity by “binge” pattern cocaine administration. Brain Res. Bull. 55, 65–70.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Subadditive withdrawal from cocaine/κ-opioid agonist combinations in Planaria Robert B. Raffa⁎, Gregory W. Stagliano, Ronald J. Tallarida Department of Pharmaceutical Sciences, Temple University School of Pharmacy (RBR, GWS) and Department of Pharmacology, Temple University Medical School (RJT), Philadelphia, PA 19140, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
We have previously developed and extensively characterized a convenient and sensitive
Accepted 13 July 2006
metric for the quantification of withdrawal responses using Planaria. Planaria are
Available online 17 August 2006
particularly valuable for these studies because of their permeable exteriors and their relevant neurotransmitter systems (e.g., dopaminergic, opioid, and serotonergic). In the
Keywords:
present study, we used this metric and mathematically rigorous joint-action analysis to
Withdrawal
investigate poly-drug withdrawal from fixed-ratio cocaine/κ-opioid agonist combinations.
Cocaine
The D50 (concentration producing half-maximal effect) for cocaine and U-50,488H was 10.3
κ-opioid
and 1.02 μg, respectively. The D50 for 19:1 or 1:19 combinations did not differ significantly
U-50,488H
(p > 0.05) from expected additive values (11.6 ± 3.0 vs. 9.9 ± 1.4 and 1.1 ± 0.2 vs. 1.5 ± 0.1,
Planaria
respectively), but the 3:1, 1:1, and 1:3 ratios did (34.5 ± 6.9 vs. 7.7 ± 1.1; 55.1 ± 10.0 vs. 5.7 ± 0.7; and 40.8 ± 8.9 vs. 3.3 ± 0.4, respectively), indicating subadditive interaction at these ratios. The finding of subadditivity in this model suggests that abstinence-induced withdrawal from the combination is less intense than that predicted from the individual drug potencies. The concept that certain combinations of drugs leads to attenuated withdrawal might generalize to humans. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Planarians have a centralized nervous system (cephalic ganglia and spinal processes) and mammalian-like neurotransmitter systems. They respond to dopaminergic agonists, antagonists, or neuronal uptake inhibitors (e.g., Welsh and Williams, 1970; Carolei et al., 1975; Algeri et al., 1983; Venturini et al., 1989; Palladini et al., 1996). Changes in 2nd-messenger levels induced by dopamine agonists (Palladini et al., 1996) suggest that the effects are mediated via dopamine receptors. The presence of an enkephalinergic system in Planaria was identified by means of radioimmunological and immunocytochemical techniques (Venturini et al., 1983) and a dopamine-opioid behavioral interaction has been described
(Passarelli et al., 1999). In a previous work, we developed a behavioral model that quantifies physical dependence and withdrawal in Planaria (Raffa and Valdez, 2001; Raffa et al., 2001, 2003). In brief, following exposure to drug, planarians display a dose-related decrease in spontaneous locomotor velocity (pLMV) when placed into drug-free water, but not drug-containing, water (i.e., abstinence-induced withdrawal) and receptor-selective antagonists decrease pLMV of drugexperienced planarians in a dose-related and enantiomericspecific manner (i.e., precipitated withdrawal) (Raffa et al., 2001). Specific withdrawal signs are noted at higher doses (Raffa and Desai, 2005). The phenomenon is not due to adverse effects of the drugs on locomotor activity or to changes in pH, temperature, or osmolarity (Umeda et al., 2004).
⁎ Corresponding author. Fax: +1 215 707 5228. E-mail address: [email protected] (R.B. Raffa). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.07.037
32
BR A IN RE S EA RCH 1 1 14 ( 20 0 6 ) 3 1 –35
The individual mechanisms of action of cocaine and opioids are relatively well known, involving modulation of biochemical processes within the central nervous system that can lead to abuse, craving, and relapse (e.g., Koob and Le Moal, 1997; Self and Nestler, 1998; Ahmed and Koob, 1998). However, little is known about possible interactions between these drugs. In the few studies that have been conducted on combinations, interactions have been reported between dopamine and opioid (rats) (e.g., Turchan et al., 1998; Woolfolk and Holtzman, 1996; Collins et al., 2001), dopamine and serotonin (rats) (e.g., Sasaki-Adams and Kelley, 2001; Filip et al., 2001), and cocaine with opioid (rats and rhesus monkeys) (Sharpe et al., 2000; Yuferov et al., 2001; Wang et al., 2001). We report a quantitative assessment of planarian withdrawal from fixed-ratio combinations of cocaine and the κopioid agonist U-50,488H (trans-(±)-3,4-Dichloro-N-methyl-N(2-[1-pyrrolidinyl]cyclohexyl)-benzeneacetamide). We used U-50,488H instead of morphine or heroin because planarians are more sensitive to this opioid (Passarelli et al., 1999). We applied ‘joint-action’ analysis to characterize the nature of the interaction between the two compounds (Tallarida, 2000).
2.
Results
2.1.
Cocaine
Consistent with previous reports (Raffa et al., 2001), cocainenaïve planarians displayed a characteristically nearly con-
Fig. 2 – Withdrawal (expressed as the 5-min cumulative mean ± SD decreased pLMV) when planarians (N = 10 each point) are placed into water following exposure to the doses indicated of cocaine alone or the opioid U-50,488H alone.
stant locomotor velocity (pLMV) of approximately 15–16 gridlines/min when tested in cocaine-free water and planarians that were exposed for 1 h to cocaine then tested in water containing the same concentration of cocaine displayed pLMV which was not significantly different from cocaine-naïve planarians tested in cocaine-free water. Hence, exposure to cocaine did not alter pLMV under these conditions. However, planarians exposed to cocaine for 1 h then placed into cocaine-free water displayed a significantly reduced pLMV. Notably, the pLMV remained constant over the 5-min observation period, even during withdrawal from the highest concentration of cocaine, suggesting a physiologic, not toxic, or local anesthetic, effect of cocaine on the planarians. The reduction in pLMV was dose-related to prior exposure to 10− 7– 10mg/ml (10− 8–10− 3 M) cocaine (Fig. 1). The D50 value (dose producing half-maximal effect) for cocaine (MW = 339.81) was 10.3 μg. The duration of withdrawal signs was previously shown to persist greater than 1 h (Raffa and Desai, 2005) followed by recovery of the animals.
Table 1 – The predicted additive and experimentally determined mean D50 ± SD (μg/ml) for each fixed-dose ratio of cocaine:U-50,488H combinations Ratio a Additive (total) D50 Experimental (total) D50 Fig. 1 – Solid lines: pLMV (expressed as the mean ± SD of the cumulative number of gridlines crossed per minute) of planarians exposed to cocaine (concentration in M indicated) for 1 h and then tested in cocaine-free water. Dotted line: cocaine-naïve animals tested in water. Dashed line: cocaine-exposed animals tested in the same concentration (1 × 10− 5 M) of cocaine. N = 10 for all groups.
1:19 1:3 1:1 3:1 19:1 a
1.5 ± 0.2 3.3 ± 0.4 5.7 ± 0.8 7.7 ± 1.1 9.9 ± 1.4
1.1 ± 0.2 40.8 ± 8.9 55.1 ± 10.0 34.5 ± 6.9 11.6 ± 3.0
Proportion of each drug's D50 value (cocaine:U-50,488H).
P n.s. <0.05 <0.05 <0.05 n.s.
BR A IN RE S E A RCH 1 1 14 ( 20 0 6 ) 3 1 –3 5
2.2.
U-50,488H
Consistent with previous reports (Raffa et al., 2003), U-50,488Hnaïve planarians displayed a characteristically nearly constant locomotor velocity of approximately 15–16 gridlines/min when tested in drug-free water and planarians that were exposed for 1h to U-50,488H then tested in water containing the same concentration of U-50,488H displayed pLMV which was not significantly different from cocaine-naïve planarians tested in drug-free water. However, planarians exposed to U-50,488H for 1h then placed into drug-free water displayed a significantly reduced pLMV. The pLMV remained constant over the 5-min observation period, even during withdrawal from the highest concentration of U-50,488H, suggesting a physiologic, not toxic, effect of U-50,488H on the planarians. The reduction in pLMV was dose-related to prior exposure to 10− 7–10mg/ml U-50,488H (Fig. 2). The D50 value for U-50,488H (MW = 465.43) was 1.02 μg.
2.3.
Cocaine/U-50,488H combinations
Planarians that were exposed to combinations of cocaine plus U-50,488H for 1 h and then placed into drug-free water displayed dose-related reduction in pLMV. The D50 value for each cocaine:U-50,488H combination is given in Table 1 and graphed as an isobologram (Fig. 3). The 19:1 and 1:19 cocaine: U-50,488H ratios yielded D50 values that were not significantly different (p > 0.05) from the expected additive values (11.6 ± 3.0 vs. 9.9 ± 1.4 and 1.1 ± 0.2 vs. 1.5 ± 0.1, respectively), indicating that there was no interaction between the two compounds at these ratios. However, the 3:1, 1:1, and 1:3 cocaine:U-50,488H
Fig. 3 – Isobologram of D50 values of cocaine and U-50,488H. All points on the line are dose pairs (additive isoboles) that give the half-maximal effect if joint action is in accord with their individual potencies. The thick diagonal line is the locus of theoretical points representing doses which in combination are additive. The actual combination ratios of 3:1, 1:1, and 1:3 produced subadditive withdrawal.
33
ratios yielded D50 values that were each significantly different (p > 0.05) from the expected additive values for these ratios (34.5 ± 6.9 vs. 7.7 ± 1.1; 55.1 ± 10.0 vs. 5.7 ± 0.7; and 40.8 ± 8.9 vs. 3.3 ± 0.4, respectively). These results indicate that there is an interaction between the two compounds on withdrawal at these ratios and that the withdrawal from these combinations is less than the expected additive amount.
3.
Discussion
Many drug abusers engage in ‘polydrug’ abuse, the co-incident use of more than one controlled substance. Of particular interest is the selection of specific combinations of substances. It is assumed that these combinations are preferred because they maximize the euphoric effect. An alternative, or additional, view is that the combination might result in less intense withdrawal during periods of abstinence. The model selected for this study involves the use of Planaria. While mammals would clearly be more clinically relevant with regard to understanding the complexities of human drug abuse, Planaria provide an advantageous model for examining questions about drug interactions. Because planarians absorb low molecular weight chemicals from their surroundings, their exposure to drugs can be expressed as a concentration, rather than a dose (a factor desirable for the mathematical analysis of drug combinations) and there are fewer sites for possible confounding pharmacokinetic drug– drug interactions. The metric used in the present study has previously been reported to demonstrate a withdrawal phenomenon in planarians (e.g., Raffa and Valdez, 2001; Raffa et al., 2001, 2003). The observed withdrawal signs (Raffa and Desai, 2005) are reversible, not due to changes in the temperature, pH, or osmolarity of the solutions, and in the case of U-50,488H are enantiomer-selective and antagonized by naloxone and the more κ-opioid receptor antagonist norBNI (nor-Binaltorphimine) (Raffa et al., 2003). ‘Joint-action’ analysis is a mathematical method used to quantify interactions between drugs. Bliss (1939) and Finney (1942) were among the first to study interactions between two drugs/toxins used in combination. Analysis of dose–response relations for each separate agent and for the combination provides information to help determine the expected response and, thereby, the nature of any interaction. The mathematical basis of joint-action analysis, development of appropriate statistical evaluations, and applications to pharmacologic endpoints have been extensively published by Tallarida (Tallarida, 1992, 2000; Tallarida et al., 1997a,b, 1999). In the present study, cocaine:U-50,488H ratios of 19:1 or 1:19 demonstrated no deviation from additivity, that is, the withdrawal was not significantly different from the expected additive effect of the two drugs. In contrast, the 3:1, 1:1, and 1:3 ratios displayed subadditive interaction at these ratios. Subadditivity in this model suggests that abstinence-induced withdrawal is less intense than predicted from the individual drug potencies. Therefore, these findings suggest that coexposure to cocaine and a κ-opioid attenuates development of physical dependence and subsequent abstinence-induced withdrawal.
34
BR A IN RE S EA RCH 1 1 14 ( 20 0 6 ) 3 1 –35
The mechanism of the observed subadditive interaction between cocaine and U-50,488H for abstinence-induced withdrawal cannot be determined from the present study. However, others have reported opioid–dopamine interaction on at least one other endpoint in Planaria (Passarelli et al., 1999) and a cocaine/opioid connection for physical dependence is supported by our previous finding that cocaine withdrawal is attenuated by co-exposure to the opioid antagonist naloxone and by more receptor subtype-selective (μ, δ, and κ) opioid antagonists (manuscript in preparation). Humans are unlikely to abuse combinations of cocaine plus a κ-opioid agonist. However, it will be important to ask if the concept suggested by our findings – i.e., that certain combinations of drugs leads to attenuated withdrawal – generalizes to humans.
4.
Experimental procedures
4.1.
Materials
Planarians (Dugesia dorotocephala) were purchased from Carolina Biological Supply Co. (Burlington, NC), maintained and acclimated to temperature-controlled (21 °C) room conditions in the jars in which they were shipped, and tested within 72 h (average weight about 2 mg). Each planarian was used only once. Cocaine and U-50,488H were purchased from Sigma Chemical Co. (St. Louis, MO).
4.2.
Behavioral measurement
To measure pLMV, planarians were placed individually into a clear plastic petri dish (14 cm diameter) containing roomtemperature tap water (treated with AmQuel® water conditioner). The dish was located over graph paper composed of gridlines spaced 0.5 cm apart. pLMV was quantified by an unbiased observer as the mean ± SD number of gridlines planarians crossed or re-crossed each minute over a 5-min observation period. Prior to measurement of pLMV, each planarian was placed into individual 0.5 ml vials containing room-temperature water or test compound(s) for 1 h. Each planarian was exposed individually for 1 h to one of the following treatments: water, U-50,488H, cocaine, or fixed-ratio mixtures (19:1, 3:1, 1:1, 1:3, 1:19) of cocaine and U-50,488H.
4.3.
Joint-action analysis
Bliss (1939) and Finney (1942) were among the first to study interactions between two drugs/toxins used in combination. The analysis has been extended by Tallarida (Tallarida, 1992, 2000; Tallarida et al., 1997a,b, 1999). The cocaine/U-50,488H combination meets a requirement of joint-action analysis for similar and independent action, since abstinence-induced withdrawal is observed for each tested individually. For a constant potency ratio R the quantity of drug B equivalent to a dose a of drug A is a / R. Hence, combination (a, b) is equivalent to a / R + b of drug B. Therefore, the a,b combination equivalent to Bx (effect level x) is such that b + a / R = Bx, or a / Ax + b / Bx = 1, which is a straight line with intercepts Ax and Bx, called the isobole of additivity. The dose
pair of the combination that experimentally produces the same effect (e.g., 50% of maximum) may plot as a point that is located either below (superadditivity) or above (subadditivity) the line of additivity. The graph containing the additive line and the plotted experimental points is an ‘isobologram’. A non-additive joint action is demonstrated when the difference between the actual effect produced by a fixed-ratio combination is significantly different from the predicted additive effect (see Tallarida, 2000).
Acknowledgments The authors thank Timothy Shickley, Ph.D. for the suggestion of Planaria as a test model. This work was supported by Grant R01-DA15378 from the NIH, NIDA (to R.B.R.) and by Grant R01DA09793 from the NIH, NIDA (to R.J.T.).
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