Distinct interactions of cannabidiol and morphine in three nociceptive behavioral models in mice.

Cannabinoid and opioid agonists can display overlapping behavioral effects and the combination of these agonists is known to produce enhanced antinociception in several rodent models of acute and chronic pain. The present study investigated the antinociceptive effects of the nonpsychoactive cannabinoid, cannabidiol (CBD) and the µ-opioid agonist morphine, both alone and in combination, using three behavioral models in mice, to test the hypothesis that combinations of morphine and CBD would produce synergistic effects. The effects of morphine, CBD, and morphine/CBD combinations were assessed in the following assays: (a) acetic acid-stimulated stretching; (b) acetic acid-decreased operant responding for palatable food; and (c) hot plate thermal nociception. Morphine alone produced antinociceptive effects in all three models of acute nociception, whereas CBD alone produced antinociception only in the acetic acid-stimulated stretching assay. The nature of the interactions between morphine and CBD combinations were assessed quantitatively based on the principle of dose equivalence. Combinations of CBD and morphine produced synergistic effects in reversing acetic acid-stimulated stretching behavior, but subadditive effects in the hot plate thermal nociceptive assay and the acetic acid-decreased operant responding for palatable food assay. These results suggest that distinct mechanisms of action underlie the interactions between CBD and morphine in the three different behavioral assays and that the choice of appropriate combination therapies for the treatment of acute pain conditions may depend on the underlying pain type and stimulus modality.

Spinal-supraspinal and intrinsic µ-opioid receptor agonist-norepinephrine reuptake inhibitor (MOR-NRI) synergy of tapentadol in diabetic heat hyperalgesia in mice.

Tapentadol is a µ-opioid receptor (MOR) agonist and norepinephrine reuptake inhibitor (NRI) with established efficacy in neuropathic pain in patients and intrinsic synergistic interaction of both mechanisms as demonstrated in rodents. In diabetic mice, we analyzed the central antihyperalgesic activity, the occurrence of site-site interaction, as well as the spinal contribution of opioid and noradrenergic mechanisms in a hotplate test. Tapentadol (0.1-3.16 µg/animal) showed full efficacy after intrathecal as well as after intracerebroventricular administration (ED50 0.42 µg/animal i.t., 0.18 µg/animal i.c.v.). Combined administration of equianalgesic doses revealed spinal-supraspinal synergy (ED50 0.053 µg/animal i.t. + i.c.v.). Morphine (0.001-10 µg/animal) also showed central efficacy and synergy (ED50 0.547 µg/animal i.t., 0.004 µg/animal i.c.v., 0.014 µg/animal i.t. + i.c.v.). Supraspinal potencies of tapentadol and morphine correlated with the 50-fold difference in their MOR affinities. In contrast, spinal potencies of both drugs were similar and correlated with their relative systemic potencies (ED50 0.27 mg/kg i.p. tapentadol, 1.1 mg/kg i.p. morphine). Spinal administration of the opioid antagonist naloxone or the α2-adrenoceptor antagonist yohimbine before systemic administration of equianalgesic doses of tapentadol (1 mg/kg i.p.) or morphine (3.16 mg/kg i.p.) revealed pronounced influence on opioidergic and noradrenergic pathways for both compounds. Tapentadol was more sensitive toward both antagonists than was morphine, with median effective dose values of 0.75 and 1.72 ng/animal i.t. naloxone and 1.56 and 2.04 ng/animal i.t. yohimbine, respectively. It is suggested that the antihyperalgesic action of systemically administered tapentadol is based on opioid spinal-supraspinal synergy, as well as intrinsic spinally mediated MOR-NRI synergy.

Revisiting the isobole and related quantitative methods for assessing drug synergism.

The isobole is well established and commonly used in the quantitative study of agonist drug combinations. This article reviews the isobole, its derivation from the concept of dose equivalence, and its usefulness in providing the predicted effect of an agonist drug combination, a topic not discussed in pharmacology textbooks. This review addresses that topic and also shows that an alternate method, called "Bliss independence," is inconsistent with the isobolar approach and also has a less clear conceptual basis. In its simplest application the isobole is the familiar linear plot in cartesian coordinates with intercepts representing the individual drug potencies. It is also shown that the isobole can be nonlinear, a fact recognized by its founder (Loewe) but neglected or rejected by virtually all other users. Whether its shape is linear or nonlinear the isobole is equally useful in detecting synergism and antagonism for drug combinations, and its theoretical basis leads to calculations of the expected effect of a drug combination. Numerous applications of isoboles in preclinical testing have shown that synergism or antagonism is not only a property of the two agonist drugs; the dose ratio is also important, a fact of potential importance to the design and testing of drug combinations in clinical trials.

'Null method' determination of drug biophase concentration.

PK/PD modeling is enhanced by improvements in the accuracy of its metrics. For PK/PD modeling of drugs and biologics that interact with enzymes or receptors, the equilibrium constant of the interaction can provide critical insight. Methodologies such as radioliogand binding and isolated tissue preparations can provide estimates of the equilibrium constants (as the dissociation constant, K value) for drugs and endogenous ligands that interact with specific enzymes and receptors. However, an impediment to further precision for PK/PD modeling is that it remains a problem to convert the concentration of drug in bulk solution (A) into an estimate of receptor occupation, since A is not necessarily the concentration (C) of drug in the biophase that yields fractional binding from the law of mass action, viz., C/(C + K). In most experimental studies A is much larger than K, so the use of administered instead of biophase concentration gives fractional occupancies very close to unity. We here provide a simple way to obtain an estimate of the factor that converts the total drug concentration into the biophase concentration in isolated tissue preparation. Our approach is an extension of the now classic 'null method' introduced and applied by Furchgott to determination of drug-receptor dissociation constants.

Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Comparison of human and porcine gastric clasp and sling fiber contraction by M2 and M3 muscarinic receptors.

To compare the gastroesophageal junction of the human with the pig, M(2) and M(3) receptor densities and the potencies of M(2) and M(3) muscarinic receptor subtype selective antagonists were determined in gastric clasp and sling smooth muscle fibers. Total muscarinic and M(2) receptors are higher in pig than human clasp and sling fibers. M(3) receptors are higher in human compared with pig sling fibers but lower in human compared with pig clasp fibers. Clasp fibers have fewer M(3) receptors than sling fibers in both humans and pigs. Similar to human clasp fibers, pig clasp fibers contract significantly less than pig sling fibers. Analysis of the methoctramine Schild plot suggests that M(2) receptors are involved in mediating contraction in pig clasp and sling fibers. Darifenacin potency suggests that M(3) receptors mediate contraction in pig sling fibers and that M(2) and M(3) receptors mediate contraction in pig clasp fibers. Taken together, the data suggest that both M(2) and M(3) muscarinic receptors mediate the contraction in both pig clasp and sling fibers similar to human clasp and sling fibers.

Combination analysis.

This chapter describes quantitative methodology that is directed toward assessing interactions between a combination of agonist drugs that individually produce overtly similar effects. Drugs administered in combination may show exaggerated, reduced or predictable effects that are dependent on the specific drug pair and the doses of t h e constituents. The basisfor quantitating these unusual interactions is the concept of dose equivalence which, in turn, is determined from the individual drug dose-effect relations. A common analytical procedure that follows from dose equivalence uses a graph termed an isobologram. We present here an overview of the isobologram, its use and certain related methods that apply to classifying various drug interactions.

Future directions.

The chapters of this book summarize much of what has been done and reported regarding cancer chemotherapy-related cognitive impairment. In this chapter, we point out some future directions for investigation.

Midazolam enhances the analgesic properties of dexmedetomidine in the rat.

OBJECTIVE: To investigate the analgesic properties of different dose combinations of midazolam and dexmedetomidine administered intraperitoneally (IP) in the rat. STUDY DESIGN: Prospective experimental trial. ANIMALS: Seventy adult male Sprague Dawley rats weighing 250-300 g. METHODS: Dexmedetomidine (D) 0.03, 0.06, 0.09, 0.12, 0.15, 0.18, 0.21 mg kg(-1) and midazolam (M) 5, 10, 25, 50 mg kg(-1) were administered IP, alone then in combinations ranging from 0.03 D:5 M to 0.18 D:30 M mg kg(-1). Analgesia was evaluated using the tail-flick test at time 0 (before injection), 15, 30, 45, 60 and 75 minutes. RESULTS: Midazolam at all doses administered (5-50 mg kg(-1)) did not significantly change tail-flick latencies from baseline values whereas D showed clear dose-dependent increases in tail-flick latency for doses administered in the range of 0.03-0.18 mg kg(-1). Tail-flick latencies in rats administered D+M combinations were significantly greater than D alone (p<0.05). CONCLUSIONS: A dose-related analgesic effect was demonstrated for D in the rat, which was enhanced by co-administration of M. CLINICAL RELEVANCE: The combination of D+M administered IP to rats at doses of 0.12:20 and 0.09:15 mg kg(-1) was shown to be a good combination to provide sedation/analgesia with a duration of action greater than 60 minutes. The onset of sedation was rapid (1-3 minutes), and onset of profound analgesia was reached within 5-10 minutes.

Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle.

Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M(2) and M(3) muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M(2) receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M(3) density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M(2) and M(3) receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M(2) and M(3) receptors to contraction is presented. Using this new method of analysis, it was found that the M(2) muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M(3) receptor predominantly mediates contraction.

Combinations of cocaine with other dopamine uptake inhibitors: assessment of additivity.

Drugs that inhibit dopamine (DA) reuptake through actions at the dopamine transporter (DAT) have been proposed as candidates for development as pharmacotherapies for cocaine abuse. Accordingly, it is important to understand the potential pharmacological interactions of cocaine with other drugs acting at the DAT. Effects of combinations of cocaine with a cocaine analog, 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (WIN 35,428), were compared quantitatively with the combinations of cocaine with the N-butyl,4',4''-diF benztropine analog, 3-(bis(4-fluorophenyl)methoxy)-8-butyl-8-azabicyclo[3.2.1]octane (JHW 007), to determine whether their effects on DA levels in the shell of the nucleus accumbens (NAC) in mice differed. Each of the drugs alone produced dose-related elevations in NAC DA levels. In contrast to the other drugs, JHW 007 was less effective, producing maximal effects that approached 400% of control versus approximately 700% with the other drugs. In addition, the JHW 007 dose-effect curve was not as steep as those for cocaine and WIN 35,428. Combinations of cocaine with its analog, WIN 35,428, were most often greater than those predicted based on dose additivity. In contrast, combinations of cocaine with JHW 007 were most often subadditive. This outcome is consistent with recent studies suggesting that structurally divergent DA uptake inhibitors bind to different domains of the DAT, which can result in different DAT conformations. The conformational changes occurring with JHW 007 binding may result in functional outcomes that alter its abuse liability and its effects in combination with cocaine.

Opioid-sparing effects of cannabinoids on morphine analgesia: participation of CB1 and CB2 receptors.

BACKGROUND AND PURPOSE: Much of the opioid epidemic arose from abuse of prescription opioid drugs. This study sought to determine if the combination of a cannabinoid with an opioid could produce additive or synergistic effects on pain, allowing reduction in the opioid dose needed for maximal analgesia. EXPERIMENTAL APPROACH: Pain was assayed using the formalin test in mice and the carrageenan assay in rats. Morphine and two synthetic cannabinoids were tested: WIN55,212-2 (WIN), which binds to both CB1 and CB2 receptors, and possibly TRPV1 channels; and GP1a, which has activity at CB2 receptors and is reported to inhibit fatty acid amide hydrolase, thus raising levels of endogenous cannabinoids. KEY RESULTS: Morphine in combination with WIN in the formalin test gave synergistic analgesia. Studies with selective antagonists showed that WIN was acting through CB1 receptors. Morphine in combination with GP1a in the formalin test was sub-additive. In the carrageenan test, WIN had no added effect when combined with morphine, but GP1a with morphine showed enhanced analgesia. Both WIN and Gp1a used alone had analgesic activity in the formalin pain test, but not in the carrageenan pain test. CONCLUSIONS AND IMPLICATIONS: The ability of a cannabinoid to produce an additive or synergistic effect on analgesia when combined with morphine varies with the pain assay and may be mediated by CB1 or CB2 receptors. These results hold the promise of using cannabinoids to reduce the dose of opioids for analgesia in certain pain conditions.

Interactions between drugs and occupied receptors.

This review has 2 parts. Part I deals with isobolographic procedures that are traditionally applied to the joint action of agonists that individually produce overtly similar effects. Special attention is directed to newer computational procedures that apply to agonists with dissimilar concentration-effect curves. These newer procedures are consistent with the isobolographic methods introduced and used by Loewe, however, the present communications provides the needed graphical and mathematical detail. A major aim is distinguishing super and sub-addictive interactions from those that are simply additive. The detection and measurement of an interaction is an important step in exploring drug mechanism and is also important clinically. Part II discusses a new use of isoboles that is applicable to a single drug or chemical whose effect is mediated by 2 or more receptor subtypes. This application produces a metric that characterizes the interaction between the receptor subtypes. The expansion of traditional isobolographic theory to this multi-receptor situation follows from the newer approaches for 2-drug combination analysis in Part I. This topic leads naturally to a re-examination of competitive antagonism and the classic Schild plot. In particular, it is shown here that the Schild plot in the multi-receptor case is not necessarily linear with unit slope. Both parts of this review emphasize the quantitative aspects rather than the many drugs that have been analyzed with isobolographic methods. The mathematical exposition is rather elementary and is further aided by several graphs. An appendix is included for the reader interested in the mathematical details.

The use of occupation isoboles for analysis of a response mediated by two receptors: M2 and M3 muscarinic receptor subtype-induced mouse stomach contractions.

Smooth muscle contains multiple muscarinic receptor subtypes, including M2 and M3. M2 receptors outnumber M3 receptors. Based on the potency of subtype selective anticholinergics, contraction is mediated by the M3 subtype. However, results from knockout (KO) mice show that the M2 receptor mediates approximately 45% of the contractile response produced by the M3 receptor. The traditional theory of one receptor mediating a response does not allow assessment of interactions between receptors when more than one receptor participates in a response. Our study was performed using a novel analysis method based on dual receptor occupancy to determine how M2 and M3 receptor subtypes interact to mediate contraction in mouse stomach. Cumulative carbachol concentration contractile responses were determined for wild-type, M2-KO, and M3-KO stomach body smooth muscle. Using affinity constants for carbachol at M2 and M3 cholinergic receptors, the concentration values were converted to fractional receptor occupation. The resulting occupation-effect relations showed maximum effects for the M2 and M3 subtypes, respectively. These occupation-effect relations allow determination of the additive (expected) isobole based on this dual occupancy, thereby providing a curve (mathematically derived) for comparison against the experimentally derived value in wild type. The actual values determined experimentally in the wild type were not statistically significantly different from that predicted by the isobole. This confirms that the interaction between these mutually occupied receptors is additive. The new method of analysis also expands the traditional Schild theory that was based on a single receptor type to which the agonist and antagonist bind.

Effects of a Cannabinoid1 receptor antagonist and Serotonin2C receptor agonist alone and in combination on motivation for palatable food: a dose-addition analysis study in mice.

The cannabinoid and serotonin systems modulate feeding behavior in humans and laboratory animals. The present study assessed whether a cannabinoid (CB)(1) receptor antagonist and a serotonin (5-HT)(2C) receptor agonist alone and in combination attenuate motivation for the liquid nutritional drink Ensure as measured by a progressive ratio (PR) schedule of reinforcement in male C57BL/6 mice. Pretreatment (15 min i.p.) with either the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716) (SR; Rimonabant or Acomplia) or the 5-HT(2C) receptor agonist m-chlorophenylpiperazine (mCPP) dose-dependently decreased the maximum ratio completed under the PR schedule (break point) in mice. ED(25) values for SR and mCPP to decrease break point were determined, and the relative potency of each drug alone was quantified. Fixed dose-ratio pairs of SR/mCPP based on their relative potency were then administered. Dose-addition analysis comparing the experimentally determined potency for SR/mCPP combinations with their predicted additive potency revealed that SR/mCPP combinations in 1:1 and 2:1 ratios based on relative potency produced significant synergistic attenuation of break point for Ensure. The ED(25) values for decreasing break point were consistently lower than ED(25) values for decreasing response rate, and synergistic effects of SR/mCPP combinations on break point were seen independent of synergistic effects on response rate. These results indicate that cannabinoid CB(1) and serotonin 5-HT(2C) receptors are involved in motivated feeding behavior in mice and that these compounds can synergistically modulate motivation for palatable food with the synergy dependent upon the ratio of SR/mCPP in the combination.

A quantitative study to assess synergistic interactions between urotensin II and angiotensin II.

Interaction between the vasoactive peptides, urotensin II and angiotensin II, could have important implications in various disease states. We examined this interaction using isolated rat aortic rings with intact adventitia and endothelium. The fixed-ratio combination we tested produced effect levels significantly greater than predicted by additivity. Thus, the interaction was synergistic, and this is illustrated in a response surface plot that shows the predicted additive effect for all possible combinations.

Intra-VTA CART 55-102 reduces the locomotor effect of systemic cocaine in rats: an isobolographic analysis.

CART (cocaine- and amphetamine-regulated transcript) peptides appear to be mediators or modulators of psychostimulant drugs. An interesting result in the nucleus accumbens has been that injection of CART peptide has no effect by itself on locomotor activity, but it reduces the locomotor activity induced by cocaine or amphetamine. However, in the ventral tegmental area (VTA), injections of CART peptide have been shown to increase locomotor activity, although to a lesser degree [Kimmel, H.L., Gong, W., Vechia, S.D., Hunter, R.G., Kuhar, M.J., 2000. Intra-ventral tegmental area injection of rat cocaine and amphetamine-regulated transcript peptide 55-102 induces locomotor activity and promotes conditioned place preference. J. Pharmacol. Exp. Ther. 294, 784-792]. This study was carried out to clarify the interaction of intra-VTA CART 55-102 and systemic cocaine on locomotor activity. The CART-cocaine interaction has been examined using the rigorous isobolographic approach. This type of analysis permits an assessment of additivity, subadditivity, or synergism of two substances. By measuring locomotor activity and using a range of doses of CART peptide and cocaine, both alone and together, with different dosing strategies, clear evidence of subadditivity was found. CART reduced the locomotor activating effects of systemic cocaine, especially at higher doses of CART. These results imply that intra-VTA CART is not simply acting in the same manner as cocaine, and is likely to oppose the action of cocaine. This has implications for the physiological significance of CART-DA (dopamine) interactions and for medications development.

Nonlinear isobologram and superadditive withdrawal from cocaine: cannabinoid combinations in planarians.

Elucidation of interactions between drugs used in polydrug abuse is especially important. However, the necessary experimental conditions for precise quantitative analysis are difficult to establish. Because withdrawal effects of cocaine and the cannabinoid receptor agonist WIN 55212-2 are easily quantified in planarians, demonstration of synergistic effects (P<0.01) of certain ratios of this combination was possible. This synergy, here analyzed with the latest (nonlinear) isobolographic methodology, is now quantitatively established for the first time.

Single and combined effects of Δ9 -tetrahydrocannabinol and cannabidiol in a mouse model of chemotherapy-induced neuropathic pain.

BACKGROUND AND PURPOSE: The non-psychoactive phytocannabinoid cannabidiol (CBD) can affect the pharmacological effects of Δ9 -tetrahydrocannabinol (THC). We tested the possible synergy between CBD and THC in decreasing mechanical sensitivity in a mouse model of paclitaxel-induced neuropathic pain. We also tested the effects of CBD on oxaliplatin- and vincristine-induced mechanical sensitivity. EXPERIMENTAL APPROACH: Paclitaxel-treated mice (8.0 mg·kg-1 i.p., days 1, 3, 5 and 7) were pretreated with CBD (0.625-20.0 mg·kg-1 i.p.), THC (0.625-20.0 mg·kg-1 i.p.) or CBD + THC (0.04 + 0.04-20.0 + 20.0 mg·kg-1 i.p.), and mechanical sensitivity was assessed on days 9, 14 and 21. Oxaliplatin-treated (6.0 mg·kg-1 i.p., day 1) or vincristine-treated mice (0.1 mg·kg-1 i.p. days 1-7) were pretreated with CBD (1.25-10.0 mg·kg-1 i.p.), THC (10.0 mg·kg-1 i.p.) or THC + CBD (0.16 mg·kg-1 THC + 0.16 mg·kg-1 CBD i.p.). KEY RESULTS: Both CBD and THC alone attenuated mechanical allodynia in mice treated with paclitaxel. Very low ineffective doses of CBD and THC were synergistic when given in combination. CBD also attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity, while THC significantly attenuated vincristine- but not oxaliplatin-induced mechanical sensitivity. The low dose combination significantly attenuated oxaliplatin- but not vincristine-induced mechanical sensitivity. CONCLUSIONS AND IMPLICATIONS: CBD may be potent and effective at preventing the development of chemotherapy-induced peripheral neuropathy, and its clinical use may be enhanced by co-administration of low doses of THC. These treatment strategies would increase the therapeutic window of cannabis-based pharmacotherapies.

Subadditive withdrawal from cocaine/kappa-opioid agonist combinations in Planaria.

We have previously developed and extensively characterized a convenient and sensitive metric for the quantification of withdrawal responses using Planaria. Planaria are particularly valuable for these studies because of their permeable exteriors and their relevant neurotransmitter systems (e.g., dopaminergic, opioid, and serotonergic). In the present study, we used this metric and mathematically rigorous joint-action analysis to investigate poly-drug withdrawal from fixed-ratio cocaine/kappa-opioid agonist combinations. The D50 (concentration producing half-maximal effect) for cocaine and U-50,488H was 10.3 and 1.02 microg, respectively. The D50 for 19:1 or 1:19 combinations did not differ significantly (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, 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.