Dose-addition Analysis Research Articles

Antinociceptive effects of α2/α3‐selective GABAA receptor positive allosteric modulators on opioid‐induced hyperalgesia

Opioids have been increasingly prescribed to treat chronic pain since the 1980s, despite evidence that long‐term use of opioids may lead to tolerance and pain sensitization called opioid‐induced hyperalgesia (OIH). OIH has been demonstrated in both preclinical models and healthy human volunteers, but is understudied and there is need for novel analgesics capable of mitigating OIH. α2/α3‐selective GABAA receptor positive allosteric modulators (PAMs) act specifically at subunits of the GABAA receptor found to mediate analgesia, and have demonstrated antinociceptive effects in models of chronic inflammatory and neuropathic pain. However, the efficacy of these compounds at relieving opioid‐induced pain hypersensitivity have not yet been investigated. This study systematically examined the antinociceptive effects of α2/α3‐selective GABAA receptor PAMs alone and in combination with acetaminophen in an OIH rat model wherein repeated treatment with the opioid fentanyl induces mechanical hyperalgesia. The von Frey test was used to measure mechanical nociception. Duration of actions of α2/α3‐selective GABAA receptor PAMs (KRM‐II‐81, NS16085, HZ‐166) alone were studied, and combinations of KRM‐II‐81 and acetaminophen were also studied at fixed ratios (1:1, 1:3, 3:1). Dose‐addition analysis was used to assess the antinociceptive interactions between KRM‐II‐81 and acetaminophen. α2/α3‐selective GABAA receptor PAMs were able to fully reverse mechanical sensitivity caused by OIH. Furthermore, KRM‐II‐81/acetaminophen combinations produced additive to supra‐additive interactions depending on the drug mixture ratios. These findings support the idea that α2/α3‐selective GABAA receptor PAMs could serve as novel analgesics for treating OIH, and may interact favorably with other non‐opioid analgesics.

Using complex behavior to understand brain mechanisms in health and disease.

Using complex behavior to understand brain mechanisms in health and disease.

Application of dose-addition analyses to characterize the abuse-related effects of drug mixtures.

Polysubstance use makes up a majority of drug use, yet relatively few studies investigate the abuse-related effects of drug mixtures. Dose-addition analyses provide a rigorous and quantitative method to determine the nature of the interaction (i.e., supraadditive, additive, or subadditive) between two or more drugs. As briefly reviewed here, studies in rhesus monkeys have applied dose-addition analyses to group level data to characterize the nature of the interaction between the reinforcing effects of stimulants and opioids (e.g., mixtures of cocaine + heroin). Building upon these foundational studies, more recent work has applied dose-addition analyses to better understand the nature of the interaction between caffeine and illicit stimulants such as MDPV and methamphetamine in rats. Inaddition to utilizing a variety of operant procedures, including drug discrimination, drug self-administration, and drug-primed reinstatement, these studies have incorporated potency and effectiveness ratios as a method for both statistical analysis and visualization of departures from additivity at both the group and individual subject level. As such, dose-addition analyses represent a powerful and underutilized approach to quantify the nature of drug-drug interactions that can be applied to a variety of abuse-related endpoints in order to better understand the behavioral pharmacology of polysubstance use.

Characterization of the Discriminative Stimulus Effects of Binary Mixtures of Mu Opioid Receptor Agonists in Rats Discriminating Fentanyl.

Drug overdose deaths involving synthetic opioids, primarily fentanyl, have risen dramatically over the past decade and are currently the driving force of the opioid epidemic in the United States. Fentanyl analogs with greater potency than fentanyl (e.g., carfentanil) pose serious risk to public health. Although fentanyl analogs are primarily encountered by humans as constituents of a mixture of drugs, research has primarily evaluated the effects of these drugs alone. The present study characterized interactions between mu opioid receptor agonists in seven male Sprague-Dawley rats trained to discriminate 10 μg/kg fentanyl from saline while responding under a fixed-ratio 10 schedule of food presentation. Dose-effect curves were determined for each drug alone and in binary mixtures (fentanyl:heroin, fentanyl:carfentanil, and heroin:carfentanil) at fixed-dose ratios of 3:1, 1:1, and 1:3 relative to the ED50 for each drug when given alone. Dose addition analyses were used to determine the nature of the drug-drug interaction for each mixture. Additive interactions were observed for all binary mixtures at each fixed dose ratio, except the 1:3 fentanyl:carfentanil mixture, which exhibited supra-additive effects at the 80% effect level. These results suggest a lack of a significant interaction between the discriminative stimulus effects of these mu opioid receptor agonists at the doses tested in this study. Future studies expanding these findings to the respiratory depressant effects of these drugs are of significant importance to rule out possible interactions directly relevant to opioid overdose that occur at doses much larger than those tested in this study. SIGNIFICANCE STATEMENT: In the United States, drug overdose deaths involving synthetic opioids, primarily fentanyls including superpotent fentanyl analogs (e.g., carfentanil), have increased 12-fold over the past decade. Although previous studies have evaluated the effects of carfentanil alone, fentanyl analogs are encountered by humans as a mixture with other drugs; this study determined the effects of mixtures of carfentanil and other opioids (fentanyl and heroin) to characterize interactions between these drugs that might contribute to their apparent increased lethality in humans.

Interactions Between Mu Opioid Receptor Agonists in Rats Discriminating Fentanyl

The opioid epidemic persists as a major public health challenge with grave consequences. In 2018 two-thirds of the 47,000 opioid overdose deaths in the US involved a synthetic opioid such as fentanyl or one of its many derivatives, e.g. carfentanil, which is reportedly 100-fold more potent than fentanyl. Clinical reports suggest that larger doses of the opioid antagonist naloxone are required to reverse opioid overdoses involving carfentanil, compared with other opioid receptor agonists. Highly potent synthetic opioids such as fentanyl and carfentanil are most frequently encountered as adulterants of another drug such as heroin, suggesting that drug interactions might underlie the reported difficulty in reversing overdoses. This study evaluated the discriminative stimulus effects of opioid mixtures to assess interactions between mu opioid receptor agonists. Eight male Sprague Dawley rats were trained to discriminate 0.01 mg/kg fentanyl under a fixed-ratio 10 schedule of food presentation. Dose-effect curves were determined for fentanyl (0.001- 0.032 mg/kg), heroin (0.032-1.0 mg/kg), and carfentanil (0.000032-0.001 mg/kg) alone, and as binary mixtures (fentanyl and heroin, fentanyl and carfentanil, heroin and carfentanil) at fixed-dose ratios of 3:1. 1:1, and 1:3 relative to the ED50 for occasioning fentanyl-appropriate responding for each drug when given alone. Fentanyl, heroin, and carfentanil dose-dependently increased responding on the fentanyl-associated lever and reduced rate of responding. The kappa opioid receptor agonist spiradoline (0.1-0.32 mg/kg) did not occasion fentanyl-appropriate responding, but dose-dependently reduced rate of responding. Dose-addition analyses were used to determine the nature of drug interactions (additive, supra-additive, subadditive) for each mixture. Some mixtures of mu opioid receptor agonists, particularly those containing carfentanil, had supra-additive discriminative stimulus effects relative to predictions based on the effects of each drug alone. These results suggest that not only the increased potency of synthetic opioids, but also interactions with other opioid drugs likely play a role in the apparent increased lethality of these mixtures in humans.

Supra‐additive effects of morphine and the α2/3 preferring GABA A receptor ligand MP‐III‐024 on mechanical hyperalgesia and thermal nociception

Opioid and GABAA receptors are both located in central nociceptive pathways and compounds that activate these receptors have pain-relieving properties. To date, the interactive effects of concurrent administration of these compounds in preclinical models of pain-like behaviors have not been assessed. The purpose of this study was to examine the interactive effects of the µ-opioid agonist morphine and the α2GABAA and α3GABAA receptor positive allosteric modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024) in preclinical models of mechanical hyperalgesia and thermal nociception. Initially, the antihyperalgesic and antinociceptive effects of morphine and MP-III-024 administered alone were assessed, followed by fixed-ratio mixtures of MP-III-024/morphine combinations. Drug interaction data were analyzed using isobolographic and dose-addition analyses. In the assay of mechanical hyperalgesia, each compound produced dose-dependent antihyperalgesic effects, whereas only morphine was effective on thermal nociception. Fixed-ratio mixtures of MP-III-024/morphine were also dose-dependently effective in both procedures. These drug combination studies revealed that morphine and MP-III-024 produced supra-additive (synergistic) effects in both assays, depending on their relative proportions. These results demonstrate an interaction between α2GABAA and α3GABAA receptor- and µ-opioid receptor-mediated signals, and suggest that combination therapy may be useful for the treatment of pain-related disorders.

Synergistic antihyperalgesic and antinociceptive effects of morphine and methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024): a positive allosteric modulator at α2GABAA and α3GABAA receptors.

Opioid and GABAA receptors are both located in central nociceptive pathways, and compounds that activate these receptors have pain-relieving properties. To date, the interactive effects of concurrent administration of these compounds in preclinical models of pain-like behaviors have not been assessed. The purpose of this study was to examine the interactive effects of the μ-opioid agonist morphine and the α2GABAA and α3GABAA receptor positive allosteric modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024) in preclinical models of mechanical hyperalgesia and thermal nociception. The antihyperalgesic and antinociceptive effects of morphine and MP-III-024 administered alone were assessed initially, followed by fixed-ratio mixtures of MP-III-024/morphine combinations. Drug interaction data were analyzed using isobolographic and dose-addition analyses. All studies were conducted in male CD-1 mice. In the assay of mechanical hyperalgesia, each compound produced dose-dependent antihyperalgesic effects, whereas only morphine was effective on thermal nociception. Fixed-ratio mixtures of MP-III-024/morphine were also dose-dependently effective in both procedures. These drug combination studies revealed that morphine and MP-III-024 produced supra-additive (synergistic) effects in both assays, depending on their relative proportions. These results demonstrate an interaction between α2GABAA and α3GABAA receptor- and μ-opioid receptor-mediated signals and suggest that combination therapy may be useful for the treatment of pain-related disorders.

Interactions between reinforcement history and drug-primed reinstatement: Studies with MDPV and mixtures of MDPV and caffeine.

Many drugs of abuse are mixed with other psychoactive substances (e.g., caffeine) prior to their sale or use. Synthetic cathinones (e.g., 3,4-methylenedioxypyrovalerone [MDPV]) are commonly mixed with caffeine or other cathinones (e.g., 3,4-methylenedioxy-N-methylcathinone [methylone]), and these "bath salts" mixtures (e.g., MDPV + caffeine) can exhibit supra-additive interactions with regard to their reinforcing and discriminative stimulus properties. However, little is known about relapse-related effects of drug mixtures. In these studies, male Sprague-Dawley rats self-administered 0.032 mg/kg/inf MDPV or a mixture of MDPV + caffeine (0.029 + 0.66 mg/kg/inf, respectively) and then underwent multiple rounds of extinction and reinstatement testing to evaluate the influence of reinforcement history and drug-associated stimuli on the effectiveness of saline (drug-paired stimuli alone), MDPV (0.032-1.0 mg/kg), caffeine (1.0-32 mg/kg), and mixtures of MDPV:caffeine (in 3:1, 1:1, and 1:3 ratios, relative to each drug's ED50 ) to reinstate responding. Dose-addition analyses were used to determine the nature of the drug-drug interaction for each mixture. MDPV and caffeine dose-dependently reinstated responding and were equally effective, regardless of reinforcement history. Most fixed ratio mixtures of MDPV + caffeine exhibited supra-additive interactions, reinstating responding to levels greater than was observed with caffeine and/or MDPV alone. Drug-associated stimuli also played a key role in reinstating responding, especially for caffeine. Together, these results demonstrate that the composition of drug mixtures can impact relapse-related effects of drug mixtures, and "bath salts" mixtures (MDPV + caffeine) may be more effective at promoting relapse-related behaviors than the constituents alone. Further research is needed to determine how other polysubstance reinforcement histories can impact relapse-related behaviors.

Morphine and Delta‐9‐Tetrahydrocannabinol Antinociceptive Interactions in Male versus Female Rats with Inflammatory Pain

Previous studies have shown that morphine is more potent in male rats compared to females with chronic inflammatory pain. In contrast, delta‐9‐tetrahydrocannabinol (THC) may be more potent in females compared to males with chronic inflammatory pain. Some studies have demonstrated that morphine and THC produce synergistic antinociceptive effects in male animals. Therefore, the purpose of the present study was to determine sex‐specific potency of morphine and THC in male versus female rats with inflammatory pain, and then to test for antinociceptive synergy between morphine and THC.Male and female Sprague Dawley rats, 60–90 days old were used. On day 1 baseline measurements were taken on the von Frey test of mechanical allodynia, incapacitance test of biased weight bearing, a 10‐min locomotor activity test, and right hindpaw thickness. After baseline testing, inflammation was induced by an intraplantar injection of complete Freund's adjuvant (CFA) to the right hindpaw. Three days later, morphine (0, 0.1, 0.32, 1.0, or 3.2 mg/kg, s.c.) or THC (0, 0.5, 1.0, 2.0, or 4.0 mg/kg, i.p.) was administered and nociceptive testing was conducted at 30, 60, 120, and 240 min post‐injection. Paw thickness was also measured at 240 min post‐injection. A three‐way ANOVA was conducted on means of the 30–120 min time point data to determine sex differences in the antinociceptive effects of morphine alone and THC alone. The ED50 was calculated for morphine and for THC in each sex using data from the von Frey test. These data were used to determine 1:1, 3:1, and 1:3 dose‐addition ratios for each sex, which were administered in Experiment 2 under the same testing protocol as in Experiment 1.In both sexes, CFA increased right hindpaw thickness to approximately 200% of baseline, as well as causing mechanical allodynia, biased weight‐bearing, and decreased locomotor activity. In Experiment 1, morphine given alone dose‐dependently reduced mechanical allodynia similarly in both sexes. Morphine also dose‐dependently reduced biased weight‐bearing in both sexes, and was more potent in males than females. Morphine did not significantly alter paw thickness or locomotor activity in either sex. THC given alone dose‐dependently reduced allodynia and decreased locomotor activity similarly in males and females, but did not alter biased weight‐bearing or paw thickness in either sex. In Experiment 2, a 1:1 combination of morphine and THC produced large anti‐allodynic effects in both sexes. A dose‐addition analysis will be conducted when data collection is complete.Results thus far indicate that THC alone may be a beneficial analgesic to treat chronic inflammatory pain in both sexes, but may also produce sedation, a negative side effect. Similar to previous reports in rats, morphine alone also alleviates persistent inflammatory pain, but with greater potency in males than females. Given the sex differences observed with each drug alone, we predict that morphine‐THC combinations will produce greater antinociception in males compared to females.Support or Funding InformationThis research was supported by funds from the National Institute on Drug Abuse (DA016644).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of lorcaserin and buspirone, administered alone and as a mixture, on cocaine self-administration in male and female rhesus monkeys.

Cocaine use disorder is a serious public health issue for which there is no effective pharmacotherapy. One strategy to speed development of medications for cocaine use disorder is to repurpose drugs already approved for use in humans based on their ability to interact with targets known to be important for addiction. Two such drugs, lorcaserin (Belviq; a drug with serotonin [5-HT]2C receptor agonist properties) and buspirone (Buspar; a drug with 5-HT1A receptor partial agonist and dopamine D3/D4 receptor antagonist properties) can produce modest decreases in cocaine self-administration in rhesus monkeys. The current study evaluated the effectiveness of mixtures of lorcaserin and buspirone (at fixed dose ratios of 3:1, 1:1, and 1:3 relative to each drug's ID50) to reduce responding for 0.032 mg/kg/inf cocaine under a progressive ratio schedule of reinforcement in 2 male and 2 female rhesus monkeys. Dose addition analyses were used to determine if the effects of the drug mixtures differed from those predicted for an additive interaction between lorcaserin and buspirone. Dose-dependent reductions of cocaine self-administration were observed when lorcaserin and buspirone were administered alone, as well as when they were administered as 3:1, 1:1, and 1:3 fixed ratio mixtures of lorcaserin + buspirone. The effects of the 1:1 mixture of lorcaserin + buspirone on cocaine self-administration were supraadditive, whereas the effects of 3:1 and 1:3 mixtures were additive. Together, these results indicate that a combination therapy containing a mixture of lorcaserin and buspirone might be more effective than either drug alone at treating cocaine use disorder. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Reinforcing Effects of Binary Mixtures of Common Bath Salt Constituents: Studies with 3,4-Methylenedioxypyrovalerone (MDPV), 3,4-Methylenedioxymethcathinone (methylone), and Caffeine in Rats

Bath salts use is associated with high rates of abuse, toxicity, and death. Bath salt preparations often contain mixtures of drugs including multiple synthetic cathinones (eg, 3,4-methylenedioxypyrovalerone (MDPV) or 3,4-methylenedioxymethcathinone (methylone)) or synthetic cathinones and caffeine; however, little is known about whether interactions among bath salt constituents contribute to the abuse-related effects of bath salts preparations. This study used male Sprague-Dawley rats responding under a progressive ratio schedule to quantify the reinforcing effectiveness of MDPV, methylone, and caffeine, administered alone and as binary mixtures (n=12 per mixture). Each mixture was evaluated at four ratios (10 : 1, 3 : 1, 1 : 1, and 1 : 3) relative to the mean ED50 for each drug alone. Dose-addition analyses were used to determine the predicted, additive effect for each dose pair within each drug mixture. MDPV, methylone, and caffeine maintained responding in a dose-dependent manner, with MDPV being the most potent and effective, and caffeine being the least potent and effective of the three bath salts constituents. High levels of responding were also maintained by each of the bath salts mixtures. Although the nature of the interactions tended toward additivity for most bath salts mixtures, supra-additive (3 : 1 MDPV : caffeine, and 3 : 1 and 1 : 1 methylone : caffeine) and sub-additive (3 : 1, 1 : 1, and 1 : 3 MDPV : methylone) interactions were also observed. Together, these findings demonstrate that the composition of bath salts preparations can have an impact on both their reinforcing potency and effectiveness, and suggest that such interactions among constituent drugs could contribute to the patterns of use and effects reported by human bath salts users.

Abuse‐Related Effects of “Bath Salts” Mixtures: Studies with MDPV, Methylone, and Caffeine in Rats.

The recreational use of synthetic cathinones has become a serious public health problem worldwide. In the US, “bath salts” products often contain MDPV (a monoamine transporter inhibitor) or methylone (a monoamine transporter substrate); however, preparations often contain mixtures of multiple synthetic cathinones, or mixtures of a synthetic cathinone and caffeine. In order to determine if the reinforcing effects of mixtures of “bath salts” constituents differed from the reinforcing effects of the constituents alone, adult male Sprague Dawley rats were trained to self‐administer either 0.032 mg/kg/inf MDPV or 0.32 mg/kg/inf methylone under a progressive ratio (PR) schedule of reinforcement. Drug mixtures were based on the concept of dose equivalence, with each pair of drugs evaluated at three ratios (3:1, 1:1, and 1:3) relative to the mean ED50 for each drug. Dose addition analyses were used to calculate predicted effect levels for an additive interaction at each dose pair of each combination of drugs using the Emax, ED50, and slope parameters derived from dose‐response curves obtained in individual rats. Although high levels of responding were maintained by each of the “bath salts” mixtures, the nature of the interaction varied depending upon the constituent drugs. For combinations containing a cathinone and caffeine (MDPV+caffeine and methylone+caffeine), the interactions were generally additive, however, when mixed at a 3:1 ratio of their ED50s the reinforcing effects of both sets of mixtures were found to be supra‐additive. Interestingly, although combining caffeine with MDPV appeared to only increase the potency of the mixture, combining caffeine and methylone significantly increased the reinforcing effectiveness of the a “bath salts” mixture beyond that which would be predicted for an additive interaction. Unlike with combinations of cathinones and caffeine, when MDPV was combined with methylone departures from additivity tended to occur in the sub‐additive direction, with the onset of toxicity observed with larger dose pairs of the MDPV+methylone mixtures. Together, these studies suggest that the reinforcing effects of binary mixtures containing common bath salts constituents (i.e., MDPV, methylone, and caffeine) are generally additive in nature; however, both supra‐additive (MDPV+caffeine and methylone+caffeine) and sub‐additive interactions (MDPV+methylone) can be observed. Although such interactions could account for the high rates of bath salts abuse, further study will be needed to determine if similar interactions exist between these and other bath salts constituents with regard to other abuse‐related and toxic effects.Support or Funding InformationThis study was supported by a National Institutes of Health (NIH) research grant (R01 DA039146) from the National Institute on Drug Abuse (NIDA), as well as the NIH Intramural Research Programs of NIDA and National Institute on Alcohol Abuse and Alcoholism (NIAAA).

Discriminative Stimulus Effects of Binary Drug Mixtures: Studies with Cocaine, MDPV, and Caffeine

Illicit drug preparations often include more than one pharmacologically active compound. For example, cocaine and synthetic cathinones [e.g., 3,4-methylenedioxypyrovalerone (MDPV)] are often mixed with caffeine before sale. Caffeine is likely added to these preparations because it is inexpensive and legal; however, caffeine might also mimic or enhance some of the effects of cocaine or MDPV. In these studies, male Sprague-Dawley rats were trained to discriminate 10 mg/kg cocaine from saline, and the discriminative stimulus effects of cocaine, caffeine, and MDPV were evaluated alone and as binary mixtures (cocaine and caffeine, MDPV and caffeine, and cocaine and MDPV) at fixed-dose ratios of 3:1, 1:1, and 1:3 relative to the dose of each drug that produced 50% cocaine-appropriate responding. Dose-addition analyses were used to determine the nature of the drug-drug interactions for each mixture (e.g., additive, supra-additive, or subadditive). Although additive interactions were observed for most mixtures, supra-additive interactions were observed at the 50% effect level for the 1:1 mixture of cocaine and caffeine and at the 80% effect level for all three mixtures of cocaine and caffeine, as well as for the 3:1 and 1:3 mixtures of cocaine and MDPV. These results demonstrate that with respect to cocaine-like discriminative stimulus effects, caffeine can function as a substitute in drug preparations containing either cocaine or MDPV, with enhancements of cocaine-like effects possible under certain conditions. Further research is needed to determine whether similar interactions exist for other abuse-related or toxic effects of drug preparations, including cocaine, synthetic cathinones, and caffeine.

Antinociceptive effects of sinomenine in a rat model of postoperative pain.

This study examined the antinociceptive effects of sinomenine in a rat model of postoperative pain. Male and female rats were subjected to a surgical incision in the right hind paw, and the von Frey filament test was used to measure mechanical hypersensitivity after drug or vehicle treatment (p.o. or i.p.). Rats were treated daily with sinomenine before or after the surgery and the AUCs of the antinociceptive effects measured during a 4h period were calculated to determine the ED50 values of sinomenine. The anti-hyperalgesic effects of different doses of a combination of sinomenine and acetaminophen (paracetamol) were assessed in another group of rats. Dose combinations were determined by using a fixed ratio dose-addition analysis method. Sinomenine (5-80mg·kg(-1) ) produced dose-dependent antinociceptive effects in rats that had been subjected to surgery and this effect lasted for 4h. The potency of sinomenine, given i.p. or p.o., did not differ between male and female rats. However, sinomenine was fourfold more potent when given i.p. than p.o. The GABAA receptor antagonist bicuculline blocked the antinociceptive effects of sinomenine. The antinociceptive effect of a daily treatment with sinomenine remained stable throughout the course of postoperative pain. Pretreatment with sinomenine did not alter the mechanical hypersensitivity post-surgery. The combination of sinomenine with acetaminophen produced an infra-additive interaction. Sinomenine demonstrated significant antinociceptive activity against postoperative pain and may be a useful novel pharmacotherapy for the management of postoperative pain.

Anti-hyperalgesic effects of imidazoline I2 receptor ligands in a rat model of inflammatory pain: interactions with oxycodone.

Emerging preclinical evidence suggests that imidazoline I2 receptor ligands may be effective analgesics. Quantitative analysis of the combined I2 receptor ligands and opioids is needed for the justification of combination therapy. This study systematically examined the anti-hyperalgesic and response rate-suppressing effects of selective I2 receptor ligands (2-BFI and phenyzoline) alone and in combination with oxycodone in rats. Von Frey filament test was used to examine the anti-hyperalgesic effects of drugs in a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Schedule-controlled responding was used to assess the rate-altering effects of study drugs. Duration of actions of individual drugs (2-BFI, phenyzoline, and oxycodone) alone or in combination was studied. Dose-addition analysis was employed to assess the anti-hyperalgesic interactions between drugs. Oxycodone (0.1-3.2 mg/kg, i.p.), 2-BFI (1-17.8 mg/kg, i.p.), and phenyzoline (17.8-56 mg/kg, i.p.) all dose-dependently produced significant antinociceptive effects. When studied as combinations, 2-BFI and oxycodone produced additive interactions while phenyzoline and oxycodone produced supra-additive interactions under all fixed ratios. The same drug combinations did not alter or significantly reduced the operant responding depending on the ratios of the drug combinations. Quantitative analysis of the anti-hyperalgesic effects of I2 receptor ligands strongly supports the therapeutic potential of I2 receptor ligands against inflammatory pain. In addition, the data reveal that phenyzoline is superior to the prototypic I2 receptor ligand 2-BFI for the management of pain and warrants further consideration as a novel analgesic.

Roles of μ-Opioid Receptors and Nociceptin/Orphanin FQ Peptide Receptors in Buprenorphine-Induced Physiological Responses in Primates

Buprenorphine is known as a μ-opioid peptide (MOP) receptor agonist, but its antinociception is compromised by the activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine-induced physiological responses in primates (rhesus monkeys). The effects of MOP antagonist (naltrexone), NOP antagonist [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)], and NOP agonists [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one (Ro 64-6198) and 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)] on buprenorphine were studied in three functional assays for measuring analgesia, respiratory depression, and itch in primates. Over the dose range of 0.01 to 0.1 mg/kg, buprenorphine dose-dependently produced antinociception, respiratory depression, and itch/scratching responses, and there was a ceiling effect at higher doses (0.1-1 mg/kg). Naltrexone (0.03 mg/kg) produced similar degrees of rightward shifts of buprenorphine's dose-response curves for all three endpoints. Mean pK(B) values of naltrexone (8.1-8.3) confirmed that MOP receptors mediated mainly buprenorphine-induced antinociception, respiratory depression, and itch/scratching. In contrast, J-113397 (0.1 mg/kg) did not change buprenorphine-induced physiological responses, indicating that there were no functional NOP receptors in buprenorphine-induced effects. More importantly, both NOP agonists, Ro 64-6198 and SCH 221510, enhanced buprenorphine-induced antinociception without respiratory depression and itch/ scratching. The dose-addition analysis revealed that buprenorphine in combination with the NOP agonist synergistically produced antinociceptive effects. These findings provided functional evidence that the activation of NOP receptors did not attenuate buprenorphine-induced antinociception in primates; instead, the coactivation of MOP and NOP receptors produced synergistic antinociception without other side effects. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics.

Roles of MOP and NOP receptors in regulating buprenorphine‐induced physiological responses in monkeys

Buprenorphine is known as a mu opioid receptor (MOP) partial agonist, but its analgesia was attenuated by activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine‐induced physiological responses in monkeys. Systemic buprenorphine (0.01–0.1 mg/kg) dose‐dependently produced antinociception, respiratory depression, and itch/scratching responses. A MOP antagonist, naltrexone 0.03 mg/kg, produced similar degrees of rightward shifts of buprenorphine's dose‐response curves for all three endpoints and confirmed that they were MOP‐mediated effects. In contrast, a NOP antagonist, J‐113397 0.1 mg/kg, did not change buprenorphine‐induced effects, indicating that there were no functional NOP receptors in buprenorphine‐induced actions. More importantly, a NOP agonist Ro 64–6198 enhanced buprenorphine‐induced antinociception, and the dose‐addition analysis revealed that systemic combination of buprenorphine and Ro 64–6198 produced superadditive antinociceptive effects. These findings provided functional evidence that co‐activation of MOP and NOP receptors produced synergistic antinociception in primates. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics (Supported by USPHS grant AR‐059193).

Effects of imidazoline I2 receptor agonists and morphine on schedule-controlled responding in rats

Accumulating evidence indicates that imidazoline I2 receptor agonists enhance the antinociceptive effects of opioids and therefore may be suitable for combination therapy with opioids for pain treatment. However, little is known of the effects of I2 receptor agonists on other behavioral effects of opioids. This study used schedule-controlled responding and dose-addition analyses to examine interactions between the μ opioid receptor agonist morphine and two imidazoline I2 receptor agonists, 2-BFI and BU224. In 8 rats responding under a fixed ratio 10 schedule of food presentation, morphine (3.2–17.8mg/kg), 2-BFI (3.2–17.8mg/kg), and BU224 (5.6–17.8mg/kg) each dose-dependently decreased responding. The addition of fixed proportions of 2-BFI or BU224 shifted the morphine dose–effect curves leftward. The interactions between morphine and 2-BFI or BU224 were infra-additive when the same proportions of morphine and I2 receptor agonists were mixed; however, the interaction between morphine and I2 receptor agonists was additive when the drugs were mixed at other proportions. These results provide quantitative evidence that I2 receptor agonists do not enhance the response rate-decreasing effect of morphine and suggest that the enhancement of morphine antinociception is selective. Together, these results further support the therapeutic potential of combining I2 receptor agonists and opioids for pain control.

Morphine-induced antinociception in the rat: Supra-additive interactions with imidazoline I2 receptor ligands

Pain remains a significant clinical challenge and currently available analgesics are not adequate to meet clinical needs. Emerging evidence suggests the role of imidazoline I(2) receptors in pain modulation primarily from studies of the non-selective imidazoline receptor ligand, agmatine. However, little is known of the generality of the effect to selective I(2) receptor ligands. This study examined the antinociceptive effects of two selective I(2) receptor ligands 2-BFI and BU224 (>2000-fold selectivity for I(2) receptors over α(2) adrenoceptors) in a hypertonic (5%) saline-induced writhing test and analyzed their interaction with morphine using a dose-addition analysis. Morphine, 2-BFI and BU224 but not agmatine produced a dose-dependent antinociceptive effect. Both composite additive curve analyses and isobolographical plots revealed a supra-additive interaction between morphine and 2-BFI or BU224, whereas the interaction between 2-BFI and BU224 was additive. The antinociceptive effect of 2-BFI and BU224 was attenuated by the I(2) receptor antagonist/α(2) adrenoceptor antagonist idazoxan but not by the selective α(2) adrenoceptor antagonist yohimbine, suggesting an I(2) receptor-mediated mechanism. Agmatine enhanced the antinociceptive effect of morphine, 2-BFI and BU224 and the enhancement was prevented by yohimbine, suggesting that the effect was mediated by α(2) adrenoceptors. Taken together, these data represent the first report that selective I(2) receptor ligands have substantial antinociceptive activity and produce antinociceptive synergy with opioids in a rat model of acute pain. These data suggest that drugs acting on imidazoline I(2) receptors may be useful either alone or in combination with opioids for the treatment of pain.

Preclinical Assessment of Drug Combinations for the Treatment of Pain: Isobolographic and Dose-Addition Analysis of the Opioidergic System

Opioid analgesics are the most frequently prescribed medications for the treatment of moderate or severe pain; however, their use is constrained by unwanted side effects. One therapeutic approach used to improve the side effect profile of opioids is the administration of a second drug in an opioid-containing mixture. Preclinical studies designed to predict the therapeutic potential of novel opioid-containing drug combinations are currently underway, and must rely on quantitative methods to assess their interactive effects. In this manuscript, an overview of isobolographic analysis is presented along with recent advances in isobolographic theory pertaining to drugs that differ in efficacy and to the statistical analysis of dose-addition. Next, studies using these analyses to assess the interactive effects of opioids and novel adjunct drugs, including selective COX-2 inhibitors, α(2)-adrenergic receptor agonists, δ-opioids, glutamate receptor antagonists and cannabinoid receptor agonists, are reviewed. Finally, comments on the future assessment of drug combinations for the treatment of pain-related disorders are made.