SR 16435 [1-(1-(Bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed...

SR 16435, A Novel Mixed NOP/μ–Opioid Receptor Partial Agonist:Analgesic and Rewarding Properties in Mice

Taline V. Khroyan, Nurulain T. Zaveri, Willma E. Polgar, Juan Orduna, Cris Olsen, FamingJiang, and Lawrence TollSRI International, Menlo Park, CA Center for Health Sciences, SRI International: T.V.K. DrugDiscovery Program, SRI International: N.T.Z., C.O., F.J. Neuropharmacology Program, SRIInternational: L.T., W.E.P., J.O.

AbstractWe identified a novel NOP/μ-opioid receptor agonist, SR 16435, with high binding affinity andpartial agonist activity at both receptors. It was hypothesized that SR 16435 would produceantinociception and yet unlike morphine, would have diminished rewarding properties and tolerancedevelopment. Antinociception was assessed in mice using the tail-flick assay, whereas behavioraland rewarding effects were assessed using the PC paradigm. PC was established by pairing druginjections with a distinct compartment. Behavioral effects were measured following acute andrepeated drug administration and the test for PC was carried out 24h following four drug- and vehicle-pairing sessions. SR 16435 produced an increase in tail flick latency but SR 16435-inducedantinociception was lower than that observed with morphine. Given that naloxone blocked SR 16435-induced antinociception, it is highly likely that this effect was mediated by μ-opioid receptors.Compared to morphine, chronic SR 16435 treatment resulted in reduced tolerance development toits antinociceptive effects. SR 16435-induced CPP was evident, an effect likely mediated via μ-opioidreceptors, as it was reversed by co-administration of naloxone. NOP receptor agonist activity wasalso present given that SR 16435 decreased global activity, and this effect was partially reversed withthe selective NOP antagonist, SR 16430. Naloxone, however, also reversed the SR 16435-induceddecrease in activity indicating that both opioid and NOP receptors mediate this behavior. In summary,the mixed NOP/μ-opioid receptor partial agonist SR 16435 exhibited both NOP and μ-opioidreceptor-mediated behaviors.

INTRODUCTIONThe NOP receptor (formerly known as the Opioid Receptor Like receptor ORL1) wasdiscovered simultaneously by several groups based upon homology with the δ-opioid receptor(Bunzow et al., 1994; Mollereau et al., 1994; Wang et al., 1994). This receptor has nucleotideand amino acid homology to the three opioid receptors, however, NOP receptors transfectedinto mammalian cells do not appear to bind opiates with high affinity. N/OFQ, the naturalligand for the NOP receptor, is a 17 amino-acid neuropeptide, (Meunier et al., 1995; Reinscheidet al., 1995). Although this peptide is in the opioid peptide family, N/OFQ has a differentbehavioral profile compared to typical opioid ligands. N/OFQ modulates nociceptiondifferentially depending upon site of administration. N/OFQ delivered ICV has been shown tohave no effect, or to be pro-nociceptive, and can block morphine-induced analgesia (Meunieret al., 1995; Reinscheid et al., 1995; Mogil et al., 1996; Tian et al., 1997). However, IT

Corresponding Author: Taline Khroyan, Ph.D. Center for Health Sciences, BN144 SRI International 333 Ravenswood Ave. Menlo Park,CA 94025, USA Telephone: (650) 859−3818 Facsimile: (650) 859−5099 E-mail: E-mail: [email protected] section: Behavioral Pharmacology

NIH Public AccessAuthor ManuscriptJ Pharmacol Exp Ther. Author manuscript; available in PMC 2009 June 4.

Published in final edited form as:J Pharmacol Exp Ther. 2007 February ; 320(2): 934–943. doi:10.1124/jpet.106.111997.

NIH

-PA Author Manuscript

NIH


NIH


administration of N/OFQ produces antinociception and potentiates morphine analgesia (Tianet al., 1997).

This complicated profile of N/OFQ and its receptor, NOP, in pain has been further examinedusing novel selective agonists and antagonists. High affinity and selective peptide antagonistssuch as [Nphe]N/OFQ(1−13)NH2 and UFP-101 are antinociceptive when injected ICV in acutethermal pain models (Calo et al., 2000; Calo et al., 2002). High throughput screening andclassical medicinal chemistry have led to the identification of several small molecule ligandsthat are active after systemic administration and could potentially be developed as analgesicmedications for therapeutic use (Zaveri, 2003). Systemic administration of highly selectiveNOP antagonists J-113397 and SB-612111, have no effect on acute thermal pain (Ozaki et al.,2000; Zaratin et al., 2004), whereas the nonselective antagonist JTC-801 (only 4-fold selectivefor NOP relative to μ–opioid receptors) produced naloxone-resistant antinociception (Shinkaiet al., 2000; Yamada et al., 2002). These discrepancies could be due to different selectivitiesfor the NOP versus opioid receptors and/or pharmacokinetic distribution in vivo. Ro 64−6198,an NOP agonist with 100-fold selectivity for NOP receptors over μ–opioid receptors, possessesanxiolytic activity but has no analgesic activity alone and inhibits morphine antinociceptionwhen given systemically (Jenck et al., 2000; Kotlinska et al., 2003)

Extended treatment with opioid analgesics is usually accompanied by undesirable side effectssuch as tolerance development and dependence. The role of NOP receptors in modulating theseopioid receptor-mediated effects has been previously examined. N/OFQ, administered ICV,attenuates tolerance development to morphine antinociception (Lutfy et al., 2001). Smallmolecule NOP receptor antagonists J-113397 (IT administration) and SB-612111 (IVadministration) also block morphine-induced tolerance to antinociception (Ueda et al., 2000;Zaratin et al., 2004). Additionally, ICV administration of N/OFQ and systemic administrationof the NOP receptor agonist Ro 64−6198 attenuate the rewarding effects of morphine, in thePC paradigm (Murphy et al., 1999; Shoblock et al., 2005). Although NOP receptor agonistscan attenuate morphine-induced CPP, they do not induce CPP or CPA when administered alone(Devine et al., 1996; Le Pen et al., 2002).

It appears, therefore, that stimulation of NOP receptors can reduce morphine-induced tolerancedevelopment and reward. Since μ-opioid receptor stimulation produces powerfulantinociception, a compound that stimulates both NOP and μ-opioid receptors may act as apotent analgesic, but be less susceptible to the development of abuse liability and tolerance toantinociception. In the course of our program to develop NOP ligands we have synthesizedcompounds showing agonist activity at both NOP and μ-opioid receptors. SR 16435 is adihydroindolinone-based small molecule ligand with high binding affinity at both NOP andμ-opioid receptors (Zaveri et al., 2004). In the [35S]GTPγS functional assay, SR 16435 is apotent partial agonist at both sites (see Table 1). Buprenorphine, a high affinity μ-opioid partialagonists has significant antinociceptive potency with reduced addiction liability (Jasinski etal., 1978; Jacob et al., 1979). Accordingly, we sought to test the hypothesis that a μ-opioidpartial agonist such as SR 16435, with equally high affinity for both NOP and μ-opioidreceptors and partial agonist activity at both receptors, could have a particularly beneficialbehavioral profile.

In the following experiments we examined whether SR 16435, a mixed NOP/ μ-opioid partialagonist, possesses analgesic activity with reduced tolerance development and diminishedreward in mice. The tail flick assay was used to examine acute and repeated effects of SR 16435on thermal nociception. The PC paradigm was used to assess whether SR 16435 had anyrewarding properties and to determine how similar and/or different the behavioral profile wasrelative to morphine.

Khroyan et al.

J Pharmacol Exp Ther. Author manuscript; available in PMC 2009 June 4.

NIH


NIH


NIH


METHODSExperimental Procedures

Animals—Male ICR mice weighing 20−25g at the start of the experiment were used. Animalswere group-housed under standard laboratory conditions and were kept on a 12:12 h day-nightcycle (lights on at 08:00). Animals were handled for 2−3 days prior to conducting theexperiments. For thermal nociception experiments, animals were transported to the testingroom and acclimated to the environment for one hour. Mice were maintained in accordancewith the guidelines of SRI International and of the “Guidelines for the Care and Use ofmammals in neuroscience and behavioral research” (National Research Council, 2003).

Drugs—SR 16435 (tested as the hydrochloride salt, synthesized at SRI, (Zaveri et al.,2004)), morphine hydrochloride (Lilly, Indianapolis, IN), and naloxone (Sigma, St. Louis, MO)were dissolved in water. SR16430 (also tested as the hydrochloride salt, synthesized at SRI)was dissolved in 1% DMSO and 0.5% Hydroxypropylcellulose. Drugs were injected in avolume of 0.1 ml/25g (SC). Controls received 0.1 ml/25g of the appropriate vehicle.

Assessing Acute Thermal NociceptionTail-Flick Assay—Acute nociception was assessed using the tail flick assay with ananalgesia instrument (Stoelting) that uses radiant heat. This instrument is equipped with anautomatic quantification of tail flick latency, and a 15 sec cutoff to prevent damage to theanimal's tail. During testing, the focused beam of light was applied to the lower half of theanimal's tail, and tail flick latency was recorded. Baseline values for tail flick latency weredetermined before drug administration in each animal. The mean basal tail flick latency was5.1±0.1 SEM.

Following baseline measures, animals received a subcutaneous (SC) injection of their assigneddose of drug and were tested for tail flick latencies at 10-, 30-, 60-min post-injection. Animalsthat were controls received an injection of vehicle prior to testing.

Drug Regimen—Animals (N=10−12/group) received SC injections of SR 16435 alone (3−30 mg/kg) or vehicle. Higher doses of SR 16435 were not tested due to drug insolubility. Ina follow-up experiment to examine whether the effects of SR 16435 were reversed by naloxone,animals (N=10/group) received SC injections of SR 16435 (10 and 30 mg/kg) co-administeredwith naloxone (1 mg/kg). The dose of naloxone was chosen because it has been shown toreverse the effects of morphine.

To compare the effects of SR 16435 with morphine, a morphine dose response curve for acutethermal antinociception was conducted using similar parameters as described above. Animals(N=10−12/group) received SC injections of morphine alone (1−30 mg/kg) or vehicle.

Statistical Analyses—Antinociception (%Maximum Potential Effect; %MPE) wasquantified by the following formula:

% MPE= 100 * [(test latency - baseline latency)/(15 - baseline latency)]. If the animal did notrespond prior to the 15-s cutoff, the animal was assigned a score of 100%.

Behavioral results were analyzed using ANOVAs with drug treatment (SR 16435, morphine,and naloxone) as between group variables and post drug-injection time (10-, 30- and 60-min)as the repeated measure followed by Student Newman-Keuls post-hoc tests where appropriate.The level of significance was set at P< 0.05.

Khroyan et al.


NIH


NIH


NIH


Assessing tolerance development to thermal antinociceptionInjection Regimen and testing—Animals received SC injections of either vehicle, SR16435 (30 mg/kg, N=15) or morphine (10 mg/kg, N=10) once daily for 9 days. The 30 mg/kgdose of SR 16435 was chosen since it produced the greatest level of antinociception of thedoses tested, whereas the 10 mg/kg dose of morphine was chosen since it produced the samelevels of antinociception as 30 mg/kg SR 16435. On days 1−5 and 9, animals were tested fortail flick latencies using the equipment and methods described above. Baseline tail flicklatencies were taken. The mean basal tail flick latencies were consistent across test days (day1, 6.0±0.2 SEM; day 2, 5.7±0.1 SEM; day 3, 5.5±0.2 SEM; day 4, 5.4±0.2 SEM; day 5, 5.5±0.2 SEM; day 9 5.3±0.2 SEM). Animals then received an injection of their assigned dose ofdrug and were tested once 30 min following their injection. The 30-min time point was chosensince the same effects were seen at the 30- and 60-min testing time points (in acute analgesiaassessment described above).

Statistical Analyses—%MPE was calculated as described above and a repeated ANOVAwith drug treatment (morphine and SR 16435) as the between group variable and injection day(days 1−5, and 9) as the repeated measure. To examine tolerance development, following asignificant overall and repeated measures ANOVAs (for each drug tested), paired t-tests wereused to compare data obtained on days 2−5, and 9 to day 1. Following significant effects,Student Newman-Keuls post-hoc tests were carried out as described above. The level ofsignificance was set at P<0.05.

Assessing the rewarding effects and behavioral effects of SR 16435 using the PC paradigmPC Apparatus—The apparatus consisted of rectangular Plexiglas chambers divided into twodistinct equal-sized compartments (19cm × 22.8cm × 18cm high), Lafayette Instruments). Onecompartment had cedar-scented bedding underneath a bar grid floor and all but the front wallswere black. The other compartment had pine-scented bedding beneath a mesh floor and all butthe front wall were white. The front walls were transparent so that the animal's behavior wasmonitored. A removable partition divided the two compartments. During conditioning, thecompartments were divided by a solid partition. On the PC test day, the solid partition wasreplaced with a partition that had an opening allowing the animal free access to bothcompartments. A video camera that was linked to a computer was mounted above the chambersand tracked the animals’ movement. Previous experiments using this experimental set-up haveindicated that the apparatus is unbiased as animals do not show a preference for onecompartment over the other (unpublished observation).

PC Training—Conditioning trials were composed of two sessions conducted over twoconsecutive days. During one conditioning session, animals received a SC injection of theirrespective drug and were confined to one of the compartments for 20 min. On the other day,the second session, animals received an injection of saline and were confined to the alternatecompartment for 20 min. Also, a group of mice received saline in both compartments andserved as controls. These two sessions were repeated over eight consecutive days such thatanimals received four drug sessions and four vehicle sessions. The particular compartmentpaired with the drug and the order of placement into the drug-paired versus saline-pairedcompartment was counterbalanced across groups.

Acute and Repeated Behavioral Measures—During conditioning, the behavioraleffects of acute and repeated drug injection were defined as follows: sniffing- directed sniffingof the floor or wall; rearing- the animal's front two paws being off the ground; and grooming-animal licking/biting or brushing itself. Each effect was measured by recording its presenceevery 10 sec throughout the session. These measures were recorded immediately following thefirst and last drug administration by an observer unaware of the animals’ treatment group. The

Khroyan et al.


NIH


NIH


NIH


overall global activity of the animals was captured by the Spontaneous Motor Recording andTracking software system (SMART; Panlab), a color image capturing system that works inreal time and can track the animal's behavior for a given amount of time via a video cameraconnected to the computer.

PC Test Day—Twenty-four h after the last conditioning session, the animals were givenaccess to both compartments simultaneously for 15 min and the amount of time the animalsspent in each compartment was recorded by the SMART tracking system

Dose Regimen—Animals were assigned to groups receiving SC injections of either vehicle(N=15), morphine (15 mg/kg; N=8), or one of two doses of SR 16435 (10 or 30 mg/kg; N=8/group) immediately before placement in the drug-paired compartment. Since SR 16435produced a significant CPP, a follow-up experiment examined whether naloxone could blockthe effects of SR 16435. Animals (N=8/group) were randomly assigned to groups receiving10 mg/kg SR 16435 alone or co-administered with 1 mg/kg naloxone (SC). Another group ofanimals served as controls and received vehicle injections. PC testing was carried out asdescribed above.

Given that acute administration of SR 16435 suppressed global activity, an effect possiblyinvolving NOP receptor activation, we tested whether SR 16430 (an NOP antagonist, seeFigure 1 and Table 1) could reverse the suppressive effects of SR 16435. Animals (N=6−8/group) were randomly assigned to groups receiving 10 mg/kg SR 16435 alone or co-administered with 30 mg/kg SR 16430 (SC). Another group of animals served as controls andreceived vehicle injections. Animals received their respective injection of drug and wereimmediately tested for global activity in the PC apparatus for 20 min.

Statistical Analyses—Drug-induced behaviors were analyzed using ANOVAs with SR16435, morphine, naloxone, and SR 16430 as between subjects measures and injection day(first versus fourth) as a repeated measure. Significant interactions were further analyzed withone way ANOVAs and post-hoc tests. To examine sensitization effects, following a significantoverall ANOVA, t-tests were used to compare data following the fourth injection relative tothe first. For the PC test day data, the percent time animals spent in their drug-pairedcompartment relative to saline-treated controls was analyzed using one way ANOVAs andsignificant effects were further analyzed with post-hoc tests. A conditioned place preference(CPP) was evident if animals spent significantly more time in their drug-paired compartmentrelative to control animals, whereas a conditioned place aversion (CPA) was evident if animalsspent significantly less time in their drug-paired compartment. The level of significance wasset at P< 0.05.

RESULTSChemical structures of the NOP partial agonist SR16435 and antagonist SR16430 used in thisstudy are shown in Figure 1. Table 1 shows in vitro binding affinities and functional activitiesof these compounds, as well as standards, morphine and N/OFQ, at the NOP, μ-opioid, andκ-opioid receptors. As seen in Table 1, SR16435 has high affinity at both NOP and μ-opioidreceptors, and partial agonist activity, as measured by [35S]GTPγS stimulation, at both sites.In contrast, SR16430 has approximately 10-fold selectivity for the NOP receptor over μ-opioidreceptors, and is an antagonist at the NOP receptor.

Effects of SR 16435 on tail-flick latency and reversal by co-administration of naloxoneThe effects of SR 16435 on tail flick latency are shown in Figure 2a. The overall ANOVAindicated that there was no significant interaction effect, and only the main effect of drug [F

Khroyan et al.


NIH


NIH


NIH


(3,48)=9.8, P<0.05] was significant, indicating that regardless of post drug-injection time (10-,30- and 60-min) SR 16435 produced the same effects. Thus, looking at the dose effectsaveraged across post drug-injection time, post-hoc tests indicated that SR 16435 (10 and 30mg/kg) produced significant increases in tail-flick latency relative to vehicle controls (P<0.05).SR 16435 produced a dose-dependent increase in tail-flick latency since animals that receivedthe 30 mg/kg dose produced a greater increase in tail-flick latency relative to animals thatreceived the 10 mg/kg dose (P<0.05).

The effects of morphine on tail flick latency are shown in Figure 2b. The overall ANOVAindicated that there was no significant interaction effect, and only the main effect of dose [F(5,62)=32.8, P<0.05] was significant indicating that regardless of post drug-injection time (10-,30- and 60-min) the effects of morphine did not change. Thus, looking at the dose effectsaveraged across post drug-injection time, post-hoc tests indicated that 3−30 mg/kg morphineproduced significant dose-dependent increases in tail-flick latency relative to vehicle (P<0.05).Compared to 15 and 30 mg/kg morphine, antinociception produced by SR 16435 never reachedmaximal levels (Figure 2).

The antinociceptive activity of SR 16435 was blocked by the opioid antagonist naloxone(Figure 3), suggesting that this activity was mediated by the μ-opioid component of SR 16435.The overall ANOVA indicated that there was a significant SR 16435 × naloxone effect [F(2,70)=3.1, P<0.05] regardless of post-injection time (10, 30, or 60 min). Averaged across post-injection time, naloxone significantly decreased SR 16435-induced antinociception at bothdoses of SR 16435 tested (10 mg/kg F(1,28)=11.28, P<0.05; 30 mg/kg F(1,24)=8.0, P<0.05).Naloxone administered alone did not alter tail flick latency since animals that receivednaloxone exhibited similar levels of %MPE relative to vehicle control animals (n.s., P>0.05).

Effects of SR 16435 on tolerance development to thermal antinociceptionThe effect of SR 16435 on thermal antinociception across repeated test days is shown in Figure4. The overall ANOVA indicated a significant drug × injection day interaction [F(10, 185)=2.64, P<0.05]. Examining the one way ANOVAs for each day tested (days 1−5, and 9) therewas a significant effect of dose. On test days 1−9, SR 16435 (30 mg/kg) produced an increasein tail-lick latency that was significantly greater than that observed in vehicle controls, whereasmorphine produced an increase in tail-flick latency on days 1−4 and not day 9 (P<0.05). Toexamine the effects of tolerance development, levels of %MPE on test days 2−5 and 9 werecompared to the first test day. As expected, tolerance developed to the antinociceptive effectsof 15 mg/kg morphine [F(5, 45)=4.3, P<0.05]. Compared to day 1 (63 %MPE), morphineproduced a significant reduction in %MPE on days 2, 4, 5, and 9 (P<0.05; 15%MPE by lasttest day). SR 16435 also produced a significant decrease in %MPE across days [F(5,70)=2.9,P<0.05), however, there was only a significant difference for days 5 and 9 when compared today 1 (P<0.05). The decrease in antinociception observed following repeated administrationof SR 16435 was not as dramatic as morphine (Day 1 = 62.5%MPE; Day 9 = 34.5%MPE).

The effects of SR 16435 on place conditioningThe effect of SR 16435 on place conditioning is shown in Figure 5a. The overall ANOVAindicated a significant main effect of dose [F(3, 34)=8.22, P<0.05]. Animals that receivedmorphine, 10 and 30 mg/kg SR 16435 exhibited a significant CPP (P<0.05) relative to vehiclecontrols. The 10 mg/kg dose of SR 16435 was also tested in combination with naloxone andthe results indicated that naloxone not only blocked the rewarding effects of SR 16435 but alsoproduced a significant CPA when co-administered with SR 16435 (P<0.05; Figure 6a).

Khroyan et al.


NIH


NIH


NIH


Behavioral effects of SR 16435 following acute and repeated administrationThe effect of SR 16435 on global activity is shown in Figure 5b. The overall ANOVA indicatedthat there was a significant dose x injection day [F(3,35)=6.45, P<0.05]. As expected, morphineadministration produced an increase in global activity following the first injection relative tovehicle controls (P<0.05). Furthermore, sensitization of morphine-induced global activity wasalso evident as an increase in activity following the fourth injection relative to the first injection(P<0.05). Conversely, SR 16435 (10.0 mg/kg) administration produced a significant decreasein global activity relative to vehicle controls following the first injection (P<0.05). Thisdecrease in global activity relative to vehicle controls was not evident following the fourthdrug injection (P>0.05).

The effect of naloxone on SR 16435-induced decrease in global activity is shown in Figure 6b.The overall ANOVA indicated that there was a significant drug × injection day interaction [F(2,27)=5.3, P<0.05]. Following the first injection, naloxone co-administered with SR 16435did not produce a decrease in activity and in fact produced a significant increase in activityrelative to vehicle controls (P<0.05). Following the fourth injection, SR 16435 alone and co-administered with naloxone was not significantly different than vehicle controls.

Further experiments were carried out to examine whether the acute suppression of globalactivity induced by SR 16435 was an NOP receptor-mediated effect. The effect of the NOPantagonist SR 16430 on SR 16435-induced decrease in global activity is given in Table 2. Theoverall ANOVA indicated that there was a significant SR 16435 × SR 16430 interaction [F(1,23)=5.8, P<0.05]. Following acute drug injection, the suppressive effects of SR 16435 werepartially reversed by SR 16430. SR 16435 co-administered with SR 16430 produced anincrease in global activity relative to animals that received SR 16435 alone, however, the levelsof activity never reached levels observed in vehicle controls (P<0.05). As seen in Table 2,levels of global activity produced by SR 16430 administration was not significantly differentrelative to vehicle controls.

Other behaviors examined included grooming, sniffing, and rearing (Table 3). The overallANOVAs indicated that there was a significant main effect of dose for grooming [F(3,35)=51.9, P<0.05], sniffing [F(3,35)=337.3, P<0.05], and a significant drug × injection dayinteraction effect for rearing [F(3,35)=7.33, P<0.05]. In comparison to vehicle controls,morphine administration produced the same incidence of sniffing, but a significantly lowerincidence of grooming and rearing (P<0.05). Following repeated administration, morphineproduced a significant increase in the incidence of rearing; however, the incidence of rearingwas still 50% less than that observed in vehicle controls. SR 16435 alone produced a dramaticdecrease in the incidence of all the behaviors examined relative to vehicle controls (P<0.05).In most of the animals receiving SR 16435 these behaviors were not present. These animalswere lying very still in one corner and in most cases had their eyes open (not quantified). Thisnonexistence in exploratory behavior/ grooming was also present following repeated drugadministration. Naloxone co-administered with SR 16435 reversed the behavior-dampeningeffects of SR 16435 and reinstated the same levels of grooming, sniffing, and rearing relativeto vehicle controls (P<0.05; Table 3).

DISCUSSIONSignificant efforts to develop NOP receptor ligands to further elucidate the role of this receptorin pain are ongoing. NOP-selective peptide antagonists administered ICV have antinociceptiveeffects to thermal stimuli (Calo et al., 2000; Calo et al., 2002), whereas systemic or ICVadministration of selective non-peptide antagonists do not (Ozaki et al., 2000; Zaratin et al.,2004). NOP receptor agonists have also been shown to have antinociceptive properties. IT butnot ICV injections of N/OFQ produce antinociception in acute pain models and are effective

Khroyan et al.


NIH


NIH


NIH


in models of chronic pain (Tian et al., 1997). High affinity hexapeptide NOP agonists are alsoeffective in animal models of chronic pain (Khroyan et al., submitted). The non-peptide smallmolecule agonist Ro 64−6198 is not antinociceptive when administered alone and reversesmorphine antinociception, yet is antiallodynic in a neuropathic pain model. (Jenck et al.,2000; Kotlinska et al., 2003; Obara et al., 2005). Thus, it still remains unclear whether NOPreceptor ligands could be developed for use as clinically relevant analgesics.

In the present study we examined the behavioral effects of a mixed NOP/μ-opioid receptoragonist, SR 16435. As seen in Table 1, SR 16435 has high affinity for both NOP and μ-opioidreceptors and is a partial agonist at both these receptors as determined by the [35S]GTPγSbinding assay (Zaveri et al., 2004). SR 16435 produced an increase in tail flick latencyfollowing acute administration. However, the slope of the dose response curve for SR 16435was considerably shallower than that of morphine, as would be expected for a compound withlow intrinsic efficacy (O'Callaghan and Holtzman, 1975). Naloxone blocked theantinociceptive effect of SR 16435, suggesting that the analgesic effect is mediated by μ-opioidreceptors.

We had hypothesized that a compound containing both NOP and μ-opioid receptor agonistactivity would have reduced tolerance development to its antinociceptive effects. SR 16435produced a significant albeit not a dramatic decrease in tolerance development to itsantinociceptive effects. SR 16435 produced a 45% decrease in %MPE on Day 9 relative toDay 1, whereas morphine produced a 76% decrease in %MPE resulting in similar levels of %MPE to controls by Day 9. Decreased tolerance development does not seem to be a functionof partial agonist activity of SR 16435 at μ-opioid receptors, since low efficacy μ-opioidagonists, such as buprenorphine, have been shown to display greater tolerance than higherefficacy compounds (Grecksch et al., 2006). Previous studies have shown that both NOPagonists and antagonists can attenuate tolerance to morphine-induced antinociception (Lutfyet al., 2001; (Rizzi et al., 2000; Zaratin et al., 2004). These conflicting studies highlight thecomplicated relationship between NOP activation and μ-opioid-mediated tolerance.

N/OFQ blocks morphine-, cocaine-, and alcohol-induced CPP (Ciccocioppo et al., 1999;Murphy et al., 1999; Ciccocioppo et al., 2002; Sakoori and Murphy, 2004) while having norewarding or aversive effects when administered alone (Devine et al., 1996). Additionally, Ro64−6198, when administered systemically 15 minutes prior to morphine, also attenuatesmorphine-induced CPP (Shoblock et al., 2005). Accordingly, in theory, if a compound hasagonist activity at both NOP and μ-opioid receptors, the NOP agonist activity would attenuatethe rewarding effects of the μ-opioid receptor-mediated component, leading to a compoundwith reduced addiction liability. However, in the present study SR 16435 still produced CPP,thereby demonstrating that even with relatively equal affinities at the two receptors, a decreasein CPP is not evident when both μ-opioid and NOP receptor partial agonist activities are presenttogether in the same molecule. There are two potential explanations for the disparate findingsas to why SR 16435 is rewarding, whereas Ro 64−6198 diminished morphine-induced reward.It is possible that the temporal relationship between μ-opioid and NOP activation is important,such that activation of the two receptors simultaneously by SR 16435 is not sufficient for theNOP receptor to attenuate the actions of the μ-opioid receptor, whereas activation of NOPreceptors by Ro 64−6198 prior to morphine administration was sufficient. Another possibilityis that SR 16435, a partial agonist at NOP, does not have high enough efficacy as Ro 64−6198,to modulate the μ-opioid receptor mediated reward. This possibility is being investigated withadditional higher efficacy compounds from our series of compounds.

Given that NOP receptor activation itself does not produce CPP, SR 16435-induced CPP islikely mediated by μ-opioid receptor activation. This is further substantiated by the observationthat naloxone reversed SR 16435-induced CPP and produced the typical CPA when co-

Khroyan et al.


NIH


NIH


NIH


administered with SR 16435. Previous studies have shown that the presence of μ-opioidreceptors is crucial for acquisition of naloxone-induced CPA since μ-opioid receptor knockoutmice do not exhibit naloxone-induced CPA (Skoubis et al., 2001). Also, it seems that partialagonist activity at μ-opioid receptors is sufficient to produce CPP since, buprenorphine alsoproduces CPP (Tzschentke, 2004). Collectively, these findings suggest that the μ-opioidreceptor is a critical component in mediating the oppositional CPP and CPA produced by opioidagonists and antagonists respectively. Additionally, activation of the NOP receptors maymodulate reward taking into consideration the temporal relationship between μ-opioid andNOP receptor activation and also efficacy at NOP receptors (Murphy et al., 1999; Shoblock etal., 2005).

SR 16435 also produced a decrease in global activity following the first injection but notfollowing repeated administration. NOP agonists administered ICV (UFP-102) andsystemically (Ro 64−6198) produce a decrease in global activity (Kuzmin et al., 2003; Carraet al., 2005). In the present study, systemic administration of the NOP antagonist, SR 16430partially reversed the inhibitory effects of SR 16435. However, naloxone also reversed theinhibitory effects of SR 16435, as well as the decrease in exploratory and grooming behavior.It is highly unlikely that SR 16435-mediated suppression of activity was due to its ability tostimulate μ-opioid receptors since morphine produces an increase in global activity in mice(Figure 5b). Although κ-opioid receptor agonists produce a decrease in locomotor activity(Kuzmin et al., 2000), SR 16435 has no κ-opioid receptor agonist activity as measured by the[35S]GTPγS binding assay (Table 1). Furthermore, a more selective NOP receptor agonist SR14150, from our series, also produced suppression in global activity following the first andfourth injection (manuscript in preparation). Therefore, it seems likely that the activity-inhibitory effects of SR 16435 are mediated through the NOP receptor.

As indicated above, SR 16435-induced decrease in global activity is naloxone-reversible. Thereversal by naloxone of the SR16435-mediated decrease in global activity is not the firstdemonstration of naloxone inhibiting an aspect of NOP-mediated behavior. Indeed, naloxoneblocks spinal N/OFQ-mediated analgesic activity in chronic pain models (Courteix et al.,2004). Furthermore, naloxone can also inhibit the hyperphagic effects of N/OFQ (Polidori etal., 2000). However, the mechanisms underlying naloxone-induced antagonism of NOP-mediated behaviors are unclear. It could be speculated that opioid receptors may interact withNOP receptors and enable NOP-mediated behaviors. However, naloxone could also have ageneral inhibitory effect independent from any system that may be controlling activity or foodintake. Further experiments with more selective agonists and antagonists could shed some lighton the interaction of NOP and opioid receptor systems.

In conclusion, the mixed NOP/μ-opioid ligand, SR 16435, has high binding affinity and is apartial agonist at both sites. SR 16435 has antinociceptive activity and has reduced tolerancedevelopment to its antinociceptive effects. However, SR 16435 induces CPP, suggesting thatthe NOP receptor-mediated component was unable to attenuate the reward induced by the μ-opioid receptor activity. It may be possible, however, to shift the functional balance betweenNOP and μ-opioid receptor stimulation to favor NOP versus μ-opioid receptor-mediatedeffects, by using a compound with full agonist activity at NOP. Such a compound may produceantinociceptive effects with reduced rewarding properties. Compounds with this profile fromour series of bifunctional NOP/μ-opioid ligands are currently being examined in ongoingstudies in our laboratory.

AcknowledgmentsThe authors thank the reviewers and Drs. Girolamo Calo and Barbara Spagnolo for their helpful suggestions.

Khroyan et al.


NIH


NIH


NIH


This research was supported by National Institute on Drug Abuse grant DA14026 to Nurulain Zaveri. Preliminaryreports of these data were presented at the annual meetings of the Society for Neuroscience and the College on Problemsof Drug Dependence.

AbbreviationsN/OFQ, Nociceptin/Orphanin FQ; NOP, receptor, ORL1, opioid receptor-like receptor; ICV,intracerebroventricular; SC, subcutaneous; IP, intraperitoneal; IT, intrathecal; IV, intravenous;PC, place conditioning; CPP, conditioned place preference, CPA, conditioned place aversion.

REFERENCESBunzow JR, Saez C, Mortrud M, Bouvier C, Williams JT, Low M, Grandy DK. Molecular cloning and

tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, deltaor kappa opioid receptor type. FEBS Lett 1994;347:284–288. [PubMed: 8034019]

Calo G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H, Bianchi C, Lambert DG, Salvadori S,Regoli D. Characterization of [Nphe(1)]nociceptin(1−13)NH(2), a new selective nociceptin receptorantagonist. Br J Pharmacol 2000;129:1183–1193. [PubMed: 10725267]

Calo G, Rizzi A, Rizzi D, Bigoni R, Guerrini R, Marzola G, Marti M, McDonald J, Morari M, LambertDG, Salvadori S, Regoli D. [Nphe(1),Arg(14),Lys(15)]nociceptin-NH(2), a novel potent and selectiveantagonist of the nociceptin/orphanin FQ receptor. Br J Pharmacol 2002;136:303–311. [PubMed:12010780]

Carra G, Rizzi A, Guerrini R, Barnes TA, McDonald J, Hebbes CP, Mela F, Kenigs VA, Marzola G,Rizzi D, Gavioli E, Zucchini S, Regoli D, Morari M, Salvadori S, Rowbotham DJ, Lambert DG,Kapusta DR, Calo G. [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selectiveagonist of the nociceptin/orphanin FQ receptor. J Pharmacol Exp Ther 2005;312:1114–1123.[PubMed: 15509719]

Ciccocioppo R, Panocka I, Polidori C, Regoli D, Massi M. Effect of nociceptin on alcohol intake inalcohol-preferring rats. Psychopharmacology (Berl) 1999;141:220–224. [PubMed: 9952048]

Ciccocioppo R, Polidori C, Antonelli L, Salvadori S, Guerrini R, Massi M. Pharmacologicalcharacterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats.Peptides 2002;23:117–125. [PubMed: 11814626]

Courteix C, Coudore-Civiale MA, Privat AM, Pelissier T, Eschalier A, Fialip J. Evidence for an exclusiveantinociceptive effect of nociceptin/orphanin FQ, an endogenous ligand for the ORL1 receptor, in twoanimal models of neuropathic pain. Pain 2004;110:236–245. [PubMed: 15275773]

Devine DP, Reinscheid RK, Monsma FJ Jr. Civelli O, Akil H. The novel neuropeptide orphanin FQ failsto produce conditioned place preference or aversion. Brain Res 1996;727:225–229. [PubMed:8842403]

Dooley CT, Spaeth CG, Berzetei-Gurske IP, Craymer K, Adapa ID, Brandt SR, Houghten RA, Toll L.Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor,ORL1. J Pharmacol Exp Ther 1997;283:735–741. [PubMed: 9353393]

Grecksch G, Bartzsch K, Widera A, Becker A, Hollt V, Koch T. Development of tolerance andsensitization to different opioid agonists in rats. Psychopharmacology (Berl) 2006;186:177–184.[PubMed: 16572262]

Jacob JJ, Michaud GM, Tremblay EC. Mixed agonist-antagonist opiates and physical dependence. Br JClin Pharmacol 1979;7(Suppl 3):291S–296S. [PubMed: 572694]

Jasinski DR, Pevnick JS, Griffith JD. Human pharmacology and abuse potential of the analgesicbuprenorphine: a potential agent for treating narcotic addiction. Arch Gen Psychiatry 1978;35:501–516. [PubMed: 215096]

Jenck F, Wichmann J, Dautzenberg FM, Moreau JL, Ouagazzal AM, Martin JR, Lundstrom K, CesuraAM, Poli SM, Roever S, Kolczewski S, Adam G, Kilpatrick G. A synthetic agonist at the orphaninFQ/nociceptin receptor ORL1: anxiolytic profile in the rat. Proc Natl Acad Sci U S A 2000;97:4938–4943. [PubMed: 10758169]

Khroyan et al.


NIH


NIH


NIH


Kotlinska J, Wichmann J, Rafalski P, Talarek S, Dylag T, Silberring J. Nonpeptidergic OP4 receptoragonist inhibits morphine antinociception but does not influence morphine dependence. Neuroreport2003;14:601–604. [PubMed: 12657894]

Kuzmin A, Sandin J, Terenius L, Ogren SO. Dose- and time-dependent bimodal effects of kappa-opioidagonists on locomotor activity in mice. J Pharmacol Exp Ther 2000;295:1031–1042. [PubMed:11082438]

Kuzmin A, Sandin J, Terenius L, Ogren SO. Acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in mice: effects of opioid receptor-like 1 receptor agonists andnaloxone. J Pharmacol Exp Ther 2003;304:310–318. [PubMed: 12490606]

Le Pen G, Wichmann J, Moreau JL, Jenck F. The orphanin receptor agonist RO 64−6198 does not induceplace conditioning in rats. Neuroreport 2002;13:451–454. [PubMed: 11930159]

Lutfy K, Hossain SM, Khaliq I, Maidment NT. Orphanin FQ/nociceptin attenuates the development ofmorphine tolerance in rats. Br J Pharmacol 2001;134:529–534. [PubMed: 11588106]

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, FerraraP, Monsarrat B, et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1receptor. Nature 1995;377:532–535. [PubMed: 7566152]

Mogil JS, Grisel JE, Zhangs G, Belknap JK, Grandy DK. Functional antagonism of mu-, delta- and kappa-opioid antinociception by orphanin FQ. Neurosci Lett 1996;214:131–134. [PubMed: 8878101]

Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C, Chalon P, Caput D, Vassart G, MeunierJC. ORL1, a novel member of the opioid receptor family. Cloning, functional expression andlocalization. FEBS Lett 1994;341:33–38. [PubMed: 8137918]

Murphy NP, Lee Y, Maidment NT. Orphanin FQ/nociceptin blocks acquisition of morphine placepreference. Brain Res 1999;832:168–170. [PubMed: 10375664]

Obara I, Przewlocki R, Przewlocka B. Spinal and local peripheral antiallodynic activity of Ro64−6198in neuropathic pain in the rat. Pain 2005;116:17–25. [PubMed: 15927383]

O'Callaghan JP, Holtzman SG. Quantification of the analgesic activity of narcotic antagonists by amodified hot-plate procedure. J Pharmacol Exp Ther 1975;192:497–505. [PubMed: 1168252]

Ozaki S, Kawamoto H, Itoh Y, Miyaji M, Azuma T, Ichikawa D, Nambu H, Iguchi T, Iwasawa Y, OhtaH. In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist. Eur J Pharmacol 2000;402:45–53. [PubMed: 10940356]

Polidori C, de Caro G, Massi M. The hyperphagic effect of nociceptin/orphanin FQ in rats. Peptides2000;21:1051–1062. [PubMed: 10998540]

Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, LangenH, Monsma FJ Jr. Civelli O. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 1995;270:792–794. [PubMed: 7481766]

Rizzi A, Bigoni R, Marzola G, Guerrini R, Salvadori S, Regoli D, Calo G. The nociceptin/orphanin FQreceptor antagonist, [Nphe1]NC(1−13)NH2, potentiates morphine analgesia. Neuroreport2000;11:2369–2372. [PubMed: 10943687]

Sakoori K, Murphy NP. Central administration of nociceptin/orphanin FQ blocks the acquisition ofconditioned place preference to morphine and cocaine, but not conditioned place aversion to naloxonein mice. Psychopharmacology (Berl) 2004;172:129–136. [PubMed: 14624329]

Shinkai H, Ito T, Iida T, Kitao Y, Yamada H, Uchida I. 4-Aminoquinolines: novel nociceptin antagonistswith analgesic activity. J Med Chem 2000;43:4667–4677. [PubMed: 11101358]

Shoblock JR, Wichmann J, Maidment NT. The effect of a systemically active ORL-1 agonist, Ro 64−6198, on the acquisition, expression, extinction, and reinstatement of morphine conditioned placepreference. Neuropharmacology 2005;49:439–446. [PubMed: 15919100]

Skoubis PD, Matthes HW, Walwyn WM, Kieffer BL, Maidment NT. Naloxone fails to produceconditioned place aversion in mu-opioid receptor knock-out mice. Neuroscience 2001;106:757–763.[PubMed: 11682161]

Suyama H, Kawamoto M, Gaus S, Yuge O. Effect of JTC-801 (nociceptin antagonist) on neuropathicpain in a rat model. Neurosci Lett 2003;351:133–136. [PubMed: 14623124]

Tian JH, Xu W, Fang Y, Mogil JS, Grisel JE, Grandy DK, Han JS. Bidirectional modulatory effect oforphanin FQ on morphine-induced analgesia: antagonism in brain and potentiation in spinal cord ofthe rat. Br J Pharmacol 1997;120:676–680. [PubMed: 9051307]

Khroyan et al.


NIH


NIH


NIH


Tzschentke TM. Reassessment of buprenorphine in conditioned place preference: temporal andpharmacological considerations. Psychopharmacology (Berl) 2004;172:58–67. [PubMed:14615874]

Ueda H, Inoue M, Takeshima H, Iwasawa Y. Enhanced spinal nociceptin receptor expression developsmorphine tolerance and dependence. J Neurosci 2000;20:7640–7647. [PubMed: 11027224]

Wang JB, Johnson PS, Imai Y, Persico AM, Ozenberger BA, Eppler CM, Uhl GR. cDNA cloning of anorphan opiate receptor gene family member and its splice variant. FEBS Lett 1994;348:75–79.[PubMed: 8026588]

Yamada H, Nakamoto H, Suzuki Y, Ito T, Aisaka K. Pharmacological profiles of a novel opioid receptor-like1 (ORL(1)) receptor antagonist, JTC-801. Br J Pharmacol 2002;135:323–332. [PubMed:11815367]

Zaratin PF, Petrone G, Sbacchi M, Garnier M, Fossati C, Petrillo P, Ronzoni S, Giardina GA, ScheidelerMA. Modification of nociception and morphine tolerance by the selective opiate receptor-like orphanreceptor antagonist (−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). J Pharmacol Exp Ther 2004;308:454–461.[PubMed: 14593080]

Zaveri N. Peptide and nonpeptide ligands for the nociceptin/orphanin FQ receptor ORL1: research toolsand potential therapeutic agents. Life Sci 2003;73:663–678. [PubMed: 12801588]

Zaveri NT, Jiang F, Olsen CM, Deschamps JR, Parrish D, Polgar W, Toll L. A novel series of piperidin-4-yl-1,3-dihydroindol-2-ones as agonist and antagonist ligands at the nociceptin receptor. J Med Chem2004;47:2973–2976. [PubMed: 15163178]

Khroyan et al.


NIH


NIH


NIH


Figure 1.The effects of a range of doses of morphine (a) and SR 16435 (b) on tail flick latency incomparison to vehicle controls. Data are mean %MPE (± SEM). An asterisk (*p<0.05; StudentNewman-Keuls test) represents a significant difference from vehicle control.

Khroyan et al.


NIH


NIH


NIH


Figure 2.The effect of naloxone (1 mg/kg) on SR16435-induced antinociception collapsed across post-injection time (10, 30, or 60 min). Data are mean %MPE (± SEM). An asterisk (*p<0.05;Student Newman-Keuls test) represents a significant difference from vehicle control, whereasa dagger (†p<0.05; Student Newman-Keuls test) represents a significant difference fromSR16435 alone.

Khroyan et al.


NIH


NIH


NIH


Figure 3.To measure tolerance development, the effect of SR 16435 (30 mg/kg) was compared tomorphine (10 mg/kg) by examining tail flick latency tested across 9 days. Data are mean %MPE (± SEM). An asterisk (*p<0.05; Student Newman-Keuls test) represents a significantdifference from vehicle controls, a dagger (†p<0.05; Student Newman-Keuls test) representsa significant difference from the first injection, and a triangle (△p<0.05; Student Newman-Keuls test) represents a significant difference from morphine alone.

Khroyan et al.


NIH


NIH


NIH


Figure 4.The effect of SR16435 alone on (a) place conditioning and (b) global activity. Data are mean(± SEM): time (s) spent in drug-paired compartment (a) or activity (cm) following first andlast drug injection. An asterisk (*) represents a significant difference from vehicle controls,whereas a dagger (†) represents a significant difference from first injection (Student Newman-Keuls test, P<0.05).

Khroyan et al.


NIH


NIH


NIH


Figure 5.The effect of naloxone (1 mg/kg) on (a) SR 16435-induced conditioned place preference and(b) inhibition of global activity. Data are mean (± SEM): (a) time (s) spent in drug-pairedcompartment or (b) activity (cm) following first and last drug injection. An asterisk (*)represents a significant difference from vehicle controls (Student Newman-Keuls test, P<0.05).

Khroyan et al.


NIH


NIH


NIH


Figure 6.

Khroyan et al.


NIH


NIH


NIH


NIH


NIH


NIH


Khroyan et al. Ta

ble

1B

indi

ng a

ffin

ities

and

fun

ctio

nal

activ

ities

of

know

n an

d SR

I co

mpo

unds

at

NO

P an

d op

ioid

rec

epto

rs c

ompa

red

to N

/OFQ

,bu

pren

orph

ine,

and

mor

phin

e.a

NO

P re

cept

orμ-

opio

id r

ecep

tor

κ-op

ioid

rec

epto

r

Com

poun

dK

iE

C50

% S

timK

iE

C50

% S

timK

iE

C50

% S

tim

N/O

FQ0.

2 ±

0.04

4.0

± 0.

110

013

3 ±

30N

.D.

N.D

.N

.D.

N.D

.

Mor

phin

eN

.DN

.D.

1.1

± 0.

0515

.6 ±

0.5

93 ±

2.8

46.9

± 1

4.5

484

± 21

362

± 7

SR 1

6435

b7.

49 ±

0.7

828

.7 ±

0.6

45.0

± 5

2.70

± 0

.05

29.5

± 1

030

± 0

31.7

4 ±

4.8

>10K

SR 1

6430

6.49

± 1

.39

>10K

60.9

5 ±

15>1

0K21

9.47

± 1

8>1

0K

Bup

reno

rphi

ne77

.42

± 16

>10K

1.5

± 0.

824

.9 ±

14

17.7

± 0

.41.

5 ±

0.25

>10K

a Rec

epto

r bin

ding

and

[35 S

]GTP

γS b

indi

ng w

as c

ondu

cted

in C

HO

cel

l mem

bran

es c

onta

inin

g th

e ap

prop

riate

hum

an re

cept

or, a

s des

crib

ed in

(Doo

ley

et a

l., 1

997)

. Bin

ding

con

stan

ts a

re sh

own

in

Ki ±

S.D

. for

eac

h co

mpo

und,

whi

ch a

re d

eriv

ed fr

om a

t lea

st tw

o in

divi

dual

exp

erim

ents

con

duct

ed in

trip

licat

e. A

n EC

50 v

alue

of >

10,0

00 fo

r [35

S]G

TPγS

bin

ding

indi

cate

s no

sign

ifica

nt st

imul

atio

nat

that

con

cent

ratio

n. N

.D. i

ndic

ates

that

it w

as n

ot d

eter

min

ed.

b Take

n fr

om Z

aver

i et a

l. (2

004)

.


NIH


NIH


NIH


Khroyan et al.

Table 2The effects of the SR 16430 on SR 16435-induced decrease in global activity

Dose of SR 16435 Dose of SR 16430

0.0 mg/kg 30.0 mg/kg

0.0 mg/kg 3430.99±290.15† 2638.98±94.12†

10.0 mg/kg 825.8±173.85* 1861.03±283.18*†

*represents a significant difference from vehicle controls

†represents a significant difference from SR 16435 alone (P<0.05).


NIH


NIH


NIH


Khroyan et al. Ta

ble

3B

ehav

iors

elic

ited

follo

win

g ac

ute

and

repe

ated

dru

g ad

min

istra

tion

Inci

denc

e of

Gro

omin

gIn

cide

nce

of S

niffi

ngIn

cide

nce

of R

eari

ng

Dos

eIn

ject

ion

1In

ject

ion

4In

ject

ion

1In

ject

ion

4In

ject

ion

1In

ject

ion

4

Veh

icle

22.6

7 ±

1.81

30.8

7 ±

3.9

63.3

3 ±

2.63

58.8

7 ±

3.42

25.2

7 ±

1.83

20.1

3 ±

2.13

Mor

phin

e (1

5 m

g/kg

)2.

38 ±

1.1

8*5.

38 ±

0.9

4*62

.75

± 1.

5269

.75

± 2.

173.

75 ±

0.7

0*9.

75 ±

1.7

3*†

SR 1

6435

(10

mg/

kg)

1.13

± 0

.88*

0.0

± 0.

0*0.

25 ±

0.2

6*0.

0 ±

0.0*

1.38

± 1

.02*

0.63

± 0

.50*

SR 1

6435

(30

mg/

kg)

0.50

± 0.

33*

0.25

± 0

.25*

0.13

± 0

.13*

2.00

± 2

.00*

1.13

± 0

.88*

0.50

± 0

.50*

SR 1

6435

(10

mg/

kg) +

Nal

oxon

e (1

mg/

kg)

26.7

5 ±

5.34

30.1

4 ±

2.64

59.0

0 ±

2.69

54.7

1 ±

3.27

26.2

5 ±

3.59

17.1

4 ±

2.11

* repr

esen

ts a

sign

ifica

nt d

iffer

ence

from

veh

icle

con

trols

† repr

esen

ts a

sign

ifica

nt d

iffer

ence

from

firs

t inj

ectio

n (S

tude

nt N

ewm

an-K

euls

test

, P<0

.05)

.


SR 16435 [1-(1-(Bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed...

Documents

Transcript of SR 16435 [1-(1-(Bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed...