preclinical pharmacology of azd2327: a highly selective agonist of

Preclinical Pharmacology of AZD2327: A Highly SelectiveAgonist of the �-Opioid Receptor

T. J. Hudzik, C. Maciag, M. A. Smith, R. Caccese, M. R. Pietras, K. H. Bui, M. Coupal,L. Adam, K. Payza, A. Griffin, G. Smagin,1 D. Song,1 M. D. B. Swedberg, and W. BrownAstraZeneca Research and Development, Wilmington, Delaware (T.J.H., C.M., M.A.S., R.C., M.R.P., K.H.B., G.S., D.S.);AstraZeneca Research and Development, Montreal, Quebec, Canada (M.C., L.A., K.P., A.G., W.B.); and AstraZeneca Researchand Development, Sodertalje, Sweden (M.D.B.S.)

Received January 18, 2011; accepted March 24, 2011

ABSTRACTIn the present article, we summarize the preclinical pharmacol-ogy of 4-{(R)-(3-aminophenyl)[4-(4-fluorobenzyl)-piperazin-1-yl]methyl}-N,N-diethylbenzamide (AZD2327), a highly potentand selective agonist of the �-opioid receptor. AZD2327 bindswith sub-nanomolar affinity to the human opioid receptor (Ki �0.49 and 0.75 nM at the C27 and F27 isoforms, respectively)and is highly selective (�1000-fold) over the human �- and�-opioid receptor subtypes as well as �130 other receptorsand channels. In functional assays, AZD2327 shows full ago-nism at human �-opioid receptors ([35S]GTP� EC50 � 24 and9.2 nM at C27 and F27 isoforms, respectively) and also at therat and mouse �-opioid receptors. AZD2327 is active in a widerange of models predictive of anxiolytic activity, including amodified Geller-Seifter conflict test and social interaction test,

as well as in antidepressant models, including learned helpless-ness. In animals implanted with microdialysis probes and thengiven an acute stressor by pairing electric shock delivery with aflashing light, there is an increase in norepinephrine release intothe prefrontal cortex associated with this acute anxiety state.Both the benzodiazepine anxiolytic standard diazepam andAZD2327 blocked this norepinephrine release equally well, andthere was no evidence of tolerance to these effects ofAZD2327. Overall, these data support the role of the �-opioidreceptor in the regulation of mood, and data suggest thatAZD2327 may possess unique antidepressant and anxiolyticactivities that could make a novel contribution to the pharma-cotherapy of psychiatric disorders.

IntroductionDespite a growing number of treatment options available

to patients with major depressive disorder, a significant pro-portion of patients remain untreated or undertreated (War-den et al., 2007). This incomplete treatment response is likelydue to a number of factors, including disease heterogeneity,and certainly to a great overlap in the mechanisms of actionof the available therapies. It is likely that if the treatmentgap is to be filled, mechanistically distinct treatment ap-proaches will be needed.

The involvement of the opioid system in emotional regula-tion has been suggested for decades (Emrich et al., 1979;

Tejedor-Real et al., 1995), although understanding of it iscomplicated by the multiplicity of receptor subtypes and en-dogenous ligands and potential hetero-oligomerization of theopioid receptors with each other as well as with other Gprotein-coupled receptors and ion channels (Walwyn et al.,2009). The three historically defined opioid subtypes are�-opioid receptors, named after the prototypic agonist mor-phine; �-opioid receptors, named after the benzomorphanopioid ketocyclazocine; and �-opioid receptors, named afterobservations of effects of agonists in a mouse vas deferensassay. All three opioid receptors are thought to affect nocice-ptive and emotional responses, albeit in qualitatively differ-ent manners. Endorphins and enkephalins [primarily Met-enkephalin (FK 33-824)] are the endogenous ligands for�-opioid receptors, and �-opioid receptor agonists produceeuphorigenic responses and robust analgesia (both spinaland supraspinal) for acute nociceptive stimuli. Limitations of

1 Current affiliation: Lundbeck Research USA, Paramus, New Jersey.Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.111.179432.

ABBREVIATIONS: FK 33-824, Met-enkephalin; AZD2327, 4-{(R)-(3-aminophenyl)[4-(4-fluorobenzyl)-piperazin-1-yl]methyl}-N,N-diethylbenz-amide; AR-M100390, N,N-diethyl-4-[phenyl(piperidin-4-ylidene)methyl]benzamide; ADL5747, [N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4’-pip-eridine]-4-yl)benzamide]; SNC-80, 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethyl-piperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethyl-benzamide; DPDYN,D-Pro10-dynorphin A(1-11); U69593, N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide; NE, norepinephrine;MHPG, 3-methoxy-4-hydroxyphenolglycol; ANOVA, analysis of variance; PTZ, pentylenetetrazole; ADL5859, N,N-diethyl-4-(5-hydroxyspiro-[chromene-2,4�-piperidin]-4-yl)benzamide; HEK, human embryonic kidney.

0022-3565/11/3381-195–204$25.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 338, No. 1Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 179432/3692920JPET 338:195–204, 2011 Printed in U.S.A.

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their therapeutic use include abuse liability and markedtolerance to the analgesic effects, requiring increases in dose,but with little or no tolerance to other, undesired effects, suchas respiratory depression or decreases in gastrointestinalmotility. The clinical use of �-opioid receptor antagonists hasbeen limited largely to rescue after opiate overdose but hasbeen shown more recently to attenuate the euphorigeniceffects of other drugs of abuse, such as alcohol, thereby fur-ther suggesting the potential involvement of the opioid sys-tem in emotional regulation. The endogenous dynorphinsbind to �-opioid receptors and are thought to negatively reg-ulate mood as well as mediate visceral and spinal analgesicresponses. Although agonists are known to produce dyspho-rigenic and, in some cases, hallucinogenic effects (Walentinyet al., 2010), there are preclinical data to suggest that �-opi-oid receptor antagonists may be useful antidepressants (Carret al., 2010). Endogenous Leu-enkephalin somewhat prefer-entially interacts with �- versus �-opioid receptors, and aconcordance of data would suggest that more selective ago-nists of the receptor will result in positive modulation ofmood, anxiolytic effects (Nieto et al., 2005), and analgesia,which may be related to these affective effects of agonists.Unlike �-opioid receptor agonists, �-opioid receptor agonistsproduce minimal effects on respiratory and gastrointestinalsystems (Porreca et al., 1984). Several studies have shownthat �-opioid receptor agonists lack or have minimal reinforc-ing effects (Negus at al., 1998; Do Carmo et al., 2009), indi-cating a low liability for abuse. However, �-opioid receptoragonists do introduce a higher risk of producing convulsionsthan agonists at the other receptor subtypes. Identification ofthe �-opioid receptor as a possible target in the treatment ofanxiety and depression began with observations of an anx-ious and depressive-like phenotype in the �-opioid receptorknockout mouse (Filliol et al., 2000). Since then, a number oflaboratories have demonstrated the efficacy of several pep-tidic and nonpeptidic opioids of varying selectivities for the�-opioid receptor in assays for antidepressant activity (re-viewed by Jutkiewicz, 2006).

4-{(R)-(3-Aminophenyl)[4-(4-fluorobenzyl)-piperazin-1-yl]methyl}-N,N-diethylbenzamide (AZD2327) is a nonpeptidic,full and highly selective agonist at the �-opioid receptor, amongonly a handful to be described in the literature over the last fewdecades since the receptor was first described (Plobeck et al.,2000; Wei et al., 2000). Its structure, along with those of a

number of other nonpeptidic �-opioid agonists, is shown inFig. 1. All of the compounds shown have been described as full�-opioid receptor agonists but nevertheless can have some im-portant differences in their pharmacologies. For example,SNC-80 (4-[(R)-[(2S,5R)-4-allyl-2,5-dimethyl-piperazin-1-yl]-(3-methoxyphenyl)methyl]-N,N-diethyl-benzamide) has beenshown to reliably stimulate locomotor activity and to produceconvulsions in a number of preclinical species (Broom et al.,2002a; Jutkiewicz et al., 2005), whereas there have been noreports of either seizure activity or alteration in locomotor ac-tivity with AR-M100390 (N,N-diethyl-4-[phenyl(piperidin-4-ylidene)methyl]benzamide) (Pradhan and Clarke, 2005;Smagin et al., 2008; T. Hudzik, unpublished observations),which may represent the opposite end of the spectrum of activ-ity. In the present article, we focus upon the biochemical andbehavioral pharmacology of a novel compound, AZD2327, andwork conducted to determine the clinical utility of the com-pound in the treatment of psychiatric disorders. The safetypharmacology of AZD2327 will be addressed in a separate report.

Materials and MethodsIn Vitro Pharmacology. Radioligands. Deltorphin II, FK 33-

824, and D-Pro10-dynorphin A(1-11) (DPDYN) were iodinated with[125I]Na and then purified by high-performance liquid chromatogra-phy to apparent homogeneity (2200 Ci/mmol) as described previously(Payza, 2003).

Cell lines and membrane preparations. As described previously(Payza, 2003), human embryonic kidney (HEK) 293S cells were usedto express all three human opioid receptor subtypes. The human�-opioid receptors were expressed with both of the prevalent single-nucleotide polymorphisms at position 27 (Adam et al., 2001): onewith cysteine (GenBank accession number U07882; C27) and theother with phenylalanine (GenBank accession number U10504;F27). Both clones were used because both are represented in thegeneral population, although the F27 isoform is more prevalent. TheC27 clone 11 cell line (Bmax 9.7 pmol/mg protein) was used forreceptor binding assays, and lower expressing clone 2 (0.69 pmol/mgprotein) was used for [35S]GTP� assays. In the case of the F27isoform, clone 4 (Bmax 3.7 pmol/mg protein) was used for both bindingand functional assays. Suspension conditions were used for the pro-duction of membranes expressing the human �-opioid receptor iso-form C27 as well as the human �- and �-opioid receptors. The cellswere grown, harvested, and processed into membrane preparationsexactly as described previously (Payza, 2003). In the case of thehuman �-opioid receptor isoform F27, culture was done under adher-

Fig. 1. Structures of representative �-opioid receptor agonists. ADL5859 [N,N-diethyl-4-(5-hydroxyspiro[chromene-2,4�-piperidin]-4-yl)benzamide]and closely related ADL5747 [N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4’-piperidine]-4-yl)benzamide] have been in clinical development as anal-gesics. AR-M100390 (sometimes referred to as ARM390) is a tool compound from AstraZeneca that has been published previously (Wei et al., 2000;Smagin et al., 2008; Pradhan et al., 2010).

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ent conditions in normal Dulbecco’s modified Eagle’s medium, 10%fetal bovine serum, 2 mM glutamine, and 600 �g/ml Geneticin,followed by membrane preparation using the same method as thatfor suspension cells. Rat brain membranes were prepared as de-scribed previously (Fraser et al., 1999).

Affinity binding assays. The Ki values of AZD2327 were deter-mined in membrane-based competitive binding assays exactly asdescribed previously (Payza, 2003). In brief, AZD2327 was tested forits ability to compete against the binding of [125I]Deltorphin II to�-opioid receptors and against [125I]FK 33-824 and [125I]DPDYN forthe �- and �-opioid receptors, respectively. Radioligands were usedat concentrations of 0.03 to 0.05 nM. In all cases, the buffer was 50mM Tris, 3 mM MgCl2, and 0.1% bovine serum albumin (pH 7.4), and10 �M naloxone was used to define nonspecific binding. Internalstandards were SNC-80 (for �), [D-Ala2,N-MePhe4,Gly-ol]-enkepha-lin (for �), and U69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrro-lidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide) (for �). All of the selec-tivity ratios were determined using high-affinity binding conditionsamong each of the receptor subtypes.

[35S]GTP� binding assays. AZD2327 was tested for agonism inmembrane-based [35S]GTP� filtration binding assays using the exactexperimental procedure and method to determine EC50 and Emax asdescribed previously for the cloned human �-opioid receptors (Payza,2003) and for endogenous �-opioid receptors in rat brain membranes(Fraser et al., 1999). In brief, the assays were performed in 96-wellplates in 50 mM HEPES, 20 mM NaOH, 200 mM NaCl, 1 mM EDTA,5 mM MgCl2, 1 mM dithiothreitol, and 0.5% bovine serum albumin(pH 7.4), with GDP added a concentration of 3 �M (for C27 and F27isoforms of the human �-opioid receptor) or 45 �M (for rat brainmembranes). Incubation was for 1 h at room temperature. SNC-80(10 �M) was used to define 100% Emax.

Electrically stimulated tissue assay. Mouse vas deferens, a stan-dard �-opioid receptor agonism assay, and guinea pig ileum, a stan-dard �-opioid receptor agonism assay, were performed at CEREP(Poitiers, France) using established methods exactly as describedpreviously (Gengo et al., 2003).

In Vivo Microdialysis. Animals. Sixty-four male Sprague-Daw-ley rats (Charles River Laboratories, Inc., Wilmington, MA) wereused in this experiment. Animals were housed in a temperature-controlled vivarium with free access to food and water.

Probe implantation and measurements. Under ketamine (60 mg/kg) and xylazine (8 mg/kg) anesthesia, animals were implanted witha guide cannula with a dummy insert (CMA/12) aimed into theprefrontal cortex using the following coordinates: AP (anterior-pos-terior) � 3.2 mm, L (lateral) 1.6 mm, V (ventral) 1.0 mm. Theguide cannulae were anchored to the skull with screws and dentalcement. Animals were allowed to recover from surgery for 6 days.Microdialysis probes (CMA/12, 4-mm membrane length) were im-planted in the brain 18 h before the experiment and were perfusedwith artificial cerebrospinal fluid (CMA Microdialysis, NorthChelmsford, MA) at a flow rate of 1.1 �l/min. Neurotransmitterlevels were assessed by high-performance liquid chromatography(model 5200A Coulochem II detector, MD-150 3 � 150 mm column,model 5041 Amperometric cell; all from ESA, Inc.) with an on-lineinjector (BAS Bioanalytical Systems, West Lafayette, IN). The mo-bile phase was 75 mM Na2HPO4, 25 mM EDTA, 1.7 mM 1-octane-sulphonic acid, 100 �l/l triethylamine, and 10% acetonitrile (pH 3.0).The potential was set at �0.65 V, and the flow rate was maintainedat 0.3 ml/min. Three 20-min samples were collected to define thebaseline, animals were given vehicle or compounds, and samplecollection was carried out for the next 5 h. Concentrations of neu-rotransmitters in three samples collected before the administrationof compounds or vehicle were averaged and defined as the baseline(100%). Concentrations of neurotransmitters in the subsequent mi-crodialysates then were expressed as percentages of baseline levels.Statistical analyses for microdialysis studies were performed usingrepeated measures multivariate analysis of variance (ANOVA) fol-lowed by Dunnett’s post hoc test.

Stress procedure. Standard avoidance chambers equipped withlights and shockers were used (MED Associates, St. Albans, VT).Boxes were placed in sound-attenuating chambers. The stress con-ditioning paradigm was conducted over 1 day. Animals were accli-mated to the chambers for 18 h before conditioning. Conditioningconsisted of turning on and off the houselight in the chamber at arate of two times per second for 2 s (conditioned stimulus), followedby delivery of electric current to the feet of the rat via the grid floorin the chambers (unconditioned stimulus: 0.5-s duration, 1.8-mAintensity, total 10 shocks). The “no stress” group was exposed tochambers with lights but did not receive shocks. Two hours after thestart of the stress paradigm, the light sequence was repeated, but noshocks were administered. The eight experimental groups were as-signed as shown in Table 1.

All of the experimental compounds and vehicle (0.9% phosphate-buffered saline) were administered orally 1 h before the stress pro-cedure. The AZD2327 doses that were tested were 0.3, 1, and 3mg/kg. Diazepam (Sigma-Aldrich, St. Louis, MO) was given at a doseof 3 mg/kg, in a vehicle of Abbott’s cocktail.

To address whether tolerance developed to the neurochemicaleffects of AZD2327, groups of 16 naive rats were administered orally1 mg/kg AZD2327 daily for either 1, 14, or 21 days, and half of theanimals in each group were conditioned as described above 1 h aftertheir last doses, and the other half were placed simply into theconditioning chamber for the same period of time as those groupsreceiving footshock. Two vehicle control groups also were included:one that received conditioning after a single administration of vehi-cle and a second that did not receive conditioning after administra-tion of vehicle. Three additional groups of four animals were treatedwith 3 mg/kg p.o. diazepam for 1, 14, and 21 days and submitted toconditioning. Forty minutes after conditioning, the brains were re-moved rapidly and snap-frozen. Medial prefrontal cortex was dis-sected from half of the brains in each treatment group, homogenized,and assayed for norepinephrine (NE) and 3-methoxy-4-hydroxyphe-nolglycol (MHPG) (a NE catabolite) in 0.1 M perchloric acid usingelectrochemical detection (Coulochem 5011; ESA, Inc., Chelmsford,MA) after separation by high-performance liquid chromatography ona reverse-phase C18 column (Ultrasphere ODS, 4.6 � 250 mm;Beckman Coulter, Inc., Fullerton, CA). Striatal membranes from theremaining brains in each treatment group (except for diazepam-treated rats) were prepared individually and assayed for agonistactivity as described above.

Anxiolytic Activity: Modified Geller-Seifter Conflict Test.Animals. Male Long-Evans rats weighing 375 to 425 g were used. Animalswere maintained at 80 to 90% of their free-feeding weights by limitedfeeding after the experimental session. For any given drug test, rats whoseresponses were most stable were chosen from a larger pool of animalstrained as described below. Several doses were tested on a given test day indifferent subjects. A minimum of eight animals was used for each datapoint.

Apparatus. Standard two-lever operant chambers were used (MEDAssociates). The chambers were fitted with two retractable responselevers and a stimulus lamp over each of the two levers. A pellet dis-penser delivered 45 mg of food pellets (Bio-Serv, Frenchtown, NJ) to acup located inside the chamber below and between the two responselevers. A lamp at the top and back of the chamber served as thehouselight. The grid floors of the operant chambers were interfaced toshock generators and scramblers (MED Associates, St. Albans, VT). Allof the events in the chambers were controlled and monitored by amicroprocessor.

TABLE 1Eight experimental groups assigned (n � 8 mice per group)

Vehicle: No Stress Vehicle: Stress

Diazepam (3 mg/kg p.o.): stress AZD2327 (0.1 mg/kg p.o.): stressAZD2327 (3 mg/kg p.o.): no stress AZD2327 (0.3 mg/kg p.o.): stressAZD2327 (3 mg/kg p.o.): stress Diazepam (3 mg/kg p.o.): no stress

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Procedure. There were two components in the procedure: 1) un-suppressed response components (unpunished) 2 min in durationand 2) suppressed response components (punished) 3 min in dura-tion. In unpunished components, the houselights and both stimuluslamps over the response levers were turned on, the lever on theleft-hand side of the chamber was extended, and a food pellet wasdelivered after an average of 17 responses on the lever in the cham-ber (range, 3–40 responses)—a variable-ratio 17 schedule. The pun-ished components followed unpunished components, and duringthese, the right-hand lever was extended into the chamber, and thestimulus lamps and houselights were turned on and off at 1-s inter-vals, in succession, which served as a cue for this component. In thepunished component, food was also available under a variable-ratio17 schedule, but in addition, electrical current (0.5-s duration) wasdelivered to the grid floor of the chamber under an independentvariable-ratio 17 schedule. The level of the current was adjusted foreach individual subject until response was reduced in the suppressedcomponent to a level that was approximately 5 to 10% that of theunpunished component and ranged from 0.2 to 0.75 mA. Unpunishedand punished components were separated by 10-s time-out periods inwhich both response levers were retracted and all of the stimuluslamps were turned off. The 2-min unpunished and 3-min punishedcomponents alternated until five of each were completed. Daily ses-sions always began with the unpunished response component.

The dependent variables recorded were the rate of response inunpunished and punished components (total responses/total timeunder the component) and the number of shocks delivered. A selec-tive anxiolytic effect is defined as an increase in response in thepunished components with relatively less or no effect on response inunpunished components. The t tests were used to compare the meanof the control group’s rate of response to vehicle for the rats used fora specific dose to the same rats’ means after delivery of each dose ofcompound (for only the rats used within each dose).

Dosages of the compound were reported as the free base. AZD2327was dissolved in distilled deionized water/85% lactic acid. Naltrin-dole was dissolved in saline and was given subcutaneously in com-bination with AZD2327 given orally. All of the compounds were givenin a dose volume of 1 ml/kg b.wt. AZD2327 was tested 1 h after drugadministration, and naltrindole was administered 20 min beforetesting. Drugs were administered on Tuesday and Friday, and vehi-cle was administered on Thursday. Animals were run in baselineoperant sessions on Mondays and Wednesdays.

Social Interaction Test. Animals. Male Long-Evans ratsweighing 250 to 300 g were used. Animals were housed individuallyduring the duration of the experiment and allowed free access to foodand water, except during experimental testing.

Apparatus. A circular steel arena (33 cm in height 33 cm, 118 cmin diameter) was used. The arena was illuminated to 1000 lux, andactivity was monitored from an adjacent room by a video camera.

Procedure. The rats were pre-exposed individually to the testunder low (�100 lux) light level for a 10-min period on each of the 2days preceding the test. The animals then were allocated to testpartners on the basis of weight (closely matched) and divided intodrug groups. On the test day, the pairs of rats were placed in thecenter of the area under high light (�1000 lux) and were scored fora 10-min period from a video monitor in an adjacent room in additionto one observer. The following behaviors were scored and classifiedas active social interaction: sniffing, following, grooming, mounting,and crawling under or over the partner. The total time spent inactive interaction then was reported. Mean scores of the drug-treated groups were compared with those of the vehicle-treatedgroups by Student t tests. AZD2327 (1 and 10 mg/kg) was adminis-tered 30 min before the session. Chlordiazepoxide (7.5 mg/kg i.p.)was administered 20 min beforehand. Both rats were administeredequivalent doses of the same compound.

Antidepressant Activity: Learned Helplessness. Animals.Sixty-four male Sprague-Dawley rats were used in the present stud-

ies. Subjects weighed on average 275 to 325 g at the start of thestudy.

Apparatus. Standard shuttle cages (20 cm in length, 16 cm inwidth, 21 cm in height) fitted with a grid floor were used. Thechambers could be partitioned with a closed partition or with anarchway that allowed the animals to pass between the two sides ofthe cage. A computer controlled and monitored all of the events inthe chamber.

Procedure. The procedure consisted of two phases: induction andavoidance training. In the induction phase, mice were enclosed intoone side of the shuttle cages, and electrical stimulation (2 mA, 9.9-sduration) was delivered to the floor of the cage every 2, 5, or 10 s(randomly selected for each trial) until 90 shocks were delivered.Subjects had no opportunity to escape or to avoid shocks. Inductionwas conducted for 2 consecutive days. In the avoidance trainingphase, testing also was conducted in the shuttle cages, except all ofthe cages then were fitted with a partition with an arch in the centerof the cage through which animals could pass between the left andright halves of the cage. The procedure used was a standard shuttleavoidance in which a compound, conditioned stimulus (a 5-s presen-tation of a tone and turning on of a lamp on the side of the cage thatthe subject was occupying) served to indicate the impending presen-tation of electrical current to the floor of the cage. Shock was pre-sented for a 5-s period, 5 s after initiation of the stimulus. Entry intothe opposite side of the shuttle cage via the arched partition beforeshock onset resulted in the end of the trial (avoidance response). If ashock was delivered, then entry into the opposite side of the cageresulted in the termination of the shock and the CS (escape re-sponse). A 30-s intertrial interval was used. Forty-minute avoidancetraining sessions consisting of 50 trials were conducted on 2 consec-utive days, beginning 48 h after the final induction session.

Drug administration and preparation. Dosages of all of the com-pounds are reported as the free base. Imipramine and AZD2327 weredissolved in distilled deionized water and were administered orallyin a volume of 1 ml/kg b.wt. Drug was administered twice dailythroughout the experiment, immediately after conditioning andtraining sessions and approximately 7 to 8 h after the first injection,as well as on the day in the middle of the study when no conditioningor training was conducted.

Data analysis. The primary dependent variable was escape fail-ures during avoidance training. In addition, because some �-opioidreceptor agonists have been shown to produce locomotor stimulation,center crossings during avoidance training also were recorded andcompared among groups, which allows a gauge of motor activity. Anincrease in center crossings with respect to the vehicle control sug-gests that locomotor stimulation may be at least partly responsiblefor the putative antidepressant effects of the compound. The t testswere used to compare the performance of the vehicle-administeredgroup to those of the drug-treated groups. The no-induction groupwas used to gauge whether learned helplessness was established bycomparison with the vehicle-treated group.

Secondary Pharmacology. All of the in vivo safety pharmacol-ogy studies described below were conducted using the standard ve-hicle (phosphate-buffered saline) with the drug delivered orally.There were no differences in the pharmacokinetics noted among thedifferent formulations used in the present report.

Locomotor Activity. Subjects. Groups of six naive maleSprague-Dawley rats (250–300 g) were used for each data point.Animals were given free access to food and water, except duringexperimental sessions.

Apparatus. An Open Field Activity System (MED Associates) wasused to assess spontaneous locomotor activity in separate groups ofanimals. The system consisted of eight locomotor activity boxes(ENV 515; MED Associates) of clear acrylic with an inside area of43.2 � 43.2 � 30.4 cm, housed in melamine sound-attenuatingcubicles (ENV 017; MED Associates) with house lights and exhaustfans. Three 16-beam infrared arrays, two placed at 2.5 cm (floorlevel) and one at 12.5 cm, were used to measure activity. All of the

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beam breaks and activity in the boxes were recorded by ActivityMonitor software (MED Associates).

Procedure. On the day of the experiment, male Sprague-Dawleyrats (Scanbur BK, Sollentuna, Sweden) weighing 200 to 250 g wereadministered the appropriate doses and placed individually into alocomotor activity box for a 60-min test session. The rats were re-moved, and the boxes were cleaned after the conclusion of the testsession. This procedure was repeated for all of the animals in theexperiment. Animals were distributed evenly over doses and time ofday. The total mean of ambulatory activity was collected in 5-minintervals during the test session, and ANOVA was used to comparemeans. Drug was administered orally.

Irwin’s Test. The rat Irwin test was performed to assess centralnervous system side effects. Six male Wistar rats were administeredorally vehicle or 0.5, 1.5, 5, 15, or 50 mg/kg AZD2327. Each animalthen was tested for signs of behavioral, autonomic, neurological, ortoxic effects at 15 and 30 min, 1, 2, 4, and 24 h after administration.

Pentylenetetrazole Seizure Thresholds. Six Wistar Han ratsweighting approximately 250 to 300 g were used for each data point.Approximately 10 min before the pentylenetetrazole (PTZ) infusion,the rat was placed in a restrainer cage, and a catheter (Neoflon 24G;BD Biosciences, Helsingborg, Sweden) was inserted into the tail veinfor the administration of PTZ. The 100 �M/kg per minute infusion ofPTZ started 60 min after the oral administration of the test doses ofAZD2327. When the first clonic convulsion was observed, the infu-sion was stopped, and the rat was sacrificed immediately with abolus infusion of a lethal dose of 100 mg/ml pentobarbital (Avlivn-ingsvatska; Apoteket AB, Stockholm, Sweden) via the Neoflon cath-eter. The volume of PTZ infused at the time of the appearance of thefirst clonic convulsion was read off the injection pump, and the doseof PTZ received was calculated and given in micromoles per kilo-gram. ANOVA was used to compare means.

Ethics. This study was conducted under protocols that have beenapproved by an ethical committee. The animals were kept and ex-periments were performed at AstraZeneca Research and Develop-ment (Wilmington, DE), which has accreditation from Association forthe Assessment and Accreditation of Laboratory Animal Care and isapproved by the AstraZeneca Global Veterinary Council for studyconduct.

ResultsReceptor Binding Assays. The activities of AZD2327 at

both isoforms of the human �-opioid receptor and at thehuman �- and �-opioid receptors are shown in Fig. 2.AZD2327 inhibited, in a dose-dependent manner, the bindingof [125I]Deltorphin II to the human �-opioid receptor isoformsC27 and F27 and rat brain �-opioid receptor with potent Ki

values; in contrast, its inhibition of [125I]FK 33-824 and[125I]DPDYN binding to human �- and �-opioid receptorswas much weaker (Table 2). The selectivity ratios ofAZD2327 for the human �-opioid receptor isoforms C27 andF27 versus human �- and �-opioid receptors were in allinstances, regardless of which clone was used, �1000-fold.AZD2327 also was tested in a broad screening panel of 130targets and was without significant activity in the samepotency range as its �-opioid receptor binding (data notshown).

Receptor Agonism Assays. AZD2327 behaved as a mod-erately potent, full agonist on both human �-opioid receptorisoforms C27 and F27 as well as on endogenous �-opioidreceptors in rat brain membranes (Fig. 3), with potency val-ues shown in Table 3. In low-affinity conditions (Fraser et al.,1999), the binding potency of AZD2327 was shifted to theright, as expected for G protein-coupled receptor agonists, by33- and 39-fold for the human �-opioid receptor isoforms F27and C27, respectively (data not shown). In the mouse vasdeferens, a standard �-opioid receptor assay, AZD2327showed full agonism with an EC50 value of 1.7 nM (data notshown). In the guinea pig ileum, a standard �-opioid receptorassay, it showed very weak activity, eliciting only 33% Emax

at a concentration of 10 �M (data not shown).Neurochemical Assays and Tolerance Development.

In microdialysis, delivery of footshock produced a 60 to 70%increase in NE content in the medial prefrontal cortex thatwas sustained for up to 3 h after the initial stressor (Fig. 4).Consequent increases in MHPG were observed �1 h after theinitial increases in NE. Pretreatment with diazepam (3 mg/kg, a dose that was chosen due to consistent efficacy notedhistorically in our laboratories) fully reversed these effects(Fig. 4). Likewise, 1 and 3 mg/kg AZD2327 reversed theneurochemical changes measured after footshock (Fig. 5). Instudies in which AZD2327 was administered for up to 21days followed by ex vivo neurochemistry and binding and

Fig. 2. Representative displacement curves for AZD2327 with variousmembrane preparations of HEK293S stable cell lines expressing thehuman �-opioid receptor F27 isoform in high-affinity (E) and low-affinityconditions (�), the human �-opioid receptor C27 isoform in high-affinity(Œ) and low-affinity conditions (f), the human �-opioid receptor (�), andthe human �-opioid receptor (�) and on membranes prepared from ratbrain (F). Points represent the means of duplicates. [125I]Deltorphin IIwas used to label �-opioid receptors under high-affinity conditions and[125I]AR-M100613 was used under low-affinity conditions, [125I]FK 33-824 was used to label �-opioid receptors, and [125I]DPDYN was used tolabel �-opioid receptors. Further details are summarized in Table 3 and inthe text.

TABLE 2AZD2327 potencies in inhibiting radioligand binding at the human �-opioid receptor isoforms C27 and F27 and human �- and �-opioid receptorsstably expressed in HEK293S cells and at rat brain �-opioid receptorsKi values (geometric means) were determined using the radioligands described under Materials and Methods. pKi � log Ki (M). n is the number of dose-response curves(each having 10 concentrations of AZD2327 in duplicate).

h�C27 h�F27 h� h� Rat Brain �

Ki (nM) 0.49 0.75 770 4500 1.2Bmax (pmol/mg) 9.7 0.62 3.71 0.72 0.29 0.04 3.4 0.59 0.076pKi S.E.M. 9.31 0.03 9.13 0.04 6.11 0.03 5.35 0.04 8.92 0.05n 14 5 14 13 6




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efficacy measurements, there was no evidence of tolerancedevelopment over the course of drug administration. NEturnover, expressed as the ratio of metabolite (MHPG) to NE,was elevated in animals that were stressed by being sub-

jected to the conditioning paradigm used for the in vivomicrodialysis studies relative to that in unstressed animals(Fig. 6A, left). This elevation in turnover was reversed by asingle administration of AZD2327 and after 7, 14, and 21 daysof administration (open squares). Diazepam, used as a positivecontrol in this study, also dampened the stress-induced changesin turnover over the entire period of administration. By 14 daysof administration, AZD2327 resulted in a lowering of the ratioto below control (unstressed) levels, although the functionalsignificance of this is not clear. In tissues taken from a subset ofanimals in this study, both potency (Fig. 6B) and efficacy (Fig.6C) were unchanged by repeated administration of AZD2327,regardless of whether they were unstressed by shock adminis-tration (Fig. 6, B and C, left) or stressed (Fig. 6, B and C, right),indicating no change in signaling through the receptor at leastup to 21 days of drug administration.

Anxiolytic Assays. In the conflict test, rates of responseaveraged 0.04 0.01 and 2.03 0.12 responses/s in the un-suppressed component under baseline conditions. Adminis-tration of �1 mg/kg AZD2327 was as effective as diazepam inproducing increases in response with respect to the control inthe suppressed component of the schedule (Fig. 7, left) anddid so with a potency that was an order of magnitude greaterthan that of diazepam. The effects of both agents upon sup-pressed response were specific to this component, becauseneither altered response in the unsuppressed component(Fig. 7, right). Evidence that the anxiolytic effects measuredin this test were in fact mediated by the �-opioid receptor isshown in Fig. 8. Pretreatment subcutaneously with 1 mg/kgof the selective �-opioid receptor antagonist naltrindole wasable to fully reverse the effects of 1 mg/kg AZD2327.

The effects of AZD2327 in the social interaction test aresummarized in Table 4. ADZ2327 produced a dose-relatedincrease in the amount of time that pairs of rats spent in

Fig. 3. Representative [35S]GTP� binding curves of AZD2327 on membranepreparations of HEK293S stable cell lines expressing the F27 isoform of thehuman �-opioid receptor (Œ) and the C27 isoform of the human �-opioidreceptor (�) and on rat brain membrane expressing endogenous �-opioidreceptor (F). Points represent the means of duplicates.

Fig. 4. Mean (S.E.) percentage of con-trol cortical NE (left) and MHPG (right)in 10 in cerebral microdialysate as a func-tion of time. Vehicle or diazepam (3 mg/kg) were administered orally (demar-cated by the arrow) 60 min beforeconditioning (footshock � light pairings,demarcated by FS � L). Sixty minuteslater, the CS (L) alone was presented. Theasterisks indicate significant differencesbetween groups by repeated-measurestwo-way ANOVA (treatment � time).

Fig. 5. Mean (S.E.) percentage of con-trol cortical NE (left) and MHPG (right)in cerebral microdialysate as a function oftime. Vehicle or compounds were admin-istered orally (demarcated by the arrow)60 min before conditioning (footshock �light pairings, demarcated by FS � L).Sixty minutes later, the CS (L) alone waspresented. The asterisks indicate signifi-cant differences from the vehicle-treatedgroup by repeated-measures two-wayANOVA (treatment � time). Dosages of 1and 3 mg/kg, but not 0.3 mg/kg, AZD2327reversed the effects of footshock uponboth NE and MHPG in cortex.

TABLE 3Agonist activity of AZD2327 in [35S]GTP� binding assays performed onmembrane preparations of HEK293S cell lines expressing human �-opioid receptor isoforms C27 and F27 and human �- and �-opioidreceptors and rat brain membranes expressing �-opioid receptorsendogenouslyEC50 values shown are the geometric means of individual results. %Emax values(arithmetic means) are relative to 10 �M SNC-80. pEC50 � log EC50 (M). n is thenumber of dose-response curves (each having 10 concentrations of AZD2327 induplicate).

h�C27 h�F27 Rat Brain �

EC50 (nM) 24 9.2 18%Emax S.E.M. 91 2 101 1 91 1pEC50 S.E.M. 7.63 0.03 8.04 0.09 7.65 0.06n 4 4 6

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social interaction, achieving a maximal effect over the dosestested of doubling the amount of social interaction at baselineconditions. These effects were comparable with those of thebenzodiazepine chlordiazepoxide.

Antidepressant Assay. The effects of AZD2327 in thelearned helplessness test for potential antidepressant activ-ity are summarized in Table 5. Inescapable shock produced,as designed, an increase in escape failures in rats treatedwith vehicle and subsequently given the opportunity to es-

cape in avoidance training. Vehicle-treated animals averaged16 escape failures in avoidance training. Treatment with thepositive control imipramine after inescapable shock resultedin a decrease in escape failures to an average of 2.3. Admin-istration of �1 mg/kg AZD2327 resulted in decreased escapefailures. Stimulation of locomotor activity, as measured bycenter crossings in the avoidance chambers during intertrialintervals, was unchanged by any treatment.

Locomotor Activity, Irwin’s Test, and PTZ SeizureThresholds. In the locomotor activity studies, there was amain effect of dose in the first and second studies [F(3,20) �20.3, P 0.0001 and F(3,20) � 35.8, P 0.0001, respectively](Fig. 9). All of the doses were active, with the exception of 1.5mg/kg, which was not significantly different from vehicle. Ingeneral, locomotor effects reached a plateau at doses �15.6mg/kg. Irwin’s test (data not shown) did not reveal any sig-nificant behavioral or physiological changes at any of thedoses tested. Most significantly, no signs of convulsions wereobserved when AZD2327 was administered alone. The dosesof PTZ [mean (S.E.M.)] required to induce clonic seizureswere 204.0 (8.0) after vehicle alone, 221.2 (7.1) after 0.6mg/kg, 187.2 (11.2) after 1.5 mg/kg, 163.8 (12.4) after 5.6mg//kg, and 137.8 (5.8) after 15.6 mg/kg. Thus, AZD2327caused a dose-dependent decrease of the threshold to PTZ-induced seizures. At doses �5.6 mg/kg, this change in seizurethreshold was significantly different from that of the vehicle-treated rats (P � 0.03 and 0.0002, respectively).

DiscussionThe suggested potential therapeutic applications of �-opioid

receptor agonists have spanned a number of different areas,including neuroprotection (Borlongan et al., 2000; Zhang et al.,2002), cardioprotection (Patel et al., 2002), Parkinson’s disease(Hudzik et al., 2000; Hille et al., 2001), and pain management(Porreca et al., 1984; Pradhan et al., 2009). Although there isclear merit as well as a high level of medical need for each ofthese therapeutic approaches, the potential use of greatest in-terest to us was an application in psychiatry, based in part uponobservations that the �-opioid receptor knockout mouse had aphenotype that was consistent with both anxiety and depres-sive-like symptoms (Filliol et al., 2000). This led us to begintesting the potential utility of agonists in psychiatric disease. Anumber of chemical approaches were undertaken to developcompounds that had a high degree of selectivity for the receptorwith otherwise drug-like properties and across several differentchemical series (Plobeck et al., 2000; Wei et al., 2000; Griffin etal., 2009). One product of this medicinal chemistry effort hasbeen AZD2327, the activity of which is reviewed in the presentarticle. Unlike the standard SNC-80, AZD2327 is active by theoral route of administration.

AZD2327 possesses equally robust anxiolytic and antidepres-sant activities. AZD2327 is active in the conflict test—a verystringent test for anxiolytic activity. The activity therein wasshown to be reversed by pretreatment with the �-opioid receptorantagonist naltrindole, strongly arguing that the effects ofAZD2327 are mediated by the �-opioid receptor. AZD2327is also active in the learned helplessness test for antidepressantactivity, an equally stringent test. Furthermore, AZD2327 isactive in the social interaction test, which reflects anxiolyticactivity, but if one considers that social withdrawal is a symp-tom of major depressive disorder as well, then it also may reflect

Fig. 6. Effects of repeated administration (1, 7, 14, and 21 days) ofAZD2327 on both ex vivo NE turnover (A) and on agonist potency andactivity (B and C, respectively). A, Veh points represent values fromstressed (Œ) or unstressed (�) rats derived from cortex taken from vehi-cle-treated animals pooled across each of the different time points ofvehicle administration because they did not differ. Points along thecurves represent the average of the cortex from four animals after eitherAZD2327 administration (1 mg/kg, �) or diazepam administration (3mg/kg, p.o., f). Asterisks indicate significant differences from the vehiclestressed condition (ANOVA, Dunnett’s post hoc test), and daggers indi-cates significant differences from vehicle-treated animals that were notstressed. B, effect of repeated administration of AZD2327 in control andstressed rats on rat �-opioid receptor potency in rat striatum. Points aremeans pEC50 values S.D. determined by a [35S]GTP� binding dose-response curve in three independent experiments. Data from each indi-vidual rat striatum is shown and categorized in respective group (VEH,vehicle; D, day; S, stressed rats). C, maximal efficacy was determined byone-point [35S]GTP� binding using 2 �M AZD2327. Fold stimulation isdefined as a ratio between maximal stimulation (2 �M) over basal con-dition (cpm with AZD2327/cpm basal condition). As in B, points aremeans of ratios from three independent experiments S.D. ANOVAswere performed separately on tissue from stressed (left sides of figures)and unstressed animals (right sides of figures). No main effect for treat-ment duration versus vehicle was noted.




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antidepressant potential. The relative potency of AZD2327 inanxiolytic tests appears to be greater than that in antidepres-sant tests, indicating specificity of the drug’s effects across thedifferent assays and therefore arguing against some secondaryeffect of the drug as the sole explanation for its effects.

The evidence that agonists for the �-opioid receptor would beantidepressants has been accruing rapidly over the last decadeand comprehensively reviewed by Jutkiewicz (2006). For exam-ple, peptidic agonists, such as [D-Pen2,D-Pen5]-enkephalin(Broom et al., 2002a), and nonpeptidic agonists, such as SNC-80

(Saitoh et al., 2004), have shown activity in antidepressantscreens, most typically the forced swim test. Although this testis useful in detecting potential antidepressant activity, demon-strating activity in additional, qualitatively different tests, suchas learned helplessness, strengthens the evidence for antide-pressant activity. The evidence that �-opioid receptor agonistsare anxiolytic is sparser. Perrine et al. (2003) showed thatSNC-80 has diazepam-like activity in the elevated plus mazetest (increased time in the open arms of the maze) as well as inthe defensive burying test and additionally showed that theseeffects were not purely due to increases in locomotion. Theeffects of AZD2327 in the conflict test, social interaction test,and stress/microdialysis paradigm in the present article furthersupport the notion that �-opioid receptor agonists are anxiolytic.After 21 days of administration, tolerance was not noted to theneurochemical effects of AZD2327, at least as determined bymeasurement of NE turnover ex vivo in response to a stressor,nor when tissue from the same set of animals was assayed foragonist potency and activity by [35S]GTP� binding. This obser-

Fig. 7. Effects of doses of AZD2327 anddiazepam on the suppressed (left) and un-suppressed (right) components of themodified Geller-Seifter conflict test foranxiolytic activity in rats. Drug was ad-ministered orally 1 h before the initiationof the operant session. The asterisks in-dicate significant difference from the ve-hicle control.

Fig. 8. Antagonism of the anxiolytic ef-fects of AZD2327 in the suppressed com-ponent (left) of the conflict test withoutaltering the effect in the unsuppressedcomponent (right). The asterisk indicatesa significant difference from the control.

TABLE 4Average S.E. amount of time (minute) engaging in social interactioncounts during a 10-min observation periodChlordiazepoxide was used as a positive control.

Mean S.D.

Vehicle 1.35 0.29AZD2327 (1 mg/kg) 2.49 0.51AZD2327 (10 mg/kg) 3.43 0.43Chlordiazepoxide (20 mg/kg) 2.83 0.75

TABLE 5Mean S.E. escape failures in the learned helplessness testEffects of doses of AZD2327 upon dependent variables in the learned helplessness paradigm. P value is associated with t test versus the inescapable shock � saline group.All of the treatments were given orally twice daily, 12 h after initial conditioning and 60 min before avoidance training (2 days after inescapable shock).

Mean EscapeFailures (S.E.M.) P Value Mean Center

Crossings (S.E.M.) P Value

Inescapable shock � saline 16.2 (3.9)No inescapable shock � saline 5.4 (1.4) 0.003* 32.9 (2.1) �0.05Imipramine (20 mg/kg) 2.3 (1) 0.002* 30.2 (1.1) 0.02*AZD2327 (0.1 mg/kg) 6.1 (3.4) 0.06 26.6 (0.85) �0.05AZD2327 (1 mg/kg) 3.1 (1.7) 0.004* 30.9 (2.9) �0.05AZD2327 (10 mg/kg) 4.9 (2) 0.01* 26.9 (0.7) 0.02*

* statistically significant values.

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vation is consistent with prior studies demonstrating a lack oftolerance development to the antidepressant effects of �-opioidreceptor agonists (Jutkiewicz et al., 2005).

Although the behavioral pharmacology strongly indicates anantidepressant and anxiolytic profile for �-opioid receptor ago-nists, the mechanistic underpinning for such a response is lessclear. Standard antidepressants such as selective serotonin re-uptake inhibitors or serotonin norepinephrine reuptake inhib-itors act, at least initially, via enhancement of monoaminergicneurotransmission. Although some synergy with dopamine ago-nists on locomotor endpoints can be noted when combined with�-opioid receptor agonists (Hudzik et al., 2000), the locomotorstimulant effects are not blocked by dopamine antagonists,suggesting a modulatory effect perhaps downstream from thedopamine receptor (Longoni et al., 1998). In the present articleas well as an earlier publication using a different �-opioid re-ceptor agonist compound with a pharmacology and structuresimilar to those of AR-M100390 (Smagin et al., 2008), no effectson basal NE, dopamine, or serotonin were noted after adminis-tration of agonists (data not shown). It could be argued that theantidepressant effects of �-opioid receptor agonists are due totheir proconvulsant effects, given the high level of efficacy oftreatments such as electroconvulsive therapy. However, not allof the agonists produce convulsions, and further, specifically,AZD2327 is markedly more potent in the efficacy tests than inproducing convulsions, because it did not produce convulsionsin rats in the present study up to doses that were �50- to100-fold higher. After repeated administration, antidepressantsevoke changes in neurotrophic factor expression and provokeassociated neurogenesis (Nibuya et al., 1995; Dranovsky andHen, 2006). Although there is somewhat mixed evidencewhether �-opioid receptor agonists may be enhancing monoam-inergic transmission, there is evidence that �-opioid receptoragonists will evoke increases in brain-derived neurotrophic fac-tor, indicating that there may be some overlap with standardantidepressants’ actions (Torregrossa et al., 2006).

The appearance of convulsions after administration of many�-opioid receptor agonists remains an obstacle to their develop-ment as a new pharmacologic class. Convulsions, when notedwith �-opioid receptor agonists are usually brief ( 1 min induration) and self-limiting (Comer et al., 1993; Broom et al.,2002b; Jutkiewicz et al., 2006). In addition, tolerance developsrapidly to the convulsant effects of SNC-80 and other agonists(Broom et al., 2002b; T. Hudzik and R. Caccese, unpublishedobservations). Furthermore, not all of the agonists have anequal liability to produce convulsions. Although SNC-80 ap-

pears to produce convulsions quite readily in rats (Broom et al.,2002b; Jutkiewicz et al., 2005), AZD2327 has been tested up todoses that are �150-fold over its “therapeutic” doses, and it doesnot produce convulsions in rats, as noted in the present article.However, AZD2327 does have proconvulsant effects (as mea-sured by decreases in PTZ seizure thresholds) after doses thatare severalfold over those indicative of anxiolytic or antidepres-sant activity. Because convulsion with �-opioid receptoragonists generally occurs within minutes of injection (Jut-kiewicz et al., 2006), it is unlikely to have been missedamong all of the present studies. Although direct convul-sion has not been noted in rats with AZD2327, it is inter-esting to note that it can be noted in other species, such asmice, dogs, and squirrel monkeys (T. Hudzik, unpublishedobservations; C. A. Paronis, personal communication). Theextensive safety pharmacology of AZD2327 with respect toseizures will be reviewed in a separate article (T. Hudzik,B. Bell, C. A. Paronis, M. Swedberg, A. Manninen, M.Quirk, K. Bui, A. Easter, M. Pietras, R. Caccese, et al.,manuscript in preparation).

The notion of differences among agonists’ pharmacologies isnot limited to convulsions. For example, some of the agonists,but not all, appear to have locomotor stimulant effects. It isnoteworthy that AZD2327 can produce both anxiolytic and an-tidepressant effects at doses 3- to 10-fold lower than thoseproducing any motor effects, thus removing this potential con-found in the interpretation of efficacy data. Significantly lessseparation of locomotor stimulant effects of SNC-80 from effectsin the forced swim test, for example, has been noted (Broom etal., 2002a; Jutkiewicz et al., 2005; Jutkiewicz, 2006). In addi-tion, many of the agonists that have been tested during thedevelopment of AZD2327 appeared to lack locomotor stimulanteffects but retain anxiolytic activity (T. Hudzik, unpublishedobservations; Gengo et al., 2003). It is interesting to speculateas to why differences among agonists can be observed. It isknown, for example, that different agonists can differentiallytraffic the receptor (Pradhan and Clarke, 2005; Pradhan et al.,2009, 2010) and that different agonists can signal differentially,as suggested by the appearance of pharmacologic subtypes ofthe receptor. It is possible that these and other mechanisms cancontribute to agonists’ differences.

Taken together, the data demonstrate that AZD2327 pos-sesses a relatively unique pharmacological profile that overlapswith those of benzodiazepines (anxiolytic-like) and antidepres-sants (e.g., monoamine oxidase inhibitors or triple reuptakeinhibitors). Its pharmacology differs from those of �- and �-opi-

Fig. 9. Ambulatory distance (in cm) trav-eled within locomotor activity chambersin a 90-min period after administration oflower doses (left) and higher doses (right)of AZD2327. All of the doses in both stud-ies were significantly different from thevehicle (Bonferroni’s post hoc test), withthe exception of 1.5 mg/kg dose.




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oid receptor agonists, which are not active in models predictiveof anxiolytic or antidepressant activity. These data support thehypothesis that �-opioid receptor agonists in general andAZD2327 in particular may have utility in the treatment ofdepression and, given the mixed anxiolytic/antidepressant pro-file, possibly depression with concomitant anxiety (“anxiousdepression”). This is significant given the very high level ofmedical need—AZD2327 could represent possibly an importantadvancement in the treatment of this group of patients. Ongo-ing clinical studies with this compound will further elucidatewhether this hypothesis is correct.

Acknowledgments

We thank Lynn Hudzik for editorial and other helpful comments on themanuscript and Mylene Gosselin, Leijla Hodzic, and Gabrielle Mankiewiczfor performing some of the in vitro pharmacology experiments.

Authorship Contributions

Participated in research design: Hudzik, Maciag, Bui, Coupal,Adam, Payza, Smagin, Swedberg, and Brown.

Conducted experiments: Hudzik, Maciag, Caccese, Pietras, Coupal,Adam, Song, and Swedberg.

Contributed new reagents or analytic tools: Payza, Griffin, andBrown.

Performed data analysis: Hudzik, Coupal, Adam, Payza, Song, andSwedberg.

Wrote or contributed to the writing of the manuscript: Hudzik,Maciag, Smith, Coupal, Adam, Payza, Smagin, and Swedberg.

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Address correspondence to: Dr. Thomas J. Hudzik, Department of Toxicology,Global Pharmaceutical Research and Development, Abbott Laboratories, 100 AbbottPark Rd., R468, Abbott Park, IL 60064. E-mail: [email protected]

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