anxiolytic and antidepressant-like activities of ufp-512, a novel selective delta opioid receptor...

Accepted Manuscript

Title: Anxiolytic- and antidepressant-like activities ofH-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novelselective delta opioid receptor agonist

Authors: Raffaella Vergura, Gianfranco Balboni, BarbaraSpagnolo, Elaine Gavioli, David G. Lambert, John McDonald,Claudio Trapella, Lawrence H. Lazarus, Domenico Regoli,Remo Guerrini, Severo Salvadori, Girolamo Calo’

PII: S0196-9781(07)00424-XDOI: doi:10.1016/j.peptides.2007.10.012Reference: PEP 67236

To appear in: Peptides

Received date: 27-7-2007Revised date: 8-10-2007Accepted date: 16-10-2007

Please cite this article as: Vergura R, Balboni G, Spagnolo B, Gavioli E, LambertDG, McDonald J, Trapella C, Lazarus LH, Regoli D, Guerrini R, Salvadori S, Calo’G, Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist, Peptides (2007),doi:10.1016/j.peptides.2007.10.012

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dx.doi.org/doi:10.1016/j.peptides.2007.10.012

dx.doi.org/10.1016/j.peptides.2007.10.012

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Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist

1Raffaella Vergura, 2,3Gianfranco Balboni, 1Barbara Spagnolo, 1Elaine Gavioli, 4David G.Lambert, 4John McDonald, 5Claudio Trapella, 6Lawrence H. Lazarus ,1Domenico Regoli, 2Remo Guerrini,

2Severo Salvadori and 1Girolamo Calo’.

1Dept. of Experimental and Clinical Medicine, Section of Pharmacology, and National Institute of

Neuroscience, University of Ferrara, Italy; 2Dept. of Pharmaceutical Sciences and Biotechnology

Centre, University of Ferrara, Italy; 3 Dept. of Toxicology, University of Cagliari, Italy; 4 Dept. of

Cardiovascular Sciences, University of Leicester, UK; 5UFPeptides s.r.l., Ferrara, Italy, 6Medicinal

Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of

Environmental Health Sciences, research Triangle Park, NC, USA

Corresponding author:

Girolamo Calo', MD PhDDept. Experimental and Clinical MedicineSection of PharmacologyUniversity of Ferraravia Fossato di Mortara, 1944100 Ferrara, ItalyPhone: +39 0532 455 221Fax: +39 0532 455 205e-mail: [email protected]

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Abstract

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor

signalling regulates emotional responses. In the present study, the in vitro and in vivo

pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was

investigated. In receptor binding experiments performed on membranes of CHO cells expressing

the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.20) and

selectivity (> 150 fold) for DOP sites. In functional studies ([35S]GTPγS binding in CHOhDOP

membranes and electrically stimulated mouse vas deferens) UFP-512 behaved as a DOP selective

full agonist showing potency values more than 100 fold higher than DPDPE. In vivo, in the mouse

forced swimming test, UFP-512 reduced immobility time both after intracerebroventricular (icv)

and intraperitoneal (ip) administration. Similar effects were recorded in rats. Moreover, UFP-512

evoked anxiolytic-like effects in the mouse elevated plus maze and light dark aversion assays. All

these in vivo actions of UFP-512 were fully prevented by the selective DOP antagonist naltrindole

(3 mg/kg, sc). In conclusion, the present findings demonstrate that UFP-512 behaves as a highly

potent and selective agonist at DOP receptors and corroborate the proposal that the selective

activation of DOP receptors elicits robust anxiolytic- and antidepressant-like effects in rodents.

Key words: UFP-512, DOP receptor, receptor binding, bioassay, forced swimming test, light-dark

aversion, elevated plus-maze test.

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1. Introduction

Classical pharmacology, receptor binding and later molecular biology studies demonstrated that

the opioid system is constituted of three opioid receptors namely the mu, delta and kappa (recently

named MOP, DOP and KOP which stands for mu, delta and kappa opioid peptide receptor,

respectively) and three precursor peptide genes coding for endorphins, enkephalins and dynorphins

[13]. Opioid receptors regulated several peripheral and central functions, including gastrointestinal

motility, chough, immune responses, pain transmission, and emotional states. The DOP receptor

system has been particularly associated with the regulation of emotional responses [20]. DOP

receptors are expressed in cortical and limbic regions [10] and DOP receptor gene knockout mice

displayed increased anxiety-like behaviors in the light–dark box and elevated plus-maze assays and

depressive-like behaviors in the forced swimming test [15]. On the other hand, preproenkephalin-

knockout mice displayed anxiety-related behaviors in the open-field and light-dark tests [33].

These transgenic studies are in line with findings from pharmacological investigations. Indeed,

Broom et al. [9] found that the nonpeptidic DOP receptor agonists (+)BW373U86 and SNC80

produce antidepressant-like effects in the rat forced swimming test. Saitoh et al. [37] demonstrated

that SNC-80, dose-dependently increased the time spent in the open arms of the plus-maze,

compared to controls. Moreover, the ability of the DOP agonist H-Dmt-Tic-NH-CH2-Bid (UFP-

502) to promote antidepressant-like effects in the forced swimming test in mice [43] and rats [42]

has been recently demonstrated. In addition, RB101, a drug that inhibits the catabolism of the

enkephalins, the endogenous DOP receptor ligands, has antidepressant effects in a learned

helplessness procedure [41]. These transgenic and pharmacological approaches converge to

indicate that DOP signalling inhibits anxiety and produces antidepressant-like actions.

In the context of structure activity studies on the Dmt-Tic pharmacophore template [38], we

previously identified the compound UFP-502 [3, 42, 43], which has high in vitro potency and

moderate DOP selectivity [43] and produced in vivo antidepressant-like effects in mice [43] and

rats [42]. However, UFP-502 was only active after intracerebroventricular (icv) administration and

its in vivo effects were only partially prevented by naltrindole [43].

In this communication we report on the in vitro and in vivo pharmacological characterization of a

novel DOP receptor ligand based on the Dmt-Tic pharmacophore template: H-Dmt-Tic-NH-

CH(CH2-COOH)-Bid (UFP-512). In vitro UFP-512 was assessed in receptor and [35S]GTPγS

binding experiments as well as in bioassay studies performed with the electrically stimulated mouse

vas deferens. In vivo this novel ligand has been tested in various assays performed in mice to

investigate its effect on locomotor activity, anxiety and mood related behaviors. Since

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antidepressant-like effects evoked by DOP receptor selective agonists represent one of the most

interesting action of this class of compounds [21], we performed forced swimming experiments

both in mice and rats to assess its robustness across species.

2. Materials and Methods

2.1 In vitro studies

Cell culture and membrane preparation - CHO cells transfected with single genes for hMOP, hKOP

and hDOP were maintained in Ham’s F12 media, containing 10% foetal bovine serum (FBS), 0.5%

Penicillin, 0.5% Streptomycin, 0.5% Fungizone and 0.4% Geneticin, for plasmid selection. Cultures

were maintained at 37°C in 5% CO2 / humidified air. When confluent, cells were harvested,

membranes prepared and used fresh each day, as previously described [23]. Confluent cells were

incubated in 10 ml of harvest buffer, containing 0.9% Saline, 0.02% EDTA, 10mM HEPES, pH of

7.4 for 5 minutes at 37°C. The resulting suspension was centrifuged at 378 RCF for 2 minutes at

20°C. The membrane pellet was resuspended in appropriate buffer, homogenised and centrifuged at

20374 RCF for 10 minutes at 4°C and repeated twice.

Radioligand displacement binding - CHO cell membranes were resuspended in approximately 4 ml

of ice-cold assay buffer: Tris-HCl (50 mM), containing, 10 µM of the peptidase inhibitors (captopril,

amastatin, bestatin, phosphoramidon), and bovine serum albumin 0.5%, pH 7.4. Approximately 25-

30 µg of membrane protein was incubated for 60 min at room temperature with 0.5 nM of [3H]-

diprenorphine. Non-specific binding was defined in the presence of 1 µM naloxone; bound and free

radioactivity was separated by rapid vacuum filtration using a Brandel cell harvester (Whatman

GF/B) presoaked in polyethyleneimine (0.5%) to reduce non-specific binding [23].

[35S]GTPγS binding assay - Assays were performed essentially as described by Berger et al. and

Kitayama et al. [6, 23]. Twenty µg of freshly prepared membranes were incubated in 0.5 ml buffer

containing Tris-HCl (50 mM), EGTA (0.2 mM), MgCl2 (1 mM), NaCl (100 mM), GDP (5 µM),

bacitracin (0.15 mM), bovine serum albumin (1 mg ml-1), peptidase inhibitors (amastatin, bestatin,

captopril, phosphoramidon; 10 µM) and [35S]GTPγS (~150 pM), for 1 h at 30°C to determine total,

non specific (in the presence of excess 10 µM GTPγS ) and stimulated (in the presence of ligand

dilution) binding. Following incubation, bound and free radiolabel were separated by vacuum

filtration onto Whatman GF/B filters.

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Electrically stimulated mouse vas deferens - Vas deferens tissues from male Swiss mice (25 – 30 g)

were prepared as previously described [7] and suspended in 5 ml organ baths containing Mg2+ free

oxygenated (95% O2 and 5% CO2) Krebs solution. The bath temperature was set at 33°C. The

tissues were continuously stimulated through two platinum ring electrodes with supramaximal

voltage rectangular pulses of 1 ms duration and 0.05 Hz frequency. Electrically evoked contractions

were measured isotonically with a strain gauge transducer (Basile 7006) and recorded with a PC

based acquisition system PowerLab 4/25 (model ML845, ADInstruments, USA). Following an

equilibration period of about 60 min, cumulative concentration response curves to DPDPE and

UFP-512 (0.5 log unit steps) in the presence and absence of naloxone and naltrindole were

performed.

2.2 In vivo studies

All experimental procedures adopted for in vivo studies complied with the standards of the

European Communities Council directives (86/609/EEC) and Italian regulations (D.L. 116/92).

Moreover, specific permission for performing the present experiments was obtained by the

University of Ferrara Ethical Committee for animal studies and by the Italian Ministry of Heath

(Prot. n. 26687/04). Male Swiss mice weighing 20 – 25 g and Sprague-Dawley rats weighing 200-

250 g were used. They were housed under standard conditions (22±1°C, 55±1% humidity, 12 h light

– dark cycle, light turned on at 7 am) with food and water ad libitum for at least 7 days before

experiments began. Mice were housed 7/8 per cage, while rats were housed 3 per cage. Each animal

was used only once. The total number of mice used in this study was 325, while the total number of

rats was 55. Drugs were administered to the animals intraperitoneally (ip) or

intracerebroventricularly (icv).

On the bases of the experience with UFP-502 [43], we first assessed the effects of UFP-512 after

icv injection and then we compared these results with those obtained after systemic administration

via the ip route. After realizing that, contrary to UFP-502, UFP-512 was active both when given icv

and ip we decided to continue its in vivo evaluation using this last route of administration.

Icv injections (2 µl per mouse) were given into the left ventricle according to the procedure

described by Laursen and Belknap [26] and routinely adopted in our laboratory [16, 36].

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Mouse forced swimming - The mouse forced swimming test, described previously [32], consisted

of placing mice, individually, in polyethylene cylinders (18.5 cm high, 12.5 cm diameter),

containing water (25±1°C, 13.5 cm deep), for two swimming sessions: an initial 15 min training

session on day 1, which was followed, 24 h later (day 2), by a 5 min test session. Results were

relative to this 5 min test session. At the end of each swimming session, the animal was removed

from the cylinder, dried with paper towels, placed in an individual cage to rest and recover, and then

it was returned to its collective home cage. The immobility time (i.e. the time spent floating in the

water without struggling) was recorded. Scoring was performed by an observer blind to the

treatment received by each animal. As antidepressant standard drugs desipramine and fluoxetine

(both at 30 mg/kg, ip) were used. UFP-512 was administered icv (0.001 – 0.1 nmol) or ip (0.01- 1

mg/kg) 5 min and 30 min prior to the test, respectively. In order to evaluate the involvement of

DOP receptors on the effects of UFP-512, mice were pretreated with naltrindole (3 mg/kg, sc) 30

min before the test. Experiments were performed between 9.00 h and 12.00 h.

Rat forced swimming test – In the measurement of antidepressant-like activity, rats were subjected

to the forced swimming assay as previously described by Detke [14]. Briefly, male Sprague-Dawley

rats (200–250 g) were placed in a plexiglas container (46 cm tall x 20 cm diameter) filled to 30 cm

with 25±1°C water. Two swimming sessions were conducted, an initial 15 min training session (day

1) followed 24 h later (day 2) by a test session lasting 5 min. Results were relative to this 5 min test

session. Rat behavior in this assay was classified as one of the followings: immobility, swimming,

or climbing [14]. Immobility was defined as floating in the water without struggling and using only

small movements to keep the head above water. Swimming was defined as moving limbs in an

active manner (more than required to keep the head above water) causing movement among

quadrants of the cylinder. Finally, climbing was defined as making active movements with

forepaws moving in and out of the water, usually directed against the side of the cylinder. The time

spent in each behavior was recorded during the 5 min test session. Cylinder water was changed after

every other rat. Following each swim period, the rats were removed from the water, towel-dried and

placed in a heated cage for 15 min. Scoring was performed by an observer blind to the treatment

received by each animal.

A dose response curve to UFP-512 (0.1, 0.3 and 1 mg/kg, ip) was performed.

Light-dark box - Mice were individually exposed for 5 min to a box consisting of a small

compartment (15x20x25 cm) with black walls and black floor dimly lit (5 lux) connected by a 4 cm

long tunnel to a large compartment (30x20x25 cm) with white walls and floor, under intense

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illumination (500 lux). Lines were drawn on the floor of lit compartment to allow measurement of

locomotor activity by counting the number of squares (5x5 cm) crossed.

The time spent in each compartment, locomotor activity and the number of visits to each zone of the

lit compartment were recorded. Each animal was placed in the lit compartment facing the tunnel at

the beginning of each session, which started 30 min after the ip injection of saline or UFP-512.

In a first experiment, the effects of UFP-512 administration (0.1 and 1 mg/kg) were evaluated and

compared with those induced by diazepam 1 mg/kg, ip. In a second experiment, the effects of

naltrindole administration (3 mg/kg, sc) vs UFP-512 (1 mg/kg) responses were evaluated. Different

groups of animals were used for each experiment. The light-dark box has been cleaned after each

animal with wet paper towels.

Elevated plus maze - The plus maze consisted of two open (30x5cm) and two wall-enclosed arms

(30x5x15 cm) connected by a central platform (5x5 cm) [25]. Light intensity in the open arms was

100 lux. The apparatus was elevated 50 cm above the floor. The floor and the walls of the enclosed

arms of the maze were constructed of black Plexiglas, while the floor of the open arms was made

from grey, non reflective plastic material. Behavioral testing was started by placing a mouse in the

central area facing an open arm in which the animal usually enters first. The number of entries into

open and closed arms, time in open and closed arms and general activity were recorded and

quantified. Entries were defined as the centre of the body of an animal entering a new zone.

The first series of experiments consisted of an evaluation of the dose response effect of UFP-512

(0.1 and 1 mg/kg, ip administered) and a comparison of the effect of the peptide with that of

diazepam 1 mg/kg, ip. In a second series of experiments the selective DOP receptor antagonist

naltrindole (3 mg/kg, sc) was tested against the effect induced by UFP-512. Different groups of

animals were used for each experiment. After each animal has been removed, the apparatus has

been cleaned with wet paper towels.

Spontaneous locomotor activity - This test was performed using Basile activity cages (Varese, Italy)

between 14.00 h and 18.00 h, as previously described [35]. Mice were tested 30 min after ip

injection of UFP-512. They were not accustomed to the cages before drug treatments and the

experiment was performed in a quiet and dimly illuminated room. The total number of impulses was

recorded for 30 min. The apparatus has been cleaned with wet paper towels after each animal.

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Open field test - The open field consisted of a red light (100 lux) illuminated squared arena (40x40

cm), made of opaque white polyethylene with the floor divided in 16 sections (4 inner squares and 12

outer squares). The parameters measured during the 5 minutes of observation were: external

ambulation (number of squares adjacent to the wall entered with the four limbs), internal ambulation

(number of squares in the central area) and frequency of rearings [12]. UFP-512 was administered ip

(0.1 and 1 mg/kg) 30 min prior to the test. The open field arena has been cleaned with wet paper

towels after each animal.

2.3 Drugs.

UFP-512 was prepared and purified using the methods previously described [4]. DPDPE, naloxone,

captopril, amastatin, bestatin, desipramine, HEPES, GDP and Tris were purchased from Sigma

Chemical Co. (Poole, U.K.); phosphoramidon from Peptide Institute (Osaka, Japan), naltrindole,

desipramine and fluoxetine from Tocris Cookson (Bristol, U.K.). All tissue culture media and

supplements were from Invitrogen (Paisley, U.K.). [35S]GTPγS (1250 Ci mmol-1) and [3H]-

diprenorphine were from NEN DuPont (Boston, U.S.A.). Bacitracin, obtained from Sigma

Chemical Co. (Poole, U.K.), was heated for 1 h at 70°C in water to inactivate any enzymatic

activity before use. Peptidase inhibitors amastatin, bestatin, phosphoramidon and captopril were

dissolved in water and frozen in 10 mM stocks. For in vitro experiments, the compounds were

dissolved in physiological buffers and stock solutions (1 mM) were kept at -20°C until use. For in

vivo studies, the substances were freshly dissolved in saline just before performing the experiment.

2.4 Terminology and data analysis.

The pharmacological terminology adopted in this paper is consistent with IUPHAR

recommendations [30]. In vitro data are expressed as mean ± s.e.m. of n separate experiments,

while in vivo data are the mean ± s.e.m. of n animals. Data were analysed using one-way ANOVA

followed by Dunnett’s test. Differences were considered statistically significant when p < 0.05.

Concentration of ligand producing 50 % displacement of specific binding (IC50) was corrected for

the competing mass of [3H]-diprenorphine to yield Ki, according to Cheng and Prusoff [11]. Curve

fitting for binding data was performed using PRISM 4.0 (GraphPad Software In., San Diego,

U.S.A.). Agonist potencies were measured as pEC50, which is the negative logarithm to base 10 of

the agonist molar concentration that produces 50 % of the maximal possible effect of that agonist.

The Emax is the maximal effect that an agonist can elicit in a given preparation. Antagonist

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potencies, expressed in terms of pA2, which is the negative logarithm to base 10 of the antagonist

molar concentration that makes it necessary to double the agonist concentration to elicit the original

submaximal response, were calculated via the Gaddum Shild equation: pA2 = CR-1/[Antagonist],

assuming a slope equal to 1.

3. Results

3.1 In vitro studies

Receptor binding - The ability of UFP-512 to bind to opioid receptors has been evaluated using

membranes from CHO cells expressing recombinant human MOP, DOP and KOP receptors and

compared to that of the DOP receptor agonist DPDPE. The standard ligands for each receptor

(DAMGO for MOP, naltrindole for DOP and nor-BNI for KOP) were included as positive controls.

The results of this series of experiments are summarized in Table 1. The novel ligand UFP-512

binds with subnanomolar affinity (pKi 10.20) to the DOP receptor showing 160 and 3500 fold

selectivity over MOP and KOP sites, respectively. DPDPE (1 µM) did not displace [3H]-

diprenorphine from MOP or KOP sites while showing moderate affinity for DOP receptors (pKi

8.48).

Functional studies - As shown in Figure 2 (left panel), DPDPE and UFP-512 stimulated [35S]GTPγS

binding to CHOhDOP membranes in a concentration dependent manner with similar maximal effects.

In this assay, UFP-512 had very high potency (pEC50 10.17), 100 fold higher than that of the

standard DOP agonist DPDPE (pEC50 8.12). DPDPE and UFP-512 inhibited the twitch response to

electrical field stimulation in the mouse vas deferens in a concentration dependent manner showing

similar maximal effects (Figure 2, right panel): UFP-512 was 1000 fold more potent than DPDPE

(pEC50 values 11.65 and 8.58, respectively). Apart from the higher potency, in vas deferens

experiments UFP-512 displayed a further difference from DPDPE worthy of mention. Indeed, the

kinetics of action of DPDPE was fast, its inhibitory effects occurred immediately after adding the

peptide to the bath and were rapidly and fully reversible after washing of the tissue, and, on

average, a complete concentration response curve to DPDPE could be obtained in approximately 8

min. In contrast, UFP-512 induced slow inhibitory effects, which reached a plateau after only 3 min

from the injection; moreover, the recovery of the tissue after washing took greater than 30 min and

was never complete. More than 25 min were needed to perform a complete concentration response

curve to this agonist. The effects of DPDPE and UFP-512 were sensitive to naloxone and

naltrindole which produced a rightward shift of the concentration response curve to agonists

without modifying their maximal effects; the pA2 values calculated for naloxone against DPDPE

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and UFP-512 were 8.08 and 8.25, respectively, while naltrindole showed a pA2 of 9.97 against

DPDPE and a pA2 of 10.54 against UFP-512.

3.2 In vivo studies

Injection of saline and UFP-512 either icv or ip or that of standard antidepressant drugs

(desipramine and fluoxetine, ip) did not produce any modification in animal gross behavior, such as

jerks, muscle tremors, convulsions, salivation, sedation, stereotyped behaviors or piloerection.

Mouse forced swimming test - Two standard antidepressants drugs, desipramine and fluoxetine

(both at 30 mg/kg, ip, given 30 min before the test session) significantly reduced the immobility

time of mice subjected to the forced swimming test (saline 180 ± 10 s; desipramine 94 ± 12 s;

fluoxetine 98 ± 17 s, n = 9 for each group). Similarly, icv and ip injections of UFP-512 dose-

dependently reduced immobility time (Figure 3), reaching levels of statistical significance at the

dose of 0.01 nmol and 0.1 mg/kg, respectively. The antidepressant effects elicited by the compound

at 0.1 mg/kg were challenged with the DOP antagonist naltrindole (3 mg/kg, sc, 30 min prior to the

test). Naltrindole per se did not produce any significant modification of mouse immobility time,

however, the DOP antagonist fully prevented the effect of UFP-512 (Figure 4).

Rat forced swimming test - Although we did not perform a complete series of experiments, a few

rats were treated with desipramine (30 mg/kg, ip) and submitted to the forced swimming test. In

these experiments we found that desipramine significantly reduced the immobility time and

increased the climbing time of rats subjected to the forced swimming test, thus confirming previous

studies from our laboratory [17].

In a similar way, UFP-512 produced antidepressant-like activity in this test. The UFP-512 dose-

effect relationship was investigated in the range 0.1-1 mg/kg, ip. The decrease in immobility

induced by UFP-512 was statistically significant beginning at 0.3 mg/kg dose and was associated

with an increase in both climbing and swimming behaviors, although these latter effects did not

reach statistical significance (Figure 5).

Light-dark box - The effects of the treatment with UFP-512 (0.1 and 1 mg/kg) were determined and

compared with those elicited by diazepam (1 mg/kg, ip) in the mouse light-dark aversion assay.

Statistical analysis revealed a significant decrease in the time spent in the dark compartment and an

increase in the time spent in the lit compartment induced by diazepam and UFP-512 administration

(saline 43 s; diazepam 1 mg/kg= 102 s; UFP-512 1 mg/kg= 110 s) (Figure 6). Pretreatment with the

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DOP receptor antagonist naltrindole (3 mg/kg, sc), which did not induce any effect when

administered alone, abolished the responses to UFP-512 1 mg/kg (Figure 7).

Elevated plus-maze test – Mice treated with UFP-512 (0.01-1 mg/kg, ip) showed a significant

increase in the percentage of time spent and visits to the open arms (Figure 8). The effects of UFP-

512 (0.1 mg/kg) were about 70% of those observed in diazepam (1 mg/kg, ip) treated animals. Both

compounds, however, had no significant effect on the percentage of time spent and visits to the

closed arms (data not shown). Naltrindole 3 mg/kg (30 min pre-treatment, sc) had no significant

effects per se, while it antagonized the effect induced by UFP-512 at 0.1 mg/kg (Figure 9).

The full ethogram of behavior displayed by mice treated with diazepam or UFP-512 and subjected

to the elevated plus maze is shown in Table 3.

Open field test - Treatment with UFP-512 (0.1 and 1 mg/kg) did not evoke any significant

modification of exploratory behavior of mice both in central locomotion (Figure 10, top panel) or in

peripheral locomotion (Figure 10, middle panel) and in general behavior, measured as number of

rearings (Figure 10, bottom panel) of the animals during 5 min observation.

Spontaneous locomotor activity - Since false positive/negative results can be obtained in behavioral

tests employing agents that affect locomotor activity, a separate series of experiments were

performed to assess the effects of UFP-512 (at 0.1 and 1 mg/kg, ip) on spontaneous locomotor

activity. The peptide did not modify this behavioral parameter either at 5 min (Figure 11, left panel)

or at 30 min observation (Figure 11, right panel).

4. Discussion

The results of the present study demonstrated that the novel pseudopeptide UFP-512 behaves in

vitro as a highly selective and extremely potent full agonist at native and recombinant DOP

receptors and is able to promote in vivo antidepressant- and anxiolytic-like effects which are solely

due to DOP receptor activation.

UFP-512 binds with subnanomolar affinity (pKi 10.20) to human recombinant DOP receptors with

160 and 3500 fold selectivity over MOP and KOP sites, respectively, and displaying similar affinity

as the potent antagonist naltrindole (pKi 10.12). This binding profile is similar to that previously

reported at opioid sites expressed in rat brain synaptosomal membranes where UFP-512 exhibited

very high DOP affinity (pKi 9.35) associated with >100 fold selectivity over MOP sites [4].

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Recently, these results were confirmed by Aguila et al. [1] who estimated a pKi value of 9.78 for

UFP-512 at DOP receptors expressed in human neuroblastoma SK-N-BE cells.

Functional biochemical and bioassay studies performed on human recombinant and mouse native

DOP receptors demonstrated that UFP-512 behaves as DOP receptor full agonist producing

maximal effects superimposable to those evoked by DPDPE. In the [35S]GTPγS binding assay

performed on CHOhDOP membranes, UFP-512 was found to be 100 fold more potent than DPDPE

while in the electrically stimulated mouse vas deferens the DPDPE / UFP-512 concentration ratio

was even larger (approx 3 log units). In line with these findings, the very high potency of UFP-512

was recently reported in SK-N-BE cells for its ability to inhibit cAMP accumulation (pEC50 9.4) as

well as to stimulate the MAP kinase pathway (pEC50 9.6) [1] .

The above mentioned in vitro effects of UFP-512 were exclusively due to the activation of DOP

receptors since they were sensitive to naltrindole. In particular the pA2 values obtained in the mouse

vas deferens assay with naloxone ( 8) and naltrindole ( 10) against UFP-512 and the standard

DOP selective agonist DPDPE were superimposable demonstrating the involvement of a single

receptor site, i.e. the DOP receptor, in the effects of all these ligands.

Collectively, results on the in vitro investigation of the pharmacological profile of UFP-512

demonstrate that this ligand behaves as an extremely potent and highly selective full agonist at DOP

receptors. Compared to our previous reference agonist UFP-502, UFP-512 displayed higher potency

associated with higher selectivity of action (present data and [43]); this prompted us to further

investigate the effects elicited by this novel ligand in vivo.

Depression and anxiety are the most prevalent and costly psychopathologies and are associated with

considerable chronicity, morbidity, and disability. The first-line pharmacologic treatments for major

depression and anxiety disorders currently include the use of tricyclic antidepressants, selective

serotonin reuptake inhibitors, and, for some types of anxiety, benzodiazepines. It should be

mentioned that low recovery rates have been reported for patients suffering from anxiety and

depression disorders. Additionally, several patients exhibit partial or no responses to these

pharmacological treatments, underscoring the need of innovative agents with broader efficacy and

fewer adverse effects to treat depression and anxiety [8, 31].

As noted in the introduction, pharmacological findings obtained with DOP receptor selective

agonists [9, 37, 40], enkephalinase inhibitors [2, 22, 39], as well as knockout studies [15, 24, 33]

have suggested that the activation of DOP receptors in the brain induced antidepressant-like effects

in rodents (for a review see [20]). Thus, we evaluated the effects of UFP-512 in the forced

swimming test, a behavioral assay widely used for the screening of antidepressant-like effects of

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drugs [32] and its effects on anxiety related behaviors by performing elevated plus maze and light

dark box experiments.

In the mouse forced swimming test UFP-512 dose-dependently reduced immobility time,

mimicking the effects of classical antidepressants such as desipramine and fluoxetine. The lowest

active dose of UFP-512 after supraspinal (icv) administration was 0.01 nmol which is 100 fold

lower than that required by UFP-502 [43]. In addition, UFP-512 but not UFP-502 (data not shown)

was also able to reduce immobility time in a dose dependent manner when given ip. The presence

of a hydroxycarbonylmethyl function (CH2-COOH) between the Dmt-Tic pharmacophore and the

1H-benzimidazole ring not only increases potency and selectivity at DOP sites but also confers to

the molecule favourable pharmacokinetic properties which allow the compound to cross the blood

brain barrier. On these bases, all subsequent in vivo experiments were performed by administering

UFP-512 systemically.

It is worthy of mention that both after icv and ip administration, UFP-512 produced bell shaped

dose response curves, similar to other DOP receptor ligands tested as antidepressant agents, such as

SNC80 [9]. The reason(s) for this phenomenon is still unclear; possible interpretations could be

agonist-induced receptor desensitization, or the appearance of DOP receptor independent

mechanisms at high ligand doses.

The dose dependent reduction of immobility time elicited by UFP-512 injected ip was also evident

in rats although slightly higher doses were required to produce statistically significant effects. This

demonstrates together with data from literature [20] that the antidepressant-like effects evoked by

DOP receptor activation represent a robust phenomenon in different rodent species. Furthermore,

our findings revealed that UFP-512 reduced immobility time in the rat forced swimming test, and

this was associated with an increase (although not statistically significant) in climbing and

swimming time. Previous results [14] demonstrated that the ethological analysis of antidepressant

drugs in the rat forced swimming test allow to discriminate between serotonergic drugs (i.e.

fluoxetine) that reduce immobility by increasing swimming and noradrenergic (i.e. desipramine) /

dopaminergic compounds that reduce immobility by increasing climbing. However, other

antidepressant drugs such as the tricyclic compound imipramine and the dual NE/5-HT reuptake

inhibitor minalcipram, dose-dependently reduce immobility time, and equally increase both

climbing and swimming behaviors [5, 34]. Interestingly enough, the DOP receptor agonist

BW373U86 produced in the rat forced swimming test a pattern of response superimposable to that

we measured in response to UFP-512 [9]. Thus, present and literature evidences suggest that DOP

receptor agonists cannot be simply categorized into either class of current antidepressant

compounds.

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Previous studies reported that DOP receptor-knockout mice displayed and anxiogenic phenotype in

the elevated-zero-maze test [15] and that preproenkephalin knockout mice had increased anxiety-

related behaviors in the open field and light-dark tests [33]. In addition, overexpression of

proenkephalin in the amygdala increased the anxiolytic effects of benzodiazepines in rats [21] and

the selective DOP receptor agonist SNC80 significantly increased the time spent in the open arms

of the rat elevated plus-maze compared to controls [37]. These findings strongly suggest an

important role for the DOP receptor in the modulation of anxiety states. Thus, the possible actions

of UFP-512 were assessed in the elevated plus maze and light-dark aversion tests in mice.

In the light-dark aversion test, the standard anxiolytic agent diazepam promoted an increase in the

time spent in the lit compartment by the animal, which was mimicked by UFP-512 that produced

statistically significant effects only at the higher dose tested (1 mg/kg). Similar results were

obtained in the elevated plus maze assay where both diazepam and UFP-512 evoked a significant

increase in the percentage of time spent and visits to the open arms; however UFP-512 was more

potent, eliciting statistically significant effects at 0.1 mg/kg and importantly produced maximal

effects, lower than those induced by diazepam administration.

Interestingly, in all the above analyses, while naltrindole (3 mg/kg, sc) did not modify animal

behavior per se, it fully prevented the antidepressant- or anxiolytic- like actions induced by UFP-

512. The implications of these findings are twofold: i) in line with in vitro results, these data

demonstrate that UFP-512 behaved as a highly selective DOP agonist. The selectivity of action of

UFP-512 is higher than that of UFP-502 whose in vivo actions were only partially sensitive to

naltrindole [43]; ii) the selective activation of DOP receptors in rodents produces both

antidepressant- and anxiolytic-like effects.

To the best of our knowledge, the brain areas responsible for the antidepressant- and anxiolytic-like

effects promoted by DOP receptor agonists are not known yet. However, rat brain autoradiography

studies have shown that DOP receptor binding sites are located in the neocortex and in several

limbic structures, such as the striatum, nucleus accumbens, claustrum, layer of bulb, amygdaloid

nucleus, pontine nuclei, and inferior colliculus [19, 29]. Additionally, several authors using in situ

hybridization techniques have shown that DOP receptor mRNA is expressed in olfactory bulb,

neocortex, and limbic structures such as caudate-putamen, nucleus accumbens, ventromedial

hypothalamus, hippocampus, amygdala, suggesting that DOP receptors are expressed in these sites

[18, 27, 28]. Taken together, these literature data suggest that UFP-512 might elicit anxiolytic- and

antidepressant-like effects by activating DOP receptors located in distinct limbic structures of the

brain.

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15

An important concern regarding the interpretation of drug induced animal behaviors is related to the

alteration of locomotor activity. In fact, drug induced locomotor depressant or stimulatory effects

may bias the interpretation of the results obtained in different behavioral assays. In the present

study, the antidepressant- and anxiolytic-like effects of UFP-512 were not associated with

modifications of locomotor activity, as assessed both in activity cages and in the open field test.

Thus, these results suggest that the behavioral actions of UFP-512 can be interpreted as genuine

antidepressant- and anxiolytic-like effects.

Finally it is noteworthy that the antidepressant like effects elicited by UFP-512 in the mouse forced

swimming test and the naltrindole sensitivity have been recently replicated in a different laboratory

[1]. Furthermore, those investigators demonstrated that the effects of UFP-512 do not produce

tolerance after 7 days subchronic treatment [1].

Collectively, our data suggest that UFP-512 represents a highly valuable research tool for

investigating the consequences of selective DOP receptor activation in vitro and in vivo. In

addition, our data corroborate the proposal of DOP receptors as therapeutic target for the treatment

of anxiety and mood disorders. Further pre-clinical and possibly clinical studies are now required to

firmly determine the advantages and disadvantages of DOP receptor selective agonists as innovative

antidepressant and anxiolytic drugs.

Acknowledgements This work was supported by the University of Ferrara (60% grant to SS) and

by Regione Emilia Romagna (PRRIITT grant to UFPeptides s.r.l., Misura 3.1 Azione A, Bando

19/07/2004 Progetto N° 93).

References

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Table 1. Displacement of [3H]-diprenorphine from CHO cell membranes expressing human opioid

receptors by UFP-512 and standard opioid receptor ligands

pKi

MOP DOP KOP

Standard ligand (DAMGO)8.91

(Naltrindole) 10.12

(nor-BNI)9.71

UFP-512 8.00 10.20 6.66

DPDPE <6 8.48 <6

Data are mean ± sem of at least 4 separate experiments.

Table 1

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Table 2. Antagonistic action of naltrindole (10 nM) and naloxone (100 nM) against DPDPE and

UFP-512 induced inhibition of electrically stimulated twitches in the mouse vas deferens.

pA2 values

Naltrindole Naloxone

DPDPE 9.97 8.08

UFP-512 10.54 8.25

Data are mean ± sem of at least 5 separate experiments.

Table 2

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Table 3. Ethological description of the behavior displayed by mice injected with diazepam (1

mg/kg, ip) or UFP-512 (0.01-1 mg/kg, ip) in the elevated plus maze test.

*p<0.05 according to ANOVA followed by the Dunnett’s test. Total arm entries: F4,49 = 11.62.

Open arm entries: F4,49 = 13.15. Percent open entries: F4,49 = 4.94. Open arm time: F4,49 = 5.70.

Closed arm time: F4,49 = 1.56. Percent open arm time: F4,49 = 2.25. Total stretched attend postures:

F4,49 = 1.04. Total head dipping: F4,49 = 5.35.

Behavioral item Control Diazepam UFP-512saline 1 mg/kg 0.01 mg/kg 0.1 mg/kg 1 mg/kg

Total arm entries 16.4±1.2 25.4±1.8* 16.8±1.0 17.7±0.6 12.7±0.8Open arm entries 4.3±0.5 12.1±1.8* 5.8±0.7 7.3±0.5* 5.3±0.6Closed arm entries 11.1±1.3 13.3±1.9 11.0±1.2 10.4±0.8 7.4±1.4Percent open entries 28.4±2.4 47.4±5.6* 35.3±4.8 42.1±2.4* 43.7±4.8*Open arm time 34.9±6.0 107.0±16.6* 47.7±5.6 71.8±6.1* 64.7±7.2*Closed arm time 161.6±8.4 111.9±17.4* 132.4±9.4 108.5±7.6* 116.9±10.1*Percent open arm time

17.5±2.7 49.2±7.9* 26.6±2.6 40.1±2.6* 35.9±4.1*

Total stretched attend postures

8.1±0.7 4.9±1.2 5.7±1.3 3.9±0.4* 4.0±1.1*

Total head dipping 3.8±0.7 8.9±1.3* 5.4±1.0 6.9±1.1* 2.1±0.8Total grooming 1.4±0.3 1.4±0.5 1.1±0.4 1.1±0.3 1.0±0.4

Table 3

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iptN

NH

OO

H2N

HO

O

OH

HN

N

Figure 1. Chemical structure of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512).

Figure 1

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-13 -12 -11 -10 -9 -8 -7 -6 -5 -4

1.0

1.2

1.4

1.6

1.8

DPDPEUFP-512

Log[ligand]

Sti

mu

lati

on

fac

tor

-16 -15 -14 -13 -12 -11 -10 -9 -8 -70

25

50

75

100

DPDPE

UFP-512

Log[ligand]

% C

on

tro

l tw

itch

Figure 2. Concentration response curves to DPDPE and UFP-512 in the [35S]GTPγS binding assay

performed using CHOhDOP cell membranes (left panel) and in the electrically stimulated mouse vas

deferens (right panel). Data are the mean ± sem of at least 5 experiments.

Figure 2

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50

100

150

200

250

*

UFP-512 (nmol)

Saline 0.001 0.01 0.1

Imm

ob

ilit

y ti

me

(s)

50

100

150

200

250

* *

Saline 0.01 0.03 0.1 0.3 1

UFP-512 (mg/kg)

Figure 3. Dose response curve to UFP-512 given icv (left panel) or ip (right panel), in the mouse

forced swimming test. Data are mean ± sem of 12-14 animals. *p<0.05 vs saline, according to

ANOVA followed by the Dunnett’s test. Left panel: F3,46 = 9.10; right panel: F5,66 = 5.07.

Figure 3

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50

100

150

200

250

*

SalineUFP-512 0.1 mg/kgNaltrindole 3 mg/kgNaltrindole vs UFP-512

Imm

ob

ilit

y ti

me

(s)

Figure 4. Effects of naltrindole (30 min pre-treatment, sc) against UFP-512 (ip) in the mouse forced

swimming test. Data are mean ± sem of 10-12 animals. *p<0.05 vs saline, according to ANOVA

followed by the Dunnett’s test. F 3,44 = 9.82.

Figure 4

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uscr

ipt

0

50

100

150

SalineUFP-512 0.1 mg/kgUFP-512 0.3 mg/kgUFP-512 1 mg/kg

*

*

immobility climbing swimming

tim

e (s

)

Figure 5. Dose response curve to UFP-512 (0.1, 0.3 and 1 mg/kg, ip) in the rat forced swimming

test. Data are mean ± sem of 12-14 animals. *p<0.05 vs saline, according to ANOVA followed by

the Dunnett’s test. Immobility: F3,33 =9.05; climbing: F3,33 = 2.13; swimming: F3,33 = 2.27.

Figure 5

of 32

Accep

ted

Man

uscr

ipt

0

50

100

150

Saline

UFP-512 0.1 mg/kgDiazepam 1 mg/kg

UFP-512 1 mg/kg

**

tim

e in

th

e li

gh

t b

ox

(s)

Figure 6. Effects of diazepam (1 mg/kg, ip) and UFP-512 (0.1 and 1 mg/kg, ip) on the time spent in

the light compartment in the light-dark aversion assay. Data are mean ± sem of 8-12 animals.

*p<0.05 vs saline, according to ANOVA followed by the Dunnett’s test. F 3,84 = 1.48.

Figure 6

of 32

Accep

ted

Man

uscr

ipt

0

50

100

150

SalineUFP-512 1 mg/kgNaltrindole 3 mg/kgNaltrindole vsUFP-512

*

tim

e in

th

e li

gh

t b

ox

(s)

Figure 7. Effects of naltrindole (30 min pre-treatment, sc) on UFP-512 (ip) response on the time

spent in the light compartment in the light-dark aversion test. Data are mean ± sem of 8-11 animals.


Figure 7

of 32

Accep

ted

Man

uscr

ipt

0

40

80

120*

**

Saline Diazepam 1 mg/kg

0.01 0.1 1

UFP-512 (mg/kg)

tim

e o

n o

pen

arm

s (s

)

Figure 8. Effects of diazepam (1 mg/kg, ip) and UFP-512 (0.01-1 mg/kg, ip) on the time spent in

the open arms in the elevated plus-maze test. Data are mean ± sem of 8-12 animals. *p<0.05 vs

saline, according to ANOVA followed by the Dunnett’s test. F 3,43 = 13.72.

Figure 8

of 32

Accep

ted

Man

uscr

ipt

0

40

80

120

SalineUFP-512 0.1 mg/kgNaltrindole 3 mg/kgNaltrindole vs UFP-512

*

tim

e o

n o

pen

arm

s (s

)

Figure 9. Effects of naltrindole (30 min pre-treatment, sc) on UFP-512 (ip) response on the time

spent in the open arms in the elevated plus-maze test. Data are mean ± sem of 8-11 animals.


Figure 9

of 32

Accep

ted

Man

uscr

ipt

0

10

20

Control

UFP-512 1 mg/kgUFP-512 0.1 mg/kg

nu

mb

er o

f cr

oss

ing

in t

he

cen

tral

are

a

0

30

60

90

ControlUFP-512 0.1 mg/kgUFP-512 1 mg/kg

nu

mb

er o

f cr

oss

ing

in t

he

per

iph

eral

are

a

0

10

20

30

40

ControlUFP-512 0.1 mg/kgUFP-512 1 mg/kg

nu

mb

er o

f re

arin

gs

Figure 10: Lack of effect of UFP-512 (0.1 and 1 mg/kg, ip) on central locomotion (top panel),

peripheral locomotion (middle panel) and number of rearings (bottom panel) in the mouse open

field test. Data are mean ± sem of 8-12 animals.

Figure 10

of 32

Accep

ted

Man

uscr

ipt

0

100

200

300

400

ControlUFP-512 0.1 mg/kg

UFP-512 1 mg/kg

nu

mb

er o

f im

pu

lses

0

500

1000

1500

ControlUFP-512 0.1 mg/kg

UFP-512 1 mg/kg

nu

mb

er o

f im

pu

lses

Figure 11: Lack of effect of UFP-512 (0.1 and 1 mg/kg, ip) on the total number of impulses in the

mouse locomotor activity assay after 5 min (left panel) and 30 min (right panel) of observation.

Data are mean ± sem of 8-12 animals.

Figure 11

anxiolytic and antidepressant-like activities of ufp-512, a novel selective delta opioid receptor...

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