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Keywords:

  • animal models of depression;
  • depression;
  • galanin;
  • galanin receptor type 2;
  • neuropeptide;
  • tail suspension test

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

Neuropeptide galanin and its three G-protein coupled receptors, galanin receptor type 1–galanin receptor type 3 (GalR1–GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression-like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide-based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant-like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant-like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.

We utilize several chemical modifications to increase in vivo usability of peptide-based ligands, acting upon CNS. Accordingly, we introduce a series of novel systemically active galanin analogues, with modest preferential binding towards GalR2, and demonstrate their ability to attenuate depression-like behavior via brain GalR2 in different mouse models of depression.

Abbreviations used
5-HT

serotonin

CHO

Chinese Hamster Ovary

DR

dorsal raphe nucleus

FST

forced swim test

GalR1

galanin receptor type 1

GalR2

galanin receptor type 2

GalR2KO

galanin receptor 2 knockout

GalR3

galanin receptor type 3

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

i.c.v.

intracerebroventricular

i.p.

intraperitoneal

LC

locus coeruleus

NA

noradrenaline

TST

tail suspension test

VTA

ventral tegmental area

Depression is a common and serious mental health problem affecting approximately 121 million people, and is estimated to be the fourth major cause of disability worldwide (WFMH 2010). It is generally accepted that the etiology of depression involves changes in the CNS monoamine system (Prange 1964; Schildkraut 1965; Coppen 1967). Accordingly, commonly used antidepressants – monoamine reuptake inhibitors – act by enhancing central biogenic amine function by targeting the monoamine transporters. However, the resultant immediate neurochemical enhancement of monoamine transmission does not match with the onset of clinical therapeutic effect, which may start even several weeks after continuous administration of the antidepressant. Therefore, the observed clinical response is often attributed to long-term compensatory effects, resulting from antidepressant treatment. These compensatory effects may include antidepressant-induced modulation of second messenger processes, transcription factors and growth factors (Morilak and Frazer 2004).

Two important suggestions can be derived from this. First, such research has led to fundamentally new hypotheses regarding the underlying pathophysiology of depression, which allow focusing on other aspects than monoamine transmission levels. For example, depression pathophysiology may be related to neuronal death or to deficiencies in specific brain regions, such as the hippocampus, and antidepressants may induce neuroprotective mechanisms or even neurogenesis to resolve such deficits (McEwen 2000; Duman et al. 2001). Second, even when considering the secondary compensatory effects derived from the prolonged changes in monoamine signaling, it does not fully explain the fact that the clinical response of antidepressants is achieved in only about two thirds of the patients. Both of these two notations imply that novel therapeutic approaches to mood disorders are needed.

Association of neuropeptides, including galanin, and classical neurotransmitters with the mechanisms of depression have been proposed already 20 years ago (Grenhoff et al. 1993; Weiss et al. 1998). The potential involvement of galanin in depression was first hypothesized (Fuxe et al. 1991) because of its co-localization with noradrenaline (NA) in the locus coeruleus (LC) and serotonin (5-HT) in dorsal raphe nucleus (DR) in the rat (Skofitsch and Jacobowitz 1985; Melander et al. 1986; Xu et al. 1998a, b). The first pharmacological proof for involvement of brain galanin in depression-like behavior was described when galanin infusion into the rat ventral tegmental area (VTA) resulted in increased immobility time in the rodent depression screen test, the forced swim test (FST) (Weiss et al. 1998). This effect was attributed to galanin mediated inhibition of dopamine signaling in nucleus accumbens, because of ‘burst’ activation of NA-ergic fibers from LC to the VTA (Weiss et al. 1998). The increased immobility time in FST test translates as ‘potentially inducing depression-like state’, and galanin-induced pro-depressive effects have since been confirmed by several groups (e.g., see ref Kuteeva et al. 2007). Similarly, it has been shown that galanin inhibits the firing rate of 5-HT neurons in the rat DR (Xu et al. 1998b) and galanin in DR inhibits 5-HT release in the rat hippocampus (Kehr et al. 2002).

The biological activity of galanin is mediated trough interactions with three different G-protein coupled receptors, galanin receptor type 1–galanin receptor type 3 (GalR1–GalR3), which are all associated with different signaling pathways. Previously, it has been shown that activation of GalR2, in contrast to GalR1 and GalR3, significantly increases 5-HT release in DR (Mazarati et al. 2005) and leads to attenuation of depression-like behavior, while activation of GalR1 and GalR3 increases it (Lu et al. 2005; Swanson et al. 2005; Barr et al. 2006; Kuteeva et al. 2008b). Taken together, all this suggests that GalR2 subtype specific ligands could potentially be used as drug candidates for treating mood disorders.

This study has been initiated by the fact that peptides, because of high receptor specificity, potency, and low toxicity, are known to have great therapeutic potential, but at the same time, their physicochemical properties strongly restrict their use as psychopharmacological tools. In addition, there is a lack of systemically active GalR2 subtype specific ligands in the galanin field. Here, we have designed an approach to increase in vivo usability of peptide ligands and we hereby demonstrate a series of systemically active novel GalR ligands with modest preferential binding toward GalR2, including more in-depth characterization of one of these peptide ligands. Furthermore, we show in side by side comparison with a common clinically used antidepressant medication imipramine their ability to attenuate depression-like behavior.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Peptide synthesis

The peptides were synthesized in a stepwise manner using small scale (0.1 mmol) 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis strategy on an automated peptide synthesizer (Applied Biosystems™ model 433A, Foster City, CA, USA). Fmoc amino acids were coupled as hydroxybenzotriazole esters to a p-methylbenzylhydrylamine (MBHA) resin (Fluka, Buchs, Switzerland) which will generate a C-terminally amidated peptide after the final cleavage. The peptides were finally cleaved from the resin using 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS) and 2.5% H2O solution for 3 h.

To obtain branched peptides with stearic acid substitution, amino acid residue Fmoc-Lys (Mtt)-OH (Iris Biotech GMBH, Marktredwitz, Germany) was used. 4-methyltrityl (Mtt) – protecting group was removed manually using 1% TFA, 2% TIS solution in dichloromethane (DCM). Stearic acid was then coupled as a hydroxybenzotriazole ester overnight.

All the peptides were purified by reverse-phase HPLC using a Jupiter 5 μ C4 300 Ǻ column with dimensions of 250 mm × 10 mm internal diameter (Phenomonex, Torrance, CA, USA) and the correct molecular weight was determined by MALDI–TOF mass spectrometry (Voyager DE Pro; Applied Biosystems).

Cell cultures

Bowes human melanoma cells (American type Culture Collection CRL-9607) were cultured in Eagle's minimal essential medium with Glutamax-1 supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% non-essential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin. Chinese Hamster Ovary (CHO) K1 cells stably expressing human GalR2 (a kind gift from Kathryn A. Jones and Tiina P. Iismaa, Sydney, Australia) were cultured in Dulbecco's modified essential medium F-12 with Glutamax supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Flp-In T-REx 293 GalR3 cell line were cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 15 μg/mL blasticidin S, and 150 μg/mL Hydromycin B (F. Hoffmann-La Roche Ltd, Basel, Switzerland). Cell cultures were grown at 37°C in a 5% CO2 incubator. Cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA) and cell plastics from Greiner Bio-One GmbH (Frickenhausen, Germany).

Galanin receptor binding assays

Cells for 125I-galanin-receptor displacement studies were seeded in 150-mm dishes and cultured 3–4 days until confluent. Cell dishes were washed and scraped into phosphate-buffered saline and centrifuged twice at 4°C, 3000 g for 5 min. The pellet was re-suspended in assay buffer (20 mM HEPES, 5 mM MgCl2, pH 7.4) supplemented with EDTA (5 mM EDTA) and incubated on ice for 45 min before centrifugation at 4°C, 8500 g for 15 min. After washing the pellet in assay buffer and repeated centrifugation, the obtained pellet was re-suspended in assay buffer supplemented with 1% protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) to a protein concentration of 1 mg/mL and stored at −80°C, until used. Protein concentration was determined according to Lowry (BioRad, Stockholm, Sweden). Displacement studies on cell membranes were performed in a final volume of 200 μL, containing 0.15 nM porcine-[125I]-galanin (2200 Ci/mmol; Perkin–Elmer Life Science, Boston, MA, USA), 30 μg cell membrane, and various concentrations of peptide (10−5–10−9 M). Peptide solutions were made in assay buffer supplemented with 0.3% bovine serum albumin using silanised (dichlorodimethylsilane; Sigma–Aldrich) tubes and pipette tips. Samples were incubated at 37°C for 30 min while shaking after which the samples were transferred and filtered through a MultiScreen-FB filter plate (Millipore, Billerica, MA, USA) pre-soaked in 0.3% polyethylenimine solution (Sigma–Aldrich) using vacuum. The filters were washed thrice with assay buffer and the retained radioactivity was determined in a β-counter (Tri-Carb Liquid Scintillation Analyzer, model 2500 TR; Packard Instrument Company, Meriden, CT, USA) using OptiPhase Supermix Cocktail (Perkin–Elmer Life Science, Boston, MA, USA) as scintillation fluid. ‘IC50’ values for the peptides were calculated using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) and converted into Ki values using the equation of Cheng–Prusoff (Cheng and Prusoff 1973).

Biodistribution studies

The stearoylated peptide J18 and non-stearoylated peptide M1145 were labeled with 125I using the Iodo-Gen method. First, 0.2 mg of peptide (5 mg/mL in H2O) was mixed with 60 μL of 10 mM HEPES-buffer (pH 7.2) in Pierce Iodo-Gen pre-coated iodination tube (Thermo-Scientific, Rockford, IL, USA). Subsequently, 5 μL of Na-125I solution (Map Medical Technologies, Tikkakoski, Finland) with radioactivity of 22.0–23.9 MBq was added and let react for 45 min at 22°C. The peptide was purified of excess 125I with Sep-Pak C-18 solid-phase extraction method (Waters Corporation, Milford, MA, USA) by following the manufacturer's instructions. In this study, 0.1% trifuoroacetic acid in H2O and 95% acetonitrile, 0.1% trifluoroacetic acid in H2O were used as eluents. The solvents were evaporated with nitrogen gas flow and the radiolabeled peptide was dissolved in H2O (0.4mg/mL). The radiochemical purity was determined using thin layer chromatography (ITLC-SG; Pall Corporation, Ann Arbor, MI, USA) with 0.1 M KI in phosphate-buffered saline as an eluent. The radiochemical purity and radioactivity for J18 and M1145 were 96.6% and 94.9%, and 17.51 MBq and 13.58 MBq, respectively. For the biodistribution studies, the peptides were mixed with non-labeled peptides (1 : 10) and diluted in 0.9% NaCl.

The animal procedures were performed according to the guidelines approved by the Ethical Committee of the National Laboratory Animal Center Finland (License number ESHL-2009-010004, Kuopio, Finland). Healthy male C57Bl/6j mice, weighing 21–29 g were used in this study. The mice received chow and water ad libitum. Isoflurane-anaesthetized mice were injected via tail vein at a dose of 0.5 mg/kg of prepared 125I-peptide solution. The radioactivity of the syringe was measured before and after injection with gamma counter (LKB-WallacClinigamma 1272, WallacOy, Turku, Finland). The radioactivity of weighted tissue samples (blood, heart, lungs, liver, spleen, kidneys, duodenum, testis, muscle, brains, and thyroid) was measured with gamma counter. Tissue-specific radioactivity is presented as percentage of injected dose per weight of the tissue sample (% ID/g), considering background radiation and decay of the radioisotope.

Behavioral characterization

Balb/c (Harlan) male and female mice (8–10 weeks old at the start of the experiment) were used for the animal behavioral experiments. The GalR2KO mice were generated on a 129/Sv genetic background (Shi et al. 2006) and backcrossed into C57BL/6 for eight generations. 8- to 10-week-old male and female animals were used. The mice were housed in a temperature-controlled room (20–22°C) under a 07.00 on and 19.00 off 12-hour light–dark cycle. Animals were provided with ad libitum access to food and water. All the animal procedures and experiments were approved by the Estonian laboratory animal ethics committee (approval no 61, dated Nov 17, 2010).

The animals to be cannulated were anesthesized with 75 mg/kg ketamine (Vetoquinol, Bioketan, France) in combination with 1.0 mg/kg dexmedetomidine administered intraperitoneally (i.p.) (Laboratorios SYVA, Dorbene, Spain). Each animal received a cannula made of stainless steel hypodermic tubing (24 gauge and 10 mm long) into the right lateral ventricle (co-ordinates from Bregma: −0.5 anterior/posterior, −2.2 dorsal/ventral, and −1.0 laterally). Two stainless screws (M 2 × 1) and dental cement (Dentalon, AgnThos, Sweden) were used to secure the small plastic tubing (which protects the cannula) in place. The anesthesia was blocked after the surgery using α2-adrenergic antagonist atipamezole hydrochloride (Antisedan, Espoo, Finland), 1.0 mg/kg, administered subcutaneously (s.c.). All the animals received tramadol 30 mg/kg i.p. immediately after the surgery and once a day for 3 days post-operatively. Mice were allowed to recover from the surgery for a minimum of 7–10 days. At the completion of the experiments, the mice were killed by decapitation under deep anesthesia and the correct placement of the cannula in the lateral ventricle was verified by visual inspection during sectioning. No incorrect cannula placements were detected.

Peptides and imipramine were dissolved in 0.9% NaCl and administered either intravenously (i.v.), i.p. or into the right lateral ventricle (i.c.v.). I.c.v. injections were performed immediately before the experiment, in a volume of 3 μL, using a Hamilton syringe connected by plastic tubing to a 31 gauge, 11 mm stainless steel injector. The i.v. and i.p. injections were performed 15 min before the experiment.

The forced swim test (FST) consisted of one episode of 6 min swimming (glass container, diameter 15 cm, height 30 cm), of which the last 4 minutes were scored for total immobility time. For tail suspension test (TST), the mice were individually suspended by the tail to the wooden beam, using adhesive tape, 1 cm from the end of the tail, for 6-min period. The duration of immobility during the last 4 min was measured. Immobility was defined as complete lack of movements besides respiration. The numbers of animals in each group are presented in Table S1.

After chronic administration experiment, where the peptide was injected daily for 17 days, whole blood of an anesthetized animal was collected by heart punction using serum separation tubes (BD Bioscience, San Jose, CA, USA). Blood sample analysis for levels of C-reactive protein and interleukin 6 was conducted by the United Laboratories at Tartu University Hospital.

Data analysis

All values are presented as mean ± SEM. Data were considered significant at *p < 0.05. The biochemical data represent at least three independent experiments performed in duplicates and analyzed using anova followed by Dunnett's multiple comparison test using Prism 5.0 (GraphPad Software Inc.). Data from behavioral experiments were analyzed by one-way anova followed by Tukey HSD post hoc (Statistica; StatSoft, Tulsa, OK, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Galanin receptor affinity of the ligands

Displacement studies using 125I-galanin with galanin and peptides, J17-J21, were performed on cell membranes from human Bowes melanoma cells endogenously expressing hGalR1, CHO cells stably transfected with hGalR2 and Flp-In T-REx 293 cells with inducible expression of hGalR3. Peptides were tested three times in independent assay experiments. In general, all tested peptides exhibited preferential binding toward GalR2 with nanomolar binding affinities (Table 1, Fig. 1). The best subtype selectivity was demonstrated by the peptide J18, showing a moderate six to seven-fold selectivity toward GalR2, compared to GalR1 and GalR3 (Table 1, Fig. 1b). The best binding affinity for GalR2 was demonstrated by J20 with a Ki of 4.9 nM, which is in the same range as for galanin.

Table 1. Amino acid sequences and affinities of peptides used or discussed in this study
NameSequenceKi GalR1 (nM)Ki GalR2 (nM)Ki GalR3 (nM)Ki GalR1/GalR2Ki GalR3/GalR2
  1. X – 6-aminohexanoic acid; O1- Ornithine;

  2. a

    Runesson et al. (2009);

  3. b

    Saar et al. (2011).

Galanin(1-29), ratGWTLNSAGYLLGPHAIDNHRSFSDKHGLT-amide1.75 ± 1.7a2.98 ± 1.4a4.49 ± 0.8a0.6a1.5a
M1153bRGRGN-WTLNSAGYLLGPK(ε-NH-C(O)CH(NH2)(CH2)2COOH) – amide587 ± 250a6.55 ± 2.7a497 ± 150a380b46b
M1145aRGRGN-WTLNSAGYLLGPVLPPPALALA - amide1890 ± 329b4.98 ± 0.81b230 ± 149b90a76a
J17RGRGN-WTLNSAGYLLGP-KKK(εNH·C(O)stearic acid)-amide156 ± 10229 ± 18125 ± 945.44.3
J18RGRGN-WTLNSAGYLLGP-kkK(εNH·C(O)stearic acid)k-amide138 ± 4620 ± 8.0112 ± 146.95.6
J19RGRGN-WTLNSAGYLLGP- O1 O1K(εNH·C(O)stearic acid) O1-amide231 ± 12083 ± 23114 ± 702.81.4
J20RGRGN-WTLNSAGYLLGP-XXK(εNH·C(O)stearic acid)K-amide25 ± 5.54.9 ± 1.613 ± 7.05.12.7
J21O1GO1GN-WTLNSAGYLLGP- O1 O1K(εNH·C(O)stearic acid) O1-amide138 ± 6041 ± 2265 ± 8.63.41.6
image

Figure 1. Galanin receptor binding studies. Displacement of porcine-[125I]-galanin from membranes by peptide J17 (a), J18 (b), J19 (c), J20 (d), and J21 (e). Membranes were from human Bowes melanoma cells expressing GalR1 (closed circle), Chinese Hamster Ovary (CHO) cells expressing galanin receptor type 2 (GalR2) (closed square) and Flp-In T-REx 293 cells expressing GalR3 (closed diamond). Calculated Ki values are summarized in Table 1.

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Mouse depression models

The effect of the peptides J17-J21 on depression-like behavior was screened by a common depression screen test, forced swim test, where decrease in immobility predicts antidepressive effect of the drug. Several of the tested peptides demonstrated an antidepressant-like effect in FST after single i.v. injection (anova F(11, 81) = 6.21). We chose one of the peptides, J18, which showed a combination of good subtype selectivity and high binding, for further behavioral characterization. Interestingly, when tested at different doses, we did not observe a linear dose–response, rather, a U-shaped curve formed, with the peak at the dose of 0.5 mg/kg in FST (Tukey post hoc p < 0.001). In addition to J18, significant reduction in immobility time was also demonstrated by the peptide J20 at the dose of 0.5 mg/kg (Tukey post hoc p < 0.001). Consistently, from all the modified peptides, J18 and J20 showed also the highest affinities toward GalR2 (Table 1). The effect exhibited by J18 and J20 was comparable to that of demonstrated by known antidepressant imipramine (Fig. 2).

image

Figure 2. Forced swim test immobility scores after single i.v. injection of different doses of the peptides. The doses (mg/kg) are represented as a numerical value after each peptide name on the X-axis. Imipramine (15 mg/kg i.p.) was used as a positive control. Number of animals is 5–15 in each group (see Table S1); experiments with saline, imipramine, J17, J18, and J20 were conducted on at least two different days. Stars represent Tukey HSD post hoc comparisons with the saline group. ***p < 0.001, *p < 0.05.

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Similar to the FST, the peptide J18 showed an antidepressant-like effect in another depression screening test, TST (anova F(4, 22) = 27.5, p < 0.001), although the magnitude of the effect was smaller than with imipramine. Again, as in case of FST, a U-shaped dose–response curve formed. The strongest effect was achieved with the dose of 0.25 mg/kg (Tukey post hoc p < 0.01), larger and smaller doses yielded slightly weaker effects (Fig. 3).

image

Figure 3. Tail suspension test immobility scores after i.v. injection of different doses of J18 (the numerical value on the X-axis represents mg/kg). Imipramine (15 mg/kg i.p.) was used as a positive control. Number of animals is four to seven in each group (see Table S1). Stars represent Tukey HSD post hoc comparisons with the saline group. ***p < 0.001, **p < 0.01, *p < 0.05.

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To assess if J18 maintains its antidepressant-like effect also in chronic administration regime, first the suitability of i.p. injection route was assessed, because this is a prerequisite for chronic low-stress administration. Accordingly, acute i.p. administration appeared to exert similar reduction in immobility as in case of i.v. administration in TST (Fig. 4, Tukey post hoc p < 0.05 for saline versus J18 i.p, anova F(3, 23) = 7.5, p < 0.01). Having confirmed the suitability of i.p, we next assessed if the effect is maintained after chronic administration of the peptide. Chronic administration of J18 (0.25 mg/kg for 17 days) still retained the reduction in immobility time in TST (Fig. 5, Tukey post hoc p < 0.05, anova F(1,25) = 5.98).

image

Figure 4. The effect of administration route: J18 was injected (0.25 mg/kg) i.v. and i.p, and the effect tested in tail suspension test (TST). J18 exerted similar antidepressant activity both for i.v. and i.p route. Stars represent Tukey HSD post hoc comparisons. **p < 0.01, *p < 0.05.

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image

Figure 5. Tail suspension test immobility scores after chronic administration of J18 (0.25 mg/kg i.p. per day for 17 days). Stars represent Tukey HSD post hoc comparisons. *p < 0.05.

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To verify that brain galanin receptors, and specifically GalR2, mediate the effects observed with J18, two approaches were used. First, a non-selective galanin receptor antagonist M35 (4 μg i.c.v.) was used concomitantly with J18 (i.p.). Indeed, i.c.v. M35 blocked the effect induced by i.p. J18 (0.25 mg/kg), supporting the hypothesis that J18 acts through brain galanin receptors (Fig. 6, Tukey post hoc p < 0.05, anova F(3, 33) = 4.98, p < 0.01). Second, GalR2 KO animals were used in TST depression screening test. Similar to normal, in genetically unmodified animals (Fig. 3), imipramine strongly reduced the immobility of the animals (comparison with the saline treatment, Fig. 7, Tukey post hoc p < 0.001, anova F(2, 26) = 47.92, p < 0.001). In contrast, J18 had no effect on the behavior of the GalR2KO animals (Fig. 7), suggesting that J18 exerts its antidepressant-like activity through GalR2.

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Figure 6. Tail suspension test immobility scores after combination administration of i.p. J18 (0.25 mg/kg) and i.c.v. galanin receptor antagonist M35 (4 ug). The analysis combines data from four independent experiments. Number of animals is seven to eight in each group (see Table S1). Stars represent Tukey HSD post hoc comparisons. *p < 0.05.

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image

Figure 7. Tail suspension test immobility scores in galanin receptor 2 knockout (GalR2KO) animals after i.p. administration of J18. Imipramine (15 mg/kg i.p.) was used as a positive control. Number of animals is 9–11 in each group (Table S1). Stars represent Tukey HSD post hoc comparisons with the saline group. ***p < 0.001.

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Biodistribution

The distribution of radiolabeled stearoylated peptide J18 was compared to a non-stearoylated analog M1145. The general tissue levels of J18 at 1 h and 3 h were significantly higher than that of the parent peptide (Fig. 8). This is probably because of higher stability to degradation and lipofilicity of J18, which arises from stearoylation and non-natural amino acids. In addition, brain levels of J18 were significantly higher than in case of M1145, reaching to about 0.25% ID/g at 1 h post-injection (Fig. 8).

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Figure 8. The biodistribution of peptides J18 and M1145. The peptides were labeled with 125I and injected i.v. at a dose of 0.5 mg/kg. Whole organs were collected and subjected to gamma-counting at different time points after injection: 1 h (a), 3 h (b), and 24 h (c). The levels of the stearoylated peptide J18 are higher in all organs at 1 and 3 h, indicating its slower elimination from the body. The levels of J18 are also significantly higher in brain (d), reaching to approximately 0.25% ID/g at 1 h.

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Toxicological analysis

We monitored the animal weights and general look during the chronic administration of J18. The weight change dynamics was identical for both sexes, and for both saline and J18 (Figure S1); no deviations were observed in general look and behavior of the animals. Blood testing after chronic administration revealed no increases in C-reactive protein or interleukin 6, indicating no inflammatory effect.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In this study, we present a series of novel systemically active GalR2 subtype preferring ligands J17-J21 (Table 1). The design of the ligands is based on two previously published GalR2 agonists M1145 and M1153 (Runesson et al. 2009; Saar et al. 2011). The N-terminal part of the novel ligands was elongated with the same four amino acid residues (Arg-Gly-Arg-Gly) from the galanin-like peptide (GALP) as was done in M1145 and M1153 (Ohtaki et al. 1999; Runesson et al. 2009; Saar et al. 2011). To disturb the ligand binding to GalR1 and GalR3, the N-terminal Gly in position 5 was replaced with Asn (Sollenberg et al. 2006; Runesson et al. 2009; Saar et al. 2011).

Blood–brain barrier (BBB) is known to act as a major obstacle to deliver macromolecular entities, such as peptides, to CNS. In addition, short half-lives and large volumes of distribution in the blood of the peptides are a concern. Therefore, two commonly known strategies were used (Kokaia et al. 2001) to improve the pharmacokinetic properties (i.e. stability to degradation) and the CNS activity of the ligands. First, the ligands were elongated with additional positively charged amino acids (cationization). Second, a stearic acid was coupled to the side chain of Lys in the C-terminal part of ligands (lipidization). Both cationization and lipidization have been shown to enhance the bioavailability of the peptide in the brain (Bulaj et al. 2008). In addition, to reduce enzymatic degradation and further increase the bioavailability, three C-terminally positioned l-lysines were substituted to d-lysines in the peptide J18.

All these modifications resulted in a slight loss of subtype specificity for GalR2 and in a decrease in binding affinity when compared to previously published GalR2 agonists M1145 and M1153 (Table 1). However, in the displacement studies, all the tested peptides still exhibited a reasonable preferential binding toward GalR2. The best subtype specificity was demonstrated by the peptide J18, with approximately six to seven-fold selectivity toward GalR2, compared to GalR1 and GalR3. Importantly, all peptides showed nanomolar binding affinities toward GalR2 ranging from 4.9 nM to 83 nM. The lowest Ki values to GalR2 were exhibited by J19 and J21, which could be because of substitution of the C-terminally positioned l-lysines with ornithine. In addition, two l-arginines were substituted with ornithines in the position 2 and 4 of the peptide J21. This modification led to a two fold increase in Ki values when peptides J19 and J20 were compared, probably because of the smaller molecular size of ornithine that might have disrupted, to a lesser degree, the binding of the ligand to GalR1-GalR3. At the same time, in the peptide J20 the substitution of two l-lysines with aminohexanoyls in the positions 18 and 19 increased the binding toward GalR2. This suggests that moving the fatty acid further away from the original peptide backbone decreases its ability to disturb the binding toward GalRs.

Nevertheless, and as discussed subsequently, somewhat decreased binding affinity and lower receptor subtype selectivity of the peptides may not indicate anything about its usefulness in vivo, because increased stability may compensate for lower affinity. The main focus of this study was to develop in vivo usable, systemically active peptide-based ligands, and to confirm the utility of these in a field which notably lacks usable compounds, but has profound clinical potential. The rationale for concentrating on galanin system was based on previous studies, suggesting that GalR2 agonists should exhibit antidepressive effect (Kuteeva et al. 2008b). Therefore, we tested the behavior of the novel galanin receptor ligands in vivo.

First, we compared the biodistribution of J18 with a previously published GalR2 subtype specific ligand M1145, which does not contain stearoyl and d-amino acids (Fig. 8). As hypothesized, the relative distribution of the peptides was notably different. The differences arise from two aspects. First, J18 clearly showed much higher half-life, which reflects its higher stability to degradation. Figure 8 shows that the levels of J18 were higher in all organs 1 h and 3 h post-injection. At 24 h post-injection, J18 had been cleared out from the organism, while M1145 had been largely eliminated already at 3 h. Second, the levels of J18 in the brain were 2.5 fold higher than that of M1145. These results suggest that the combination of increased stability and lipofilicity increased the ability of J18 to penetrate the BBB.

We also demonstrate the antidepressant activity of the peptides by performing FST. The first notable thing is that we are able to see a CNS-derived behavioral effect upon systemic administration of a (modified) peptide, which, according to classical pharmacology, would not be able to reach the brain. We showed that i.v. administration of several peptides reduced the immobility time in FST. These results are in concordance with a previous publication demonstrating that GalR2(R3) agonist AR-M1896 decreased immobility, while GalR2 antagonist M871 increased immobility time in the FST (Kuteeva et al. 2008b) – the effects that have been observed upon i.c.v. administration of the drugs. The highest effect in this study was obtained by the peptide J18 at the concentration of 0.5 mg/kg, comparable to the effect presented by the clinically relevant antidepressant medication imipramine. Remarkably, the effective doses of J18 of 0.5 and 0.25 mg/kg are equal to 192 and 96 nmol/kg, which reflect strikingly high potency of the peptides.

The U-shaped dose–response relationship peaking at 0.5 mg/kg shown by J18 could possibly be explained by increased binding to GalR1 and GalR3 at higher concentrations, an assumption, which is supported by the binding profile (see Table 1) of the J-peptides, mostly showing modest specificity toward GalR2. Previous research has shown that the activation of GalR1 and GalR3 can increase the pro-depressive effect of galanin (Kuteeva et al. 2008b). Accordingly, the activation of GalR1 and GalR3 caused by the higher concentrations of the ligand could possibly counterattack the effect mediated through GalR2, the one that is known to lead to the attenuation of depression-like behavior (Kuteeva et al. 2008a). Further studies with lipidated peptides with higher receptor subtype selectivity might confirm this hypothesis; if this is the case, higher selectivity toward GalR2 should exhibit more linear dose–response curve in depression animal models.

Nevertheless, it should be noted that high receptor subtype selectivity in itself may not always yield the best-behaving pharmaceutical compound. For example, the same antidepressant drug used in this article, imipramine, has different binding sites and directly modulates several neurotransmitter levels in the brain (including serotonin, noradrenalin, and dopamine). Therefore, simultaneous binding to several receptor subtypes may contribute to the unique behavioral effect (or clinical effect) of the compound (in this case, antidepressive effect).

We also used another depression model (tail suspension test) to further confirm systemic activity of the novel peptides (Fig. 3). As expected, similar to the effect in FST, J18 exhibited potent systemic activity, with slight shift in the dose curve, the dose of 0.25 mg/kg being most efficient in this test paradigm. In addition, we show that the antidepressive effect of J18 is retained also via i.p. administration route, which is important for animal studies, as it permits easy, stress-free handling, and experimental setup with chronic administration. Antidepressive drug screening animal models utilize acute administration of a drug (two injections for rats and one injection for mice in TST or FST). However, it is generally known that chronic administration for 2–4 weeks is required for SSRIs to see antidepressive effect in clinics. Therefore, we investigated if it is possible to use J18 in other depression models which use repeated administration of a drug, as theoretically, different feedback mechanisms or receptor desensitization may decrease the pharmacological potency of the compound. Chronic administration of J18 (0.25 mg/kg i.p. for 17 days) still retained its antidepressant-like effect in TST (Fig. 5), suggesting that further characterization of J18 and other currently presented peptides in different models is possible.

Considering that the behaviors observed and quantified in mouse depression paradigms are purely motor (differentiation between locomotion vs. immobility), and not necessarily because of central effects, we confirmed, using pharmacological tools, that the effect of the novel peptides is really CNS-derived, and mediated by GalR2. Accordingly, i.c.v. infusion of a known galanin receptor antagonist blocked the antidepressive effect of systemically administered J18 (Fig. 6), suggesting that indeed, brain galanin receptors are activated by J18. Moreover, as J18 failed to elicit antidepressant-like effect in GalR2KO animals supports the hypothesis that J18 acts through GalR2. The mechanism by which GalR2 stimulation leads to antidepressant-like effect may be mediated by interactions between galanin and 5-HT/DR and NA/LC – the same CNS areas that mediate pro-depressive effects through GalR1 activation (Weiss et al. 1998). It is hypothesized that galanin is released under stressful conditions (Lundberg and Hokfelt 1986), creating a tonic galanin transmission. Accordingly, it has been shown that activation of GalR1 and R2 mediate opposing effects, where i.c.v. galanin, GalR1 agonist, or GalR2 antagonist induced pro-depressive behavior, and GalR2(R3) agonist induced antidepressive behavior (Kuteeva et al. 2008b). However, antidepressant treatment may augment the GalR2 tone (Kuteeva et al. 2008b), by increasing galanin levels and GalR2 binding sites (Lu et al. 2005), leading to relative shift from normally major pro-depressive GalR1 to now dominating antidepressive GalR2. It is known that GalR1 mainly activates Gi/o types of G-proteins, mediating inhibition via adenylate cyclase (Habertortoli et al. 1994) and inhibition of monoamine system in LC and DR (Kuteeva et al. 2008a). In contrast, the GalR2 subtype may transmit stimulatory effects of galanin, activating Gq/11 types of G-proteins (Wang et al. 1998) and thus may mediate antidepressive effects through LC (Kuteeva et al. 2008b) and DR (Lu et al. 2005).

In our study, i.c.v infusion of non-specific galanin antagonist M35 blocked the antidepressive activity of GalR2 agonist J18, but did not, by itself, reduce immobility in a rodent depression test, as has been shown previously for M35 (Kuteeva et al. 2007), or for other non-specific antagonists (for example, see ref Weiss et al. 1998). However, there are other works that have, similar to the current one, not observed this effect (for example Lu et al. 2005). One possible explanation that may explain this is species differences between mouse and rat (Larm et al. 2003).

Taken together, we demonstrate a set of chemical modifications that alter peptide pharmacokinetics and biodistribution, increasing its stability to degradation and penetration to brain. Hence, we characterize a series of novel systemically active GalR2 agonists and demonstrate their potent antidepressant effect at notably low doses in several common rodent screening models of depression-like behavior.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Kathryn A. Jones and Tiina P. Iismaa, Neurobiology Program, Garvan Institue of Medical Research, Sydney, Australia for Chinese Hamster Ovary cells stably transfected with the human GalR2 and Linda Lundström and Silvia Gatti-McArthur, F. Hoffmann-La Roche AG, Basel, Switzerland for Flp-In T-REx 293 cell line stably transfected with the human GalR3. This study was supported by national scholarship program Kristjan Jaak, which is funded and managed by Archimedes Foundation (IS); by the Helge Ax:son Johnsons foundation, Sven & Dagmar Saléns foundation and Olle Engkvist Byggmästares foundation (JRu); by the EU through the European Regional Development Fund through the Center of Excellence in Chemical Biology, Estonia; by the targeted financing SF0180027s08 from the Estonian Government; by grants from Estonian Ministry of Education and Research Grant SF0180064s08 (JJ), by The National Doctoral Program in Nanoscience (NGS-nano) and the strategic funding of University of Eastern Finland (JRy), by the grant ETF8679 from the Estonian Science Foundation and Swedish Science Foundation (VR-Med), the Knut and Alice Wallenberg Foundation and Swedish Center for Biomembrane Research (ÜL). The authors declare no competing financial interests.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12274-sup-0001-FigureS1.tifimage/tif857KFigure S1. Animal weight dynamics during the chronic administration of the peptide J18 in male (M) and female (F) animals.
jnc12274-sup-0002-TableS1.docxWord document12KTable S1. Animal numbers used in in vivo experiments

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