Galanin (1-15) and fluoxetine in the treatment of treatment-resistant depression

Abstract

Galanin (1-15) and fluoxetine in the treatment of treatment-resistant depression, GAL (1-15) in combination with a seratonin reuptake inhibitor, preferably fluoxetine, for use in the treatment of treatment-resistant depression, and a composition comprising same.

Description

[0001] DESCRIPTION

[0002] Galanin (1-15) and Fluoxetine in the treatment of treatment-resistant depression

[0003] TECHNICAL FIELD

[0004] The present invention falls within the pharmaceutical sector and is intended for the treatment of psychiatric disorders. Specifically, it focuses on the combined use of galanin (1-15) and selective serotonin reuptake inhibitors, such as fluoxetine, in the treatment of treatment-resistant depression (TRD).

[0005] PRIOR ART

[0006] Major depressive disorder (MDD) is the most prevalent psychiatric disorder and the leading cause of suicide worldwide. According to estimates from the World Health Organization, it will become the leading cause of disability by 2030. Furthermore, following the 2019 coronavirus disease (COVID-19) pandemic, an estimated 53.2 million additional cases of MDD have been diagnosed globally.

[0007] Major depressive disorder (MDD) is characterized by hopelessness, anhedonia, heightened guilt, and painful physical symptoms, often leading to suicidal tendencies. Medications for MDD primarily regulate the neurotransmission of serotonin (5-hydroxytryptamine or 5-HT) and norepinephrine. The most common initial choice is selective serotonin reuptake inhibitor (SSRI) antidepressants due to their high efficacy, tolerability, and adherence. However, 50% of patients do not respond to two or more medications or psychotherapeutic treatments, a condition known as treatment-resistant depression (TRD). Therefore, it is imperative to find innovative solutions to provide effective, faster, and longer-lasting relief of depressive symptoms in patients with TRD.

[0008] In this context, certain members of the galanin (GAL) neuropeptide family are implicated in the development of depression, offering a promising opportunity to enhance the efficacy of antidepressants by combining them with these neuropeptides. GAL is involved in various physiological processes and diseases in animal models, including mood regulation and depression, through its three GAL receptors (GALRs). Activation of GALR1 and GALR3 is associated with depressive behaviors, while stimulation of GALR2 has antidepressant effects. Chronic use of the SSRI fluoxetine (FLX) and electroconvulsive therapy increase GAL mRNA levels in the dorsal raphe nuclei (DR) of rats. The DR is the main source of serotonergic innervation to the forebrain and is involved in modulating the neural network implicated in anxiety.This increase is accompanied by an increase in GALR2 receptor binding sites in this monoaminergic nucleus, while GALR1 receptor binding sites are unaffected.

[0009] The N-terminal fragment GAL(1-15) exhibits differential biological activity compared to the full peptide in modulating mood in animal models. GAL(1-15), acting through GALR1-GALR2 heteroreceptor complexes, particularly in the dorsal hippocampus and right dorsum (RD), induces prodepressant and anxiogenic effects in animal models. Interestingly, GAL(1-15) potentiated the antidepressant effects induced by the 5-HT1AR agonist (5-hydroxythyroxine, a subtype of 5-HT receptor that binds to endogenous serotonin neurotransmitters), 8-OH-DPAT (±-8-hydroxy-2-dipropylaminotetraline), in the forced swim test (FST) [1], an effect that was significantly greater than that induced by GAL. The mechanism of this action involved alterations in the binding characteristics and mRNA levels of 5-HT1AR in the dorsal hippocampus and the RD.

[0010] In subsequent studies with rats, it was observed that GAL(1-15) enhances the antidepressant effects and reverses the memory impairment induced by FLX [2] with 5-HT1AR being involved in the hippocampus and prefrontal cortex (PFC), respectively

[0011] Based on these findings, the use of GAL(1-15) has been proposed as a combination treatment with SSRIs against MDD, among other disorders related to extracellular serotonin levels [ES2659092]. Behavioral changes were observed in rats administered GAL(1-15) and FLX compared to those administered only the SSRI FLX.

[0012] The interaction between GAL(1-15) and FLX has also been investigated in a depression model using olfactory bulbectomy (OBX) rats [3]. GAL(1-15) enhances the antidepressant effect of FLX in despair and anhedonia tests. This process depends on 5-HT1AR in the hippocampus at the plasma membrane and transcriptional levels. GAL(1-15) also potentiates the effect of escitalopram in depressive behavior tests in OBX rats, which involves 5-HT1AR activation. This effect is inhibited when 5-HT1AR is downregulated using siRNA (silencing RNA) [4]. Two networks are involved in the effect of GAL(1-15) on Escitalopram activity: one involves the lateral (LHb) and medial (mHb) habenula, the dorsal raphe (RD) and the ventral tegmental area (VTA), and the other includes the dentate gyrus (DG) and the prefrontal cortex (PFC).

[0013] DESCRIPTION OF THE INVENTION

[0014] The present invention investigates the effects of co-administration of GAL(1-15) and FLX in the treatment of DDT. For this purpose, the Wistar-Kyoto (WKY) rat model, which is genetically similar to endogenous depression [5], has been selected. This strain meets the criteria for a depression model and reflects the irregularities of the monoamine, glutamate, and GABA systems, as well as the dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis observed in patients with major depressive disorder (MDD). Standard antidepressants, especially SSRIs, do not elicit responses in these WKY rats, making them a suitable model for DDT.

[0015] To evaluate the impact of combined administration of GAL(1-15) and FLX on the behavior of WKY rats, tests measuring despair (FST and TST) were conducted. The effectiveness of the GAL(1-15) + FLX treatment is particularly evident in these despair behavioral tests.

[0016] The involvement of GALR2 was also explored using the antagonist M871 (a peptide antagonist that selectively recognizes the galanin receptor type 2) and an in vivo 5-HT1A knockdown siRNA model. The results indicate the possible involvement of GALR2, as demonstrated by the fact that M871 blocks the actions of GAL(1-15) in the FST, and highlight the role of 5-HT1AR in the interaction between GAL(1-15) and FLX, as demonstrated by the siRNA-induced downregulation of 5-HT1AR, which blocked the potentiation of the antidepressant effects of GAL(1-15) + FLX observed in WKY rats.

[0017] We explored whether the effects of GAL(1-15) on FLX were related to 5-HT1AR, by analyzing the binding of 5-HT1AR in brain regions (RD, GD, CA1 area of ​​the hippocampus, ventral hippocampus and medial prefrontal cortex - CPFm) of WKY rats treated with GAL(1-15)+FLX.

[0018] Using Principal Component Analysis (PCA), the relationship between the FST test results (immobility, swimming) and the Kd / Bmax data obtained from autoradiography with the 5-HT1AR agonist radioligand was evaluated, [ 3 H]-8-OHDPAT.

[0019] The mRNA expression levels of glucocorticoid (GR) and mineralocorticoid (MR) receptors, 5-HT1AR, GALR1 and GALR2, brain-derived neurotrophic factor (BDNF), and tropomyosin kinase receptor B (TrkB) were measured in WKY rats treated with GAL(1-15) + FLX in different brain regions of interest. The data obtained reveal that GAL(1-15) potentiates the antidepressant effects of FLX in WKY animals, offering an effective treatment for TRD. The results indicate a potent effect of the GAL(1-15) / SSRI combination in reversing resistance in the depressive WKY strain to conventional antidepressants. These results support the use of GAL(1-15) in combination with SSRIs as a novel strategy for the treatment of TRD.

[0020] Therefore, a first aspect of the invention describes the compound GAL(1-15), or a pharmaceutically acceptable salt thereof, in combination with at least one serotonin reuptake inhibitor for use in the treatment of treatment-resistant depression or treatment-refractory depression (TRD).

[0021] In this document, GAL(1-15) is defined as the compound with the general formula (SEQ ID NO: 1): Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-Ala or GWTLNSAGYLLGPHA.

[0022] For therapeutic use, GAL(1-15) or its salts may be in a substantially pure or pharmaceutically acceptable form, i.e., with a pharmaceutically acceptable purity level and excluding usual pharmaceutical additives such as diluents and vehicles, and without including materials considered toxic at usual dosage levels. The purity levels of the active ingredient are preferably greater than 50%, more preferably greater than 70%, and even more preferably greater than 90%. In a preferred embodiment, the levels are greater than 95% in GAL(1-15) or its salts.

[0023] The term "selective serotonin reuptake inhibitor" (SSRI) means a monoamine transporter inhibitor that has a stronger inhibitory effect on the serotonin transporter than on the dopamine and norepinephrine transporters. Many antidepressants with serotonin reuptake inhibitory effects have been described in the literature. The present invention describes the combination of GAL(1-15) or its salts with at least one serotonin reuptake inhibitor, and there may be one, two, three, or more serotonin reuptake inhibitors involved. Any pharmacologically active compound that primarily or partially exerts its therapeutic effect through the inhibition of serotonin reuptake in the central nervous system (CNS) may benefit from the combined effect with GAL(1-15).Serotonin reuptake inhibitors that may benefit from potentiation with GAL (1-15) would be: FLX, R-FLX, citalopram, escitalopram, sertraline, paroxetine, fluvoxamine, venlafaxine, duloxetine, dapoxetine, nefazodone, imipramine, imipramine N-oxide, desipramine, pirandamine, dazepinil, nefopam, befuralin, fezolamine, femoxetine, clomipramine, cyanomipramine, lithoxetine, cericlamine, seproxetine, WY 27587, WY 27866, imeldine, ifoxetine, tiflucarbine, viqualine, milnacipran, bazinaprine, YM 922, S 33005, F 98214-TA, OPC 14523, alaproclate, cyanodothepine, trimipramine, quinupramine, dotiepine, amoxapine, nitroxazepine, McN 5652, McN 5707, 01 77, Org 6582, Org 6997, Org 6906, amitriptyline, amitriptyline N-oxide, nortriptyline, CL 255.663, pirlindole, indatraline, LY 113.821, LY 214.281, CGP 6085 A, RU 25.591, napamezole, diclofensine, trazodone, EMD 68.843, BMY 42.569, NS 2389, serchloremine, nitroquipazine, ademethionine, sibutramine and clovoxamine.The compounds mentioned above can be used in their basic form or as a pharmaceutically acceptable acidic addition salt thereof. The above list of serotonin reuptake inhibitors and other compounds that cause an increase in extracellular serotonin levels should not be interpreted as limiting.

[0024] The particularly preferred serotonin reuptake inhibitor according to the present invention is fluoxetine (FLX), or a pharmaceutically acceptable salt thereof. In this specification, fluoxetine or FLX is defined as the compound with the IUPAC name (RS)-N-Methyl-3-phenyl-3-(4-trifluoromethylphenoxy)propylamine, CAS number 54910-89-3, and the following general formula:

[0025] Formula I

[0026] Preferably, FLX is found in the form of fluoxetine hydrochloride.

[0027] GAL(1-15), or a pharmaceutically acceptable salt thereof, as well as serotonin reuptake inhibitors, especially FLX, preferably in the form of fluoxetine hydrochloride, are also referred to as the “active compounds” or “active ingredients” of the invention. As used in this invention, the term “active compound” means any component that potentially provides a pharmacological or other effect in the diagnosis, cure, palliation, treatment, or prevention of a disease, or that affects the structure or function of the human body or the bodies of other animals. The term includes those components that promote a chemical change during the manufacture of the drug and are present in it in a modified form intended to provide a specific activity or effect.

[0028] "Combined use" refers to the administration of the active ingredients in association with each other but not necessarily simultaneously. GAL (1-15) or its pharmaceutically acceptable salts and the serotonin reuptake inhibitor, specifically FLX, may be administered simultaneously as described above. Alternatively, the active ingredients may be administered sequentially, for example, in two separate unit-dose forms. GAL (1-15) or its pharmaceutically acceptable salts may be administered before, during, or after the administration of the serotonin reuptake inhibitor, preferably after the administration of the serotonin reuptake inhibitor, provided that the time between the administration of GAL (1-15) or its pharmaceutically acceptable salts and the administration of the serotonin reuptake inhibitor is such that the ingredients are permitted to act synergistically in the CNS.

[0029] The term "treatment", as used herein, refers to the ability of the molecules of the invention in their combined use or of the pharmaceutical composition of the invention, to prevent, reduce or completely eliminate the effects on patient behavior caused by or resulting from the DRT.

[0030] As previously defined, treatment-resistant depression, or TRD, is that in which the patient does not respond to two or more drugs or psychotherapeutic treatments.

[0031] A second aspect of the invention relates to a composition comprising GAL(1-15) or a pharmaceutically acceptable salt thereof, in combination with at least one serotonin reuptake inhibitor for use in the treatment of DRT.

[0032] Preferably, the serotonin reuptake inhibitor is fluoxetine (FLX), or a pharmaceutically acceptable salt thereof, and more preferably, is in the form of fluoxetine hydrochloride. In another preferred embodiment of this aspect of the invention, the composition comprises two, three, or more serotonin reuptake inhibitors simultaneously. Preferably, at least one of them is FLX.

[0033] In a preferred embodiment of this aspect of the invention, the composition of the invention is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to any substance used for the diagnosis, prevention, relief, treatment, or cure of a disease in humans or animals. The pharmaceutical composition may comprise a single composition or separate compositions. The pharmaceutical composition of the invention may be used alone or in combination with other pharmaceutical compositions, preferably comprising another compound useful in the treatment of TRD, in which case it may also comprise a single composition or separate compositions. In one particular embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier or excipient.

[0034] The term "pharmaceutically acceptable excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the composition of the invention, stabilizes said pharmaceutical composition, or assists in its manufacture by providing consistency, shape, flavor, or any other specific functional characteristic. Thus, excipients may have the function of binding the ingredients together, such as starches, sugars, or cellulose; a sweetening function; a coloring function; a protective function, such as isolating it from air and / or moisture; a filler function for a tablet, capsule, or any other form of formulation, such as dibasic calcium phosphate; or a disintegrating function to facilitate the dissolution of the components and their absorption, without excluding other types of excipients not mentioned in this paragraph.

[0035] A "pharmaceutically acceptable carrier" (or "pharmacologically acceptable") refers to any substance, or combination of substances, known in the pharmaceutical industry, used in the manufacture of dosage forms and includes, among others, solids, liquids, solvents, or surfactants. The carrier may be an inert substance or have an action similar to any of the compounds of the present invention, serving to facilitate the incorporation of the drug, as well as other compounds, allowing for improved dosage and administration, or providing consistency and form to the pharmaceutical composition. When the dosage form is liquid, the carrier is the diluent. The term "pharmacologically acceptable" refers to the fact that the compound in question is permitted and evaluated to be harmless to the organisms to which it is administered.

[0036] The pharmaceutical composition of the invention can be administered via any route of administration, and as such, said composition shall be formulated in the pharmaceutical form appropriate to the chosen route of administration. Thus, the pharmaceutical composition of the invention can be administered orally, nasally, ocularly, topically, intradermally, intracranially, intravenously, or intraperitoneally. Preferably, it is administered orally.

[0037] The composition of the invention may include an effective amount of GAL(1-15) and FLX. The term "effective amount" used herein refers to an amount sufficient to effectively treat the DRT. The effective amount may be appropriately selected by a person skilled in the art based on the individual being treated, and factors such as age, health conditions, duration of administration, route of administration, excretion rate, and others may be involved, including the use of a drug in combination with or concurrently with the pharmaceutical composition, and other factors known in the field of medicine. Therapy is considered "personalized" when the compound administered to an individual to treat a disease is specifically tailored to the genotypic and phenotypic characteristics of the individual being treated, thereby avoiding the waste of time with ineffective therapies.

[0038] A final aspect of the invention relates to a kit comprising GAL(1-15) or a pharmaceutically acceptable salt thereof, in combination with at least one serotonin reuptake inhibitor, preferably FLX, or the compositions as described herein, for use in the treatment of treatment-resistant depression. Preferably, the kit is adapted for simultaneous or sequential administration of the active compounds.

[0039] The term "patient," "individual," or "subject," as used herein, refers to a mammal and includes, but is not limited to, domestic and farm animals, primates, and humans, e.g., humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as rats and mice. In a preferred embodiment, the subject is a human being.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which this invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice of the present invention. Throughout the description and claims, the word "comprises" and its variations are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will be evident to those skilled in the art upon examination of the description or may be discovered through the practice of the invention. The following examples and drawings are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0041] DESCRIPTION OF THE FIGURES

[0042] Figure 1: Schematic of the experimental program. After brain cannulation, all animals underwent a recovery period before the behavioral tests. A) The effects of different pharmacological treatments on the forced swim test (FST) were evaluated in different groups of WKY rats. One week after the behavioral tests, autoradiography experiments were performed. 5-HT1AR knockdown WKY animals were also used to evaluate the pharmacological treatments in the FST. B) In other groups of animals, the effects of different pharmacological treatments on the tail suspension test (TST) were evaluated. One hour after the TST, blood and brain samples were collected from the animals for qPCR-rt experiments to determine blood corticosterone levels.

[0043] Figure 2: Behavioral effects of co-administration of FLX (10 mg / kg) alone or in combination with GAL (1-15)(1 nmol) or GAL (1-15)+M871 (3 nmol) on the FST test. A) immobility time and B) swimming time in the FST. FLX was administered 23, 5, and 1 h before testing, and GAL (1-15) or GAL (1-15)+M871 was injected 15 min before testing. Vertical bars represent mean ± SD (n=11-6). *p<0.05 vs. the other groups, #p<0.05 vs. the Control and FLX (10 mg / kg)+GAL (1-15)(1 nmol)+M871 (3 nmol) groups. p<0.05 versus the FLX(10mg / kg) and FLX(10mg / kg)+GAL(1-15)(1nmol) groups. C) Behavioral effects of co-administration of FLX(10mg / kg) alone or in combination with GAL(1-15)(1nmol) on immobility time in the TST. & p<0.01 versus the FLX(10mg / kg) group according to one-way ANOVA followed by Fisher's minimum significant difference test.

[0044] Figure 3: A) 5-HT1AR immunoreactivity in the dorsal raphe of WKY rats injected ICV14 days later with a single dose of SI5HT1A (red) or vehicle-derived immunosorbent. Vertical bars represent mean ± SD. *** p < 0.05 vs. the WKY group according to Student's t-test. B) Representative photomicrographs of 5-HT1AR immunofluorescence in tissue sections of the dorsal raphe. Scale bar 100 µm.

[0045] Figure 4: (A) Principal component analysis of the FST and Kd parameters of the autoradiographic experiment. (B) Subjects' factor score on component 1: 1-way ANOVA, F2 5 = 6.532, P = 0.0091. (C) Subjects' factor score on component 2: 1-way ANOVA, F2 5 = 11.85, P = 0.0008. (D) Subjects' factor score on component 3: 1-way ANOVA, F2 5 = 3.411, P = 0.061. Data represent the mean factor score of subjects on each component ± SD in the Control, FLX(10mg / Kg) and FLX(10mg / Kg)+GAL(1-15)(1nmol) groups (n=6). *p<0.05 compared to all other groups according to one-way ANOVA followed by Fisher's least significant difference test.

[0046] Figure 5: Overall dimensions derived from FST parameters and Bmax in the autoradiographic experiment. (A) PCA revealed two independent components indicative of Bmax values ​​in the PFC, dorsal raphe (DR), ventral hippocampus (vHip), and dentate gyrus (DG) and CA1 of the dorsal hippocampus (C4, variance explained -44.4%) and FST performance (C5, variance explained -25.6%). (B, C) Data represent the mean factor score of subjects on each component ± SD in the Control, FLX(10 mg / kg), and FLX(10 mg / kg)+GAL(1-15)(1 nmol) groups (n=6). ***p<0.001 vs. the control group, #p<0.05 vs. the FLX(10 mg / kg) group according to one-way ANOVA followed by Fisher's least significant difference test.

[0047] Figure 6: Plasma corticosterone levels (ng / ml) after administration of FLX (10 mg / kg) alone or in combination with GAL (1-15) (1 nmol) in WKY rats. FLX was administered 23, 5, and 1 h before the TST, and aCSF or GAL (1-15) ICV was injected 15 min before. Blood was drawn 1 h after the test. Vertical bars represent the mean ± SD (n = 7-6 rats per group). *p < 0.05 compared to the other groups according to one-way ANOVA followed by Fisher's least significant difference test.

[0048] DETAILED DESCRIPTION OF THE INVENTION

[0049] Animals

[0050] Male Wistar Kyoto (WKY) and Wistar (CRIFFA, Barcelona, ​​Spain) rats were housed individually under standard laboratory conditions (12-hour dark / light cycle, 55%-60% humidity, and 22±2°C temperature) with free access to food and water. All experimental procedures were approved by the Institutional Animal Ethics Committee of the University of Málaga, Spain.

[0051] Stereotactic surgery

[0052] The animals were implanted with a fixed cannula using stereotactic surgery prior to the experimental procedures. Following the previously described protocol [6], the rats were anesthetized with 5% isoflurane (ISOFLO® ZOETIS ABOTT Liquid Gas Anesthetic, 100% Isoflurane) in an induction chamber and maintained with a 1.5–2.5% isoflurane mask placed on a stereotactic instrument. In all animals, a stainless steel guide cannula was implanted in the right lateral cerebral ventricle (+1.4 mm lateral, -1 mm posterior to the bregma, and 3.6 mm below the skull surface). After surgery, the animals were housed individually and had a 7-day recovery period, after which they were randomly assigned to different experimental groups for the behavioral tests. The sample size was estimated based on previous studies [1, 4].

[0053] For 5-HT1AR knockdown in WKY rats immediately after guide cannula implantation in stereotactic surgery, rats received an intracerebroventricular (ICV) injection of 5 pg (0.35 nmol) of either Accell Smart pool siRNA 5HT1A (Dharmacon) or five pl of vehicle (Acell siRNA Delivery Media). In this experiment, animals had a recovery period of 14 days after cerebral cannulation before the behavioral test, the time required to reduce 5HT1A receptor levels, as previously described [7].

[0054] Administration of GAL(1-15) and FLX

[0055] Intracytoplasmic reticulum (ICV) administration of GAL(1-15)(1 nmol) alone or in combination with the GALR2 antagonist M871 (3 nmol) (TOCRIS, Bristol, UK) was administered 15 minutes before the start of behavioral testing. Both preparations used artificial cerebrospinal fluid (aCSF). ICV injection was performed as previously described [6] using the fixed injection cannula connected to a Hamilton syringe via PE-10 tubing; 5 pl were injected over 1 minute with care, ensuring a smooth flow without overflow. All rats receiving ICV injections were assumed to have proper cannula placement, and no rats were excluded.

[0056] Fluoxetine hydrochloride (FLX) (Sigma-Aldrich, St. Louis, USA) or vehicle was injected subcutaneously (SC) three times at a dose of 10 mg / kg. The FLX solution was prepared using distilled water to improve solubility in a volume of 2 mL / kg. The injections were administered 23, 5, and 1 hour before the start of the behavioral tests. This injection regimen produced clinical effects similar to chronic treatment in humans [8].

[0057] Two sets of experiments were performed, summarized in Figure 1. Experiment 1 (Fig. 1 A)

[0058] In WKY animals, the effects of administering FLX (10 mg / kg) alone or in combination with GAL(1-15) (1 nmol) were evaluated in the FST test. FLX or vehicle was injected in a subchronic administration schedule 23, 5 and 1 hour subcutaneously (SC) before the start of the tests and GAL(1-15) 15 min before by intracerebroventricular (ICV) injection based on previous work [4, 9].

[0059] Forced swim test (FST): Animals were placed individually in a vertical glass cylinder (50 cm high, 20 cm in diameter) containing water (25°C) to a height of 30 cm. Immobility was defined as passively floating upright in the water with only minor movements necessary to keep the head above the water's surface. All tests were performed between 13:00 and 16:00 h during the light cycle [7]. The total duration of immobility, climbing, and swimming behavior was recorded during the second 5 min. Drug administration was performed between two sessions: a 15-min pre-test followed 24 h later by a 5-min test.

[0060] Tail suspension test (TST): The rat was hung upside down using adhesive tape to fix its tail to a string through an eyelet at 60 cm. The animal was considered immobile when it made no movements or struggles, attempts to grasp the adhesive tape, body twists or jerks [7]. The total duration of immobility was recorded during the 6 min test.

[0061] Quantitative Autoradiography: One week after the behavioral assessment, rats were administered FLX (10mg / kg) or GAL(1-15) (1 nmol) alone or in combination, and their brains were collected 30min later for the autoradiographic experiment, as previously described [9].

[0062] The rats were euthanized by decapitation, and the brains were rapidly extracted and frozen in isopentane at -40°C. Coronal sections (14 pm thick) were cut in a chonostat at bregma levels (dorsal hippocampus: 3.14 mm - 3.8 mm), thawed and mounted on gelatin-coated slides and stored at -20°C until further processing.

[0063] The saturation experiments were performed using [ 3 H]-8-OH-DPAT (specific activity: 142 Ci / mmol, Perkin Elmer) at concentrations ranging from 0.24 to 10.8 nM, as previously described [3]. In each saturation experiment, two groups of ten sections were prepared for total and nonspecific binding. Nonspecific binding was defined as the binding of [ 3H]-8-OH-DPAT in the presence of 10-5M 5-HT (Sigma Aldrich, St. Louis, CA). Brain sections were pre-incubated for 30 minutes at room temperature in 50mM Tris-HCl buffer (pH 7.6), containing 4mM CaCh, 0.01% ascorbic acid, and 10mM pargyline. The sections were then incubated for 60 minutes at room temperature with [ 3 H]-8-OH-DPAT in the same solution as before. The sections were then washed twice for 5 minutes in ice-cold buffer and rinsed briefly in ice-cold distilled water before being dried under a stream of cold air. The dried sections were exposed to [ 3 H]- Kodak Biomax-MR (Sigma Aldrich) for 6 weeks along with radiolabeled plastic standard strips with [ 3 H]- (Amersham, United Kingdom).

[0064] On the other hand, the involvement of GALR2 and 5HT1AR in the behavioral effects of the GAL(1-15)+FLX combination in the FST in WKY rats was also studied.

[0065] To analyze the role of GALR2, M871 was used, in combination with GAL(1-15) via ICV to block the effect of GAL(1-15) on FLX-mediated action, as previously described [3, 4, 9].

[0066] To analyze the role of 5HT1AR, 5HT1AR knockdown WKY rats were generated as previously described [4, 7, 10]. In these knockdown animals, the effects of FLX (10 mg / kg) and GAL(1-15)(1 nmol) alone or in combination were also tested.

[0067] 5-HT1A Immunofluorescence: Ninety minutes after drug administration, rats were anesthetized with sodium pentobarbital (Mebumal; 100 mg / kg body weight, i.p.) and intracardially perfused with 200 ml of ice-cold phosphate-buffered isotonic saline (PBS) followed by 200 ml of fixative containing 4% (w / v) paraformaldehyde in 0.1 M PBS buffer (pH 7.4). Brains were removed, post-fixed in the same fixative for 12 hours, and cryoprotected in 30% sucrose at 4°C. Finally, 30 µm dorsal raphe sections were obtained in a cryostat (-20°C). The procedures were carried out as previously described [7]. Primary antibodies directed to the polyclonal anti-5HT1AR antibody (rabbit, AB15350, Sigma-Aldrich, 1 / 500) were incubated for 12h at 4°C and detected with the secondary anti-rabbit goat red antibody DyLight 649 (Jackson immunoResearch Laboratories, 1 / 100).The sections were mounted on slides with fluorescent mounting medium (Dako) and visualized using a Leica SP8 confocal microscope. Immunofluorescence was performed on WKY dorsal raphe sections with 5-HT1A siRNA (knockdown) and untreated rats without 5-HT1A siRNA (control). Experiment 2 (Fig. 1B).

[0068] In the second experimental group, the behavioral effects of the GAL(1-15)+FLX combination were analyzed in the TST test. FLX and GAL(1-15) were injected using the same administration pattern and dosage described in Experiment 1. One hour after the test, the animals were euthanized. Blood samples were collected from the trunk and brains for subsequent analysis of circulating corticosterone levels, and the brains were processed for RT-qPCR analysis.

[0069] Corticosterone assay: Blood samples obtained from animals euthanized by decapitation one hour after TST were rapidly collected in EDTA-containing tubes, cooled on ice, and centrifuged in a refrigerated centrifuge (4°C, 3000 rpm, 15 minutes). Plasma was then isolated and stored at -80°C until further analysis. Plasma corticosterone levels were measured using the Corticosterone ELISA kit (ArborAssay, Ann Arbor, MI) according to the manufacturer's instructions.

[0070] RT-qPCR: Rats were euthanized by decapitation one hour after the TST test. Brains were rapidly removed from the skull and frozen in isopentane at -40°C. Nuclei were dissected as briefly described below. Brains were sliced ​​over the brain matrix (1 mm) and maintained at -20°C until each region of interest entered the plane of the cut. Tissues of interest were dissected using a punch with an internal diameter of 2 mm and collected in Eppendorf tubes. Total RNA was isolated from the hypothalamus, dorsal hippocampus, and prefrontal cortex of Wistar and WKY rats using the RNeasy Lipid Tissue Kit (Qiagen, Hilden, Germany). The purity of the isolated RNA was assessed by measuring absorbance ratios at 260 / 230 nm and 260 / 280 nm using a nanodrop (Thermo Scientific, Waltham, USA).To remove residual DNA, the samples were treated with a DNase kit (Takara Inc., Shiga, Japan), and cDNA was obtained using a Reverse Transcriptase Core kit (Eurogentec, Seraing, Belgium). These three steps were performed according to the manufacturer's instructions.

[0071] All analyses were performed in triplicate using LightCycler® Multiplex Masters on a LightCycler 96 PCR machine (Roche®), and relative gene expression was determined using the double delta Ct method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA measurements. The primers used in this study were purchased pre-designed from Sygma-Aldrich® and had been previously used. GAPDH was used as a reference. The efficiency of the primers used was verified in previous experiments [3] and all were found to be in the range of 1.9 to 2.1.

[0072] Statistical analysis

[0073] Data are presented as mean ± SD, and the number of samples (n) is indicated in the figure legends. All data were analyzed using GraphPad PRISM 8.0 (GraphPad Software, Inc., San Diego, CA, USA). Two-tailed, unpaired Student's t-tests were performed to compare two experimental conditions. One-way ANOVAs were performed to compare more than two groups. Fisher's least significant difference (LSD) test was performed after comparison when the F-ratio in the one-way ANOVA was statistically significant. Differences were considered statistically significant at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001).

[0074] In the saturation experiments, the values ​​of the dissociation constant (Kd) and the maximum number of binding sites (Bmax) were determined by fitting the data to the equation Y = Bmax*X / (X+Kd) using nonlinear regression with the Prism program, version 8 (GraphPad Software).

[0075] A principal component analysis (PCA) with varimax rotation was also performed to extract independent dimensions (i.e., components) from the FST and autoradiographic data. An eigenvalue > 1 was chosen as the criterion for component extraction, and a PC score (i.e., a standardized value indicating the relative position of each animal in each component) was calculated using regression (SPSS Statistics 20, IBM Corporation, Armonk, NY, USA). Loadings greater than 0.6 (in absolute value) were chosen as the criterion for including variables in a given component.

[0076] Effects on behavior of co-administration of GAL(1-15) and FLX

[0077] Co-administration of GAL(1-15) and FLX induces an antidepressant effect in WKY rats in behavioral tests.

[0078] Before examining the behavioral effects of the FLX+GAL(1-15) combination in WKY animals, the depressive behavioral profile of these animals was established for the tests used and compared with Wistar rats, used as a control.

[0079] In the FST, WKY animals showed a statistically significant increase in immobility time (tis = 3.004, p = 0.0076) and a decrease in climbing time (tis = 4.927, p < 0.001) compared to Wistar animals (Table 1). In the TST, WKY animals showed a longer immobility time (tw = 2.864, p = 0.0168) compared to Wistar rats (Table 1). The FST results were consistent with the TST results, a widely used assessment of desperation behavior in rodents.

[0080] Table 1: Behavioral comparison between Wistar and WKY strain rats in the parameters of immobility, swimming and climbing time in the FST and immobility time in the TST.

[0081] Data are represented as mean ± SD (n=10-6). * p< 0.05, **p<0.01 , ***p<0.001 versus Wistar-saline group according to Student's t-test.

[0082] In both the FST and TST, WKY animals showed increased immobility time and decreased climbing compared to Wistar strain animals. These findings reinforce the WKY strain's predisposition toward desperation behavior.

[0083] Subsequently, the effect of co-administration of GAL(1-15) and FLX was analyzed in WKY rats.

[0084] In the FST, only the WKY animal group administered GAL(1-15)-FLX showed an antidepressant effect, with a statistically significant decrease in immobility time (FS,33 = 5,800, P = 0.0027; Fisher's post hoc LSD: P < 0.05) (Figure 2A) and an increase in swimming time (FS,33 = 5,159, P = 0.0049; Fisher's post hoc LSD: P < 0.05) (Figure 2B) compared to the WKY control group. The FLX dose and administration schedule used in this study did not show an antidepressant effect in the FST. Furthermore, the FLX doses and administration schedule used in this study did not induce antidepressant-like effects in any of the tests, highlighting the resistance of the WKY strain to conventional antidepressants.

[0085] The impact of the GAL(1-15)-FLX combination on the behavior of WKY animals was corroborated in the TST, where co-administration of GAL(1-15) with FLX led to a reduction in immobility time compared to FLX alone (F2,2o =4,475, P=0.0248; Fisher's LSD post hoc: P<0.01 ) (Figure 2C).

[0086] Therefore, in WKY rats, only the GAL(1-15)+FLX combination effectively relieves behavioral despair.

[0087] Role of GALR2 and 5HT1AR in the antidepressant effects induced by the GAL(1-15)-FLX combination

[0088] The role of GALR2 in the antidepressant effects induced by the GAL(1-15)-FLX combination was tested using the antagonist M871. M871 (3nmol) blocked the reduction in immobility time (Fisher's LSD post hoc: P<0.05) (Figure 2A) and the increase in swimming time (Fisher's LSD post hoc: P<0.05) (Figure 2B) found after co-administration of GAL(1-15) and FLX in the FST.

[0089] GAL(1-15)(1 nmol) and M871 (3nmol) administered alone had no effect on FST in WKY animals (Table 2).

[0090] Table 2: Effects of GAL(1-15)(1 nmol) or M871 (3 nmol) administered alone 15 min before FST in WKY rats. No statistically significant differences were observed in immobility or swimming time.

[0091] The results suggest the involvement of GALR2 in the effects mediated by GAL(1-15), since the GALR2 antagonist M871 blocked the potentiation by GAL(1-15) of the antidepressant effects of FLX in WKY rats.

[0092] The role of 5HT1AR in this interaction was also studied using WKY knockdown animals with 5HT1AR siRNA.

[0093] WKY rats injected with a single dose of 5HT1A siRNA showed reduced 5-HT1AR immunoreactivity in the RD (tu = 4.665, p < 0.001) 14 days post-injection (Figure 3), as previously described in both naive and OBX animals [4]. The reduction of 5HT1AR siRNA in WKY rats validates the involvement of 5-HT1AR in GAL(1-15)-induced effects. The decrease in 5-HT1AR was sufficient to block GAL(1-15) potentiation of FLX-mediated antidepressant effects in the FST (Table 3). Thus, co-administration of GAL(1-15) + FLX had no effect on immobility and swimming time in the FST.

[0094] Table 3: Behavioral effects of co-administration of FLX(10mg / kg) alone or in combination with GAL(1-15)(1nmol) in WKY Knock-down 5HT1A rats in FST.

[0095] No statistically significant differences were observed in immobility or swimming time in this animal model.

[0096] Therefore, the fundamental role of 5-HT1AR in the effects induced by GAL(1-15) was also confirmed at the behavioral and neurochemical levels. Thus, reducing 5-HT1AR levels by knocking down 5-HT1AR siRNA in WKY rats blocked the potentiation by GAL(1-15) of FLX-mediated antidepressant effects, consistent with previous findings.

[0097] Autoradiography with [ 3 HJ-8-OHDPAT, 5-HT1AR agonist radioligand

[0098] In this study, the neurochemical effects on the binding characteristics of [ 3 H]-8-OH-DPAT in the prefrontal cortex (mPFC), RD, GD, and CA1 of the dorsal and ventral hippocampus of WKY rats treated with the GAL(1-15)+FLX combination. FLX was administered 23, 5, and 1.25 hours before rat euthanasia. aCSF or GAL(1-15) was injected intravenously 30 minutes before rat euthanasia. Saturation experiments were performed with 10 concentrations of [ 3H]-8-OHDPAT (0.26-10 nM) was applied to different sections of each brain region. Non-specific binding was defined as binding in the presence of 10 mM of serotonin. Table 4 describes the effects of the different treatments on the Kd or Bmax parameters in all areas analyzed.

[0099] Table 4: Effects of FLX(10mg / kg) administration alone or in combination with GAL(1-15)(1nmol) on the binding characteristics of the 5-HT1AR agonist [ 3 H]-8-OHDPAT in the medial prefrontal cortex (PFC), dorsal raphe (RD), CA1 and dentate gyrus (GD) of the dorsal hippocampus and ventral hippocampus (v-Hyp) of WKY rats.

[0100] Data are represented as mean ± SD (n=6) of Kd and Bmax values. *p<0.05, **p<0.01 ***p<0.001 compared to control groups, # p<0.001 compared to control group and FLX(10 mg)+GAL(1-15)(1 nmol), & p<0.001 compared to control group and FLX(10 mg) according to one-way ANOVA followed by Fisher's least significant difference test.

[0101] In the CPF, a decrease in Kd value was observed as a result of administration of FLX or GAL(1-15)+FLX (F2,IS = 5.736, P < 0.0141). Furthermore, an increase in Bmax was observed in this region (F2,IS = 95.49, P < 0.001). However, the group treated with GAL(1-15)+FLX showed a smaller increase compared to the WKY animals treated with FLX (Fisher's LSD post hoc: P < 0.01).

[0102] In RD, an increase in Bmax induced by the administration of FLX or GAL(1-15)+FLX was observed (F2,15 = 9.045, P = 0.0026; Fisher's post hoc LSD: P < 0.01) without any changes observed in the Kd value.

[0103] Conversely, in the CA1 region of the dorsal hippocampus, both FLX and the combination of GAL(1-15)+FLX resulted in a decrease in Bmax (F2,IS = 7.409, P = 0.0058), with no observed changes in the Kd value. In the GD, only the combination of GAL(1-15)+FLX produced a decrease in Bmax (F2,IS = 4.725, P = 0.0256). Regarding the Kd value, the reduction was induced only by the FLX group (F2,IS = 59.68, P < 0.001).

[0104] In the ventral hippocampus (v-Hyp), both FLX and the combination of GAL(1-15)+FLX caused a reduction in Bmax (F2,15 =7,741 , P=0.0049), along with a significant decrease in Kd (F2,IS =115,3, P<0.001 ).

[0105] Previous autoradiography experiments in naive and OBX animals revealed that the combination of GAL(1-15) and FLX modified the Kd and Bmax of the 5-HT1AR in the CPFm, a critical region involved in the interaction between emotional processing and cognition, and in the CA1 and GD regions of the dorsal hippocampus. The data obtained here also show modifications in Bmax and Kd in all the regions analyzed. However, in the CPFm and the GD region of the dorsal hippocampus, the administration of GAL(1-15)+FLX induces differences compared to other experimental groups.

[0106] Therefore, regarding the impact of treatment with GAL(1-15)+FLX on the binding characteristics of [ 3 H]-8-OH-DPAT in the CPFm, RD, GD, CA1 of the dorsal and ventral hippocampus of WKY rats, the role of 5-HT1AR in GAL-mediated effects was validated(1-15).

[0107] Principal component analysis (PCA)

[0108] Two PCAs were performed to analyze the interrelation between FST data (immobility and swimming time) and the autoradiographic parameters Kd / Bmax.

[0109] The first PCA included FST measurements and Kd autoradiographic parameters as variables. Sample adequacy tests revealed that the data were adequate for the PCA (Kaiser-Meyer-OIkin, KMO=0.584; Bartlett's sphericity test: X 2 (21 )=43,165, P=0.003). Three independent components were extracted that explained 78.8% of the total variance (Table 5).

[0110] Table 5: Principal component analysis of the FST and Kd parameters of the autoradiographic experiment.

[0111] Component 1 (variance explained -35.6%) was representative of FST performance and PFC Kd values, with low PC scores indicating less immobility and more swimming during the FST task and lower PFC Kd values. Component 2 (variance explained -28.5%) included the RD, ventral hippocampus, and dorsal hippocampal GD Kd, with low PC scores indicating high Kd levels in hippocampal regions and low Kd values ​​in the RD. Component 3 (variance explained -14.7%) included the CA1 Kd (Figure 4A).

[0112] To determine whether the experimental groups differed in the PC scores extracted in the analysis, a one-way ANOVA was performed on each of the components. Regarding Component 1, a significant reduction in PC scores was observed in the group of animals treated with GAL(1-15)+FLX (F2,IS =6.532, P=0.0091) (Figure 4B), indicating that the animals in this group showed less desperation behavior along with lower Kd values ​​in PFC.

[0113] In Component 2, a reduction in PC score was found in animals treated with FLX (F2 5 =11 ,85, P=0.008), indicating that rats in this group showed higher Kd values ​​in hippocampal regions along with lower Kd values ​​in RD (Figure 4C).

[0114] In Component 3, no significant differences were observed between groups in PC scores between groups (Figure 4D).

[0115] A second PCA was performed, including FST measurements and Bmax autoradiographic parameters.

[0116] Table 6: Principal component analysis of the FST and Bmax parameters of the autoradiographic experiment.

[0117] Once again, the sample adequacy tests revealed that the data were adequate for PCA (KMO=0.544; Bartlett's test of sphericity: X 2(21)=60,154, P<0.001). The analysis revealed two independent components that explained 70% of the total variance (Figure 5A). Component 1 (variance explained -44.4%) was representative of Bmax values ​​in all brain areas analyzed, with high PC scores indicating high Bmax levels in hippocampal regions and low Bmax values ​​in the RD and PFC. Component 2 (variance explained -25.6%) was representative of FST performance, with low PC scores indicating less immobility and more swimming during the task.

[0118] One-way ANOVA analysis of the PC scores obtained in the second PCA revealed that, in Component 1, the groups treated with FLX and GAL(1-15)+FLX showed significantly lower PC scores than the control group (F2,IS =42.97, P<0.001) (Figure 5B), while in Component 2, the animals treated with GAL(1-15)+FLX showed significantly lower PC scores compared to the rest of the groups (F2.15 =5.034, P=0.0212) (Figure 5C).

[0119] The results of the PCA analysis indicate that only the decrease in Kd in the CPF coincides with the effects induced by the GAL(1-15)-FLX combination in the FST, which involve a reduction in immobility and an increase in swimming time. Furthermore, FLX administration caused alterations in Kd in the RD, the ventral hippocampus (GD), and modifications in the Bmax parameter in all regions examined.

[0120] Therefore, the prefrontal cortex (PFC) was the only region associated with FST performance. Lower Kd values ​​in the CPFm, indicated by low PC scores, corresponded to less immobility and greater swimming during the FST task. A significant decrease in PC scores was detected in the group of animals treated with GAL(1-15)+FLX, suggesting that this group exhibited reduced desperation behavior and lower Kd values ​​in the CPFm.

[0121] Hormonal effects

[0122] Corticosterone levels were examined in WKY animals one hour after the TST. Abnormally elevated corticosterone levels were detected in control WKY rats tested one hour after the TST. FLX administration did not significantly affect corticosterone levels in WKY rats under ordinary or stress conditions. Only co-administration of GAL(1-15) and FLX induced a statistically significant reduction in corticosterone levels (F2,16 = 4.141, P = 0.0355; Fisher's LSD post hoc: P < 0.001), approximately 30% compared to the control group and the FLX-treated WKY group (Figure 6).

[0123] This suggests possible underlying mechanisms involving regulation of the HPA axis.

[0124] Effects on GALR1 and GALR2 expression levels of glucocorticoids (GR) and mineralocorticoids (MR) BDNF, TrkB, 5HT1A

[0125] Studies BY

[0126] The impact on mRNA expression of proteins associated with the GAL(1-15)-FLX interaction mechanism in the hypothalamus, ventral and dorsal hippocampus, and prefrontal cortex (PFC) was described. Table 7 summarizes the mRNA expression levels of WKY rats treated with FLX or GAL(1-15)+FLX. The analysis includes not only a group of saline WKY rats but also a group of Wistar animals in all brain regions of interest.

[0127] Table 7: Effect of FLX (10 mg / kg) administration alone or in combination with GAL(1-15) (1 nmol) on the relative mRNA expression of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), GalR1, GalR2, 5-ht1a, brain-derived neurotrophic factor (BDNF), tropomyosin kinase B receptor (TrkB), p11, and Horner's (both proteins that make up metabotropic glutamate receptor 5 or mGluR5) in the hypothalamus (HPT), ventral (vHyp) and dorsal (dHyp) hippocampus, and prefrontal cortex (PFC) of WKY rats compared to untreated Wistar rats. FLX was administered 23, 5, and 1 h before the TST, and aCSF or GAL(1-15) ICV was injected 15 min before. The brains were extracted and properly dissected for PCR experiments 1h after testing.

[0128] . Wi...istar Contro ili WA / iK / vY r FiLXv(n1n0mg / / iKg ») F „LX(1 Omg / Kg)+ ..

[0129] ' a GAL(1-15)(1nmol)

[0130] HPT GR 0,57 ± 0,06 0,8 ±0,063# 0,79 ± 0,06 # 0,7 ± 0,03

[0131] MR 0,84 ±0,16 1,09 ±0,14 0,95 ±0,12 1,07 ±0,12

[0132] GalR1 0,69 ±0,06 0,99 ±0,12* 0,97 ± 0,07 * 0,64 ± 0,08

[0133] GalR2 1 ,01 ± 0,25 0,79 ± 0,07 0,86 ± 0,08 0,82 ± 0,06

[0134] 5ht1a 1 ,01 ± 0,25 0,79 ± 0,07 0,86 ± 0,08 0,82 ± 0,06 vHyp GR 1,24±0,25 0,81 ±0,19 0,85 ± 0,2 1,02±0,37

[0135] MR 0,83 ±0,08 0,69 ±0,14 1,03 ±0,12 0,81 ± 0,08

[0136] GalR1 0,47 ±0,21 1,18 ±0,24 0,65 ±0,17 0,52 ± 0,28

[0137] GalR2 3,53 ±0,52 1,57 ±0,23# 1,93 ±0,44# 0,53 ± 0,32 &

[0138] 5ht1a 1,24±0,25 0,81 ±0,19 0,85 ± 0,2 1,02±0,37

[0139] BDNF 2,32 ±0,5 1,22 ±0,3 1,34 ±0,3 0,99 ±0,16

[0140] P11 0,59±0,11 0,56±0,13 1,24±0,35 1,1 ±0,5 dHyp GR 0,82 ± 0,46 0,94 ± 0,08 0,84 ± 0,35 0,87 ± 0,43

[0141] MR 0,96±0,15 1,22±0,11 1,17±0,2 1,21 ±0,34

[0142] GalR1 1,47 ±0,45 1,67 ±0,9 0,86 ± 0,42 1,8 ±1,4

[0143] GalR2 1,25 ±0,27 1,31 ±0,46 1,46 ±0,7 1,98 ±1

[0144] 5ht1a 1,01 ±0,25 0,78±0,11 0,61 ±0,23 0,58±0,16

[0145] BDNF 1,66 ±0,35 0,72 ±0,1# 0,89 ± 0,31 # 0,39 ± 0,04 #

[0146] TrkB 0,96 ±0,16 1,88 ±0,27 1,77 ±0,44 1,92 ±0,61

[0147] PFC GR 0,83 ±0,11 0,75 ±0,25 0,83 ± 0,3 0,71 ±0,15 GalR1 0,82 ± 0,2 0,64 ± 0,22 0,67 ± 0,23 0,83 ± 0,22

[0148] GalR2 1 ,6 ± 0,28 1 ,23 ± 0,22 1 ,4 ± 0,22 1 ,28 ± 0,31

[0149] 5ht1a 0,8 ± 0,08 0,59 ± 0,11 0,56 ± 0,05 0,61 ± 0,1

[0150] BDNF 1 ,5 ± 0,15 1 ,73 ± 0,39 1 ,35 ± 0,33 1 ,09 ± 0,1

[0151] Hornería 0,69 ± 0,04 0,73 ± 0,06 0,59 ± 0,06 0,71 ± 0,04

[0152] Data are represented as the mean ± SD (n=6-4 rats per group) of relative gene expression and are expressed as arbitrary units. *p<0.05 vs. Wistar and FLX(10mg / Kg)+GAL(1-15)(1 nmol). # p<0.05 vs. Wistar group, & p<0.05 vs. Wistar and FLX(10mg / Kg) groups according to one-way ANOVA followed by Fisher's least significant difference test.

[0153] The expression levels of GALR1 and GALR2 in WKY rats, compared to those in Wistar rats, revealed alterations in expression within two specific regions: the hypothalamus and the ventral hippocampus. In particular, GALR1 showed elevated mRNA expression in the hypothalamus of WKY rats compared to Wistar rats (Fs. = 3.87, P = 0.028). Conversely, in the ventral hippocampus, there was a significant reduction in GALR2 expression in WKY rats compared to Wistar rats (Fs. = 8.763, P = 0.0016).

[0154] Interestingly, elevated GALR1 levels in the WKY hypothalamus returned to baseline values ​​after co-administration of GAL(1-15)+FLX (Fisher's post hoc LSD: P<0.05), whereas FLX alone had no effect. In the ventral hippocampus, co-administration of GAL(1-15)+FLX reduced GALR2 levels compared to FLX alone (Fisher's post hoc LSD: P<0.05). In the other brain areas analyzed, no differences were observed in GALR1 or GALR2 mRNA levels induced by either treatment.

[0155] These results highlight the role of GALR1 and GALR2 in the effects mediated by GAL(1-15).

[0156] All these data reinforce the hypothesis of the existence of a trimeric GALR1-GALR2-5-HT1AR heteroreceptor complex [which could be a key point for understanding the effects of the GAL(1-15)-SSRI interaction in WKY]. In this complex, altered receptor-receptor allosteric interactions can develop with the ability of the GALR1-GALR2 component to enhance 5-HT1AR protomer signaling.

[0157] The analysis also included an examination of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA levels. Specifically, a statistically significant increase in GR levels was observed in the hypothalamus of the WKY control and FLX-treated groups compared to the Wistar strain (Fs. = 4.781, P = 0.0145). This increase was reduced with co-administration of GAL(1-15) + FLX in the WKY animals.

[0158] The results described in this report show high levels of glucocorticoid receptor (GR) mRNA expression in WKY rats compared to Wistar strain rats.

[0159] BDNF and TrkB mRNA expression levels were also assessed. In the dorsal hippocampus, a marked and statistically significant reduction in BDNF levels was observed in all WKY groups compared to Wistar animals, consistent with previous findings (Fs. = 4.654, P = 0.014). This decrease was similarly observed in the ventral hippocampus.

[0160] No alterations in 5HT1A mRNA expression were detected in any of the analyzed regions.

[0161] References

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[0164] Narváez JA, Fuxe K, Santín L, Díaz-Cabiale Z. Galanin (1-15)-fluoxetine interaction in the novel object recognition test. Involvement of 5-HT1A receptors in the prefrontal cortex of the rats. Neuropharmacology, Volume 155, 2019, Pages 104-112,

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[0166] 3. Flores-Burgess A, Millón C, Gago B, García-Durán L, Cantero-García N, Puigcerver A, Narváez JA, Fuxe K, Santín L, Díaz-Cabiale Z, Galanin (1-15) Enhances the Behavioral Effects of Fluoxetine in the Olfactory Bulbectomy Rat, Suggesting a New Augmentation Strategy in Depression, International Journal of Neuropsychopharmacology, Volume 25, Issue 4, April 2022, Pages 307-318, https: / / doi.org / 10.1093 / ijnp / pyab089

[0167] 4. García-Duran L, Flores-Burgess A, Cantero-García N, Puigcerver A, Narváez JÁ, Fuxe K, Santín L, Millón C, Díaz-Cabiale Z. Galanin(1-15) Potentiates the Antidepressant-like Effects Induced by Escitalopram in a Rat Model of Depression. Int J Mol Sci. 2021 Oct 7;22(19):10848. doi: 10.3390 / ¡jms221910848. PMID: 34639188; PMCID: PMC8509384.

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[0175] 10. Flores-Burgess A, Millón C, Gago B, Narváez JA, Fuxe K, Díaz-Cabiale Z Small interference RNA knockdown rats in behavioral functions: GALR1 / GALR2 heteroreceptor in anxiety and depression-like behavior. In: Receptor-receptor interactions in the central nervous system Fuxe K, Borroto-Escuela D, eds , 2018 pp. 133-148. New York: Humana Press.

Claims

CLAIMS 1. Compound GAL(1-15) or a pharmaceutically acceptable salt thereof, in combination with at least one serotonin reuptake inhibitor for use in the treatment of treatment-resistant depression.

2. Compound GAL(1-15) in combination with a serotonin reuptake inhibitor for use according to the preceding claim, characterized in that the serotonin reuptake inhibitor is fluoxetine.

3. A composition comprising the compounds of claim 1 for use in the treatment of treatment-resistant depression.

4. A composition for use according to the preceding claim, characterized in that the serotonin reuptake inhibitor is fluoxetine.

5. Composition for use according to any of claims 3 or 4 characterized in that it is a pharmaceutical composition.

6. Kit comprising the compounds according to any of claims 1 or 2 or the compositions according to any of claims 3 to 5 for use in the treatment of treatment-resistant depression.

7. Kit for use according to the preceding claim, characterized in that it is adapted for simultaneous or sequential administration of the active compounds.

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