C-terminal fragment of tetanus toxin (HC) for the treatment of depression

Abstract

To provide an antidepressant with a broader range of therapeutic effects, fewer side effects, and a faster onset of action. [Solution] The C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) provides therapeutic effects in motor dysfunction associated with Parkinson's disease (PD), and provides long-lasting antidepressant effects, and is therefore useful in treating and alleviating depression, particularly PD-comorbid depression. A method for treating and alleviating depression involves administering an effective amount of the C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) to a subject in need. A method for treating or alleviating motor dysfunction associated with Parkinson's disease (PD) involves administering an effective amount of the C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) to a subject in need.

Description

Technical Field

[0001] (Statement regarding Federally Sponsored Research or Development) Some aspects of the present invention were made under grant NIH / NIAAA R03AA022479 awarded by the National Institutes of Health, and accordingly, the government has certain rights in some aspects of the present invention.

[0002] The method relates to the treatment or alleviation of depression, particularly the co - morbidity of depression - Parkinson's disease (PD). In particular, in the treatment or alleviation of depression, it relates to a method comprising administering the carboxy - terminal domain of the heavy chain of tetanus toxin (Hc - TeTx) or any non - toxic fraction of tetanus toxin, or the coding sequence of Hc - TeTx.

Background Art

[0003] The toll of clinical depression, characterized by feelings of sadness, loss of interest in pleasurable activities, guilt, lethargy, and difficulty concentrating, is a major medical concern. This is due to its relatively high morbidity. In the United States alone, approximately 16 million people, or 7% of adults, suffer from major depressive disorder, which can also include abnormal appetite and sleep, loss of productivity, and suicidal ideation. The actual suicide rate, estimated at 1 million worldwide, affects not only the sufferers themselves, but also their families, friends, and sometimes society as a whole (Non - Patent Document 1).

[0004] While our understanding of the highly complex neurobiological networks involved in mood regulation remains far from complete, it is known that depressive symptoms are diverse and vary greatly from patient to patient. Furthermore, numerous medications developed over the past 60 years, such as tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), norepinephrine reuptake inhibitors (NRIs), and selective serotonin reuptake inhibitors (SSRIs), have provided significant relief to at least some patients (Non-Patent Literature 2). However, these drug therapies are based on the bioamine theory of depression, which assumes that a decrease in these neurotransmitters is the primary cause of the disorder, and have several major drawbacks. These include limited efficacy, slow onset of action, and a variety of undesirable side effects, some of which can be persistent (Non-Patent Literature 3 and 4). Therefore, there is an urgent need for antidepressants with broader efficacy, fewer side effects, and a faster onset of action.

[0005] In recent years, the significant contributions of neurotrophic factors and inflammatory processes to mood regulation / dysregulation have been elucidated, pointing to new approaches in the development of more effective antidepressants. In this regard, several natural and synthetic compounds with anti-inflammatory and neurotrophic factor-increasing properties, particularly brain-derived neurotrophic factor (BDNF), have been proposed as potential novel antidepressants (Non-Patent Documents 5-8). However, no research has been conducted on the C-terminal domain of the tetanus toxin heavy chain (Hc-TeTx) as a potential antidepressant. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] PE Greenberg, AA Fournier, T. Sisitsky, et al., The economic burden of adults with major depressive disorder in the United States, (2005 and 2010) J Clin Psychiatry, 76 (2015), pp. 155-162 [Non-Patent Document 2] MR Levinstein, BA Samuels, Mechanisms underlying the antidepressant response and treatment resistance, Front Behav Neurosci, 8 (2014), pp. 208 [Non-Patent Document 3] AJ Rush, Targeting treatments for depression: what can our patients tell us? Epidemiol. Psychiatr Sci, 26 (2017), pp. 37-39 [Non-Patent Document 4] J. Ben-Sheetrit, D. Aizenberg, AB Csoka, et al., Post-SSRI sexual dysfunction: clinical characterization and preliminary assessment of contributory factors and dose-response relationship. J Clin Psychopharmacol, 35 (2015), pp. 273-278 [Non-Patent Document 5] LL Hurley, Y. Tizabi, Neuroinflammation, neurodegeneration, and depression. Neurotox. Res, 23 (2013), pp. 131-144 [Non-Patent Document 6] O. Kalejaiye, B. Getachew, CL Ferguson, et al., Alcohol-Induced Increases in Inflammatory Cytokines Are Attenuated by Nicotine in Region-Selective Manner in Male Rats. J Drug Alcohol Res, (2017), pp. 6: 236036 [Non-Patent Document 7] CN Bodnar, JM Morganti, AD Bachstetter, Depression following a traumatic brain injury: uncovering cytokine dysregulation as a pathogenic mechanism. Neural Regen Res., 13 (2018), pp. 1693-1704 [Non-Patent Document 8] RS Duman, BDNF, 5-HT, and anxiety: identification of a critical periadolescent developmental period. Am. J. Psychiatry, 174 (2017), pp. 1137-1139 [Overview of the project]

[0007] The inventors have conducted extensive research and discovered that the C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) has antidepressant effects in animal models of depression and may be effective in treating depression, particularly depression associated with Parkinson's disease (PD). The inventors have found that Hc-TeTx results in a dose-dependent reduction in immobility scores, while not affecting open-field spontaneous motor activity (OFLA). In addition to the behavioral effects, the inventors discovered that in two regions closely related to mood regulation, namely the hippocampus and the prefrontal cortex, central brain-derived neurotrophic factor (BDNF) increased while tumor necrosis factor (TNF-alpha) decreased (J. Jin and S. Maren, Prefrontal-hippocampal interactions in memory and emotion. Frontiers Systems Neuroscience, 9 (2015), pp. e170; Getachew, SR Hauser, AB Csoka et al., Role of cortical alpha-2 adrenoceptors in alcohol withdrawal-induced depression and tricyclic antidepressants. Drug Alcohol Depend., 175 (2017), pp. 133-139). These results demonstrate the long-lasting antidepressant effect of Hc-TeTx and suggest its potential usefulness in depression, particularly in PD-comorbid depression.

[0008] The above-mentioned features, other features, and advantages of the present invention are made apparent by the following description and accompanying drawings. [Brief explanation of the drawing]

[0009] [Figure 1]Figures 1A and 1B show the effects of various doses of Hc-TeTx on immobility (1A) and open-field spontaneous motility (1B) in WKY rats during a forced swim test. Animals were tested 24 hours after a single intramuscular injection. Values ​​are mean ± SEM. N=6 / group. *p<0.05, **p<0.01 compared to control. [Figure 2] Figures 2A and 2B show the effects of various doses of Hc-TeTx on immobility during the forced swimming test in WKY rats. Animals were tested one week (2A) and two weeks (2B) after a single intramuscular injection. Values ​​are mean ± SEM. N=6 / group. *p<0.05, **p<0.01 compared to control. [Figure 3] This figure shows the effect of Hc-TeTx on BDNF levels in the hippocampus (Hippo) and frontal cortex (FCX) of WKY rats treated with 60 μg / kg of Hc-TeTx. Animals were sacrificed 24 hours after a single intramuscular injection of Hc-TeTx. Values ​​are mean ± SEM. N=6 / group. **p<0.01** compared to control. [Figure 4] This figure shows the effect of 60 μg / kg of Hc-TeTx on TNF-alpha levels in the hippocampus (Hippo) and frontal cortex (FCX) of WKY rats treated with Hc-TeTx. Animals were sacrificed 24 hours after a single intramuscular injection of Hc-TeTx. Values ​​are mean ± SEM. N=6 / group. **p<0.01** compared to control. [Modes for carrying out the invention]

[0010] Tetanus toxin is a synthetic monopeptide of approximately 150 kDa, consisting of 1315 amino acid residues. Endogenous Clostridium protease forms a double-chain activated molecule composed of a heavy chain (HC) and a light chain (LC) linked by disulfide bonds. The catalytic domain of this toxin is located in the LC, while the rearrangement domain and receptor-binding domain are located in the HC. The C-terminal domain of the tetanus toxin heavy chain (Hc-TeTx) is a non-toxic fragment of TeTx and has been demonstrated to protect against cell death induced by various neurotoxins, including methamphetamine (I. Chaib-Oukadour, C. Gil, J. Rodriguez-Alvarez, et al., Tetanus toxin H(C) fragment reduces neuronal MPP+ toxicity. Mol Cell Neurosci, 41 (2009), pp. 297-303; L. Mendieta, B. Venegas, N. Moreno, et al., The carboxyl-terminal domain of the heavy chain of tetanus toxin prevents dopaminergic degeneration and improves motor behavior in rats with striatal MPP(+)-lesions. Neurosci Res 65 (2009), pp. 98-106; A. Sanchez-Gonzalez, L. Mendieta, V. Palafox, et al., The restorative effect of intramuscular injection of tetanus toxin C-fragment in hemiparkinsonian rats. Neurosci Res, 84 (2014), pp. 1-9.L;Mendieta, N. Granado, J. Aguilera, et al., Fragment C domain of tetanus toxin mitigates methamphetamine neurotoxicity and its motor consequences in mice. Int J Neuropsychopharmacol, 19 (2016), pyw021;L. Radenovic, V. Selakovic, S. Olivan, et al., Neuroprotective efficiency of tetanus toxin C fragment in model of global cerebral ischemia in Mongolian gerbils. Brain Res Bull, 101 (2010), pp. 37-44;MC Sozbilen, M. Ozturk, G. Kaftan, et al., Neuroprotective effects of C-terminal domain of tetanus toxin on rat brain against motorneuron damages after experimental spinal cord injury. Spine, (Phila Pa 1976) 43 (2018), pp. E327-E333). A strong correlation has been demonstrated between neurodegenerative diseases (e.g., Parkinson's disease) and neuropsychiatric disorders (e.g., depression) from the perspective of neurobiological substrates and drug therapies (Y. Tizabi, Duality of Antidepressants and Neuroprotectants. Neurotox Res, 30 (2016), pp. 1-13).

[0011] The inventors first conducted a study to investigate the potential antidepressant effects of Hc-TeTx in an animal model of depression. They found that Hc-TeTx resulted in a dose-dependent reduction in immobility scores, but did not affect open-field spontaneous motor activity (OFLA). Immobility in the forced swim test (FST) is a measure of helplessness or depressive behavior (WP Pare, Open field, learned helplessness, conditioned defensive burying, and forced-swim tests in WKY rats. Physiol Behav, 55 (1994), pp. 433-439). In addition to the behavioral effects, the inventors found that in two areas closely related to mood, namely the hippocampus and the prefrontal cortex, central brain-derived neurotrophic factor (BDNF) increased while tumor necrosis factor (TNF)-alpha (TNF-alpha) decreased. These results demonstrate the long-lasting antidepressant effect of Hc-TeTx and suggest its potential usefulness in depression, particularly in PD-comorbid depression.

[0012] One example of the embodiments of the disclosed subject matter provides an agent for the treatment or alleviation of depression, containing the C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) as an active ingredient.

[0013] Another example of an embodiment of the disclosed subject matter provides a method for treating or alleviating depression, comprising administering an effective amount of Hc-TeTx to a patient in need.

[0014] In one aspect, depression is depression associated with Parkinson's disease (PD) or PD-depressive disorder.

[0015] In one manifestation, Hc-TeTx increases central brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex.

[0016] In one aspect, Hc-TeTx reduces tumor necrosis factor (TNF)-alpha (TNF-α) in the hippocampus and prefrontal cortex.

[0017] In one aspect, Hc-TeTx has a long-lasting effect, thereby making it possible to reduce the frequency of drug administration.

[0018] The formulation of Hc-TeTx and the method of administering Hc-TeTx can be appropriately selected. For example, Hc-TeTx can be formulated in a liquid suitable for administration by injection or nasal spray. This formulation can be produced by well-known methods.

[0019] Injectable or nasal spray preparations contain a solution of a solid agent that is dissolved in a solvent before use. Injectable or nasal spray preparations are used, for example, by dissolving the active ingredient in a solvent. Examples of solvents include distilled water for injection, physiological saline, and the like. Furthermore, injectable or nasal spray preparations may contain stabilizers, solubilizers, suspending agents, emulsifying agents, sedatives, buffers, preservatives, and the like. Such injectable preparations are produced by sterilization in the final step or by adopting an aseptic process.

[0020] [[ID=I6]]The dose of Hc-TeTx used varies depending on factors such as age, body weight, symptoms, treatment effect, administration method, treatment time, and the like. For example, the dose of Hc-TeTx per adult is usually 150 μg to 600 μg per dose, once every two weeks by parenteral administration (most often intramuscular injection or applicable by single intranasal dose administration), in one aspect 180 μg to 540 μg per dose, and in another aspect 180 μg to 360 μg per dose. Needless to say, as described above, the dose used varies depending on various conditions. Therefore, there may be cases where the minimum dose specified above is sufficient, and there may also be cases where a dose larger than the range specified above is required.

[0021] Hc-TeTx may be administered in combination with other pharmaceuticals (e.g., well-known agents for the treatment of depression) for the purposes of (1) supplementing and / or enhancing the therapeutic effect; (2) improving kinetics, improving absorption, and reducing the dose; and / or (3) eliminating adverse reactions of the compound. [Examples]

[0022] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples.

[0023] The inbred Wistar Kyoto (WKY) rat was initially developed as a normal blood pressure control for spontaneously hypertensive rats, and was later found to exaggerate immobility in the forced-swim test (FST), a measure of helplessness or depressive behavior (WP Pare, Open field, learned helplessness, conditioned defensive burying, and forced-swim tests in WKY rats. Physiol Behav, 55 (1994), pp. 433-439). Furthermore, these rats were found to be unresponsive to selective serotonin reuptake inhibitors (SSRIs) and therefore could be considered a model of treatment-resistant depression (C. Lopez-Rubalcava, I. Lucki, Strain-dependent modification of behavior following antidepressant treatment. Prog Neuropsychopharmacol Biol Psychiatry, 27 (2003), pp. 7-14; CC Will, EE Aird, F., Redei, selectively bred Wistar-Kyoto rats: an animal model of depression and hyper-responsiveness to antidepressants. Mol Psychiatry, 8 (2003), pp. 925-932).

[0024] Adult male WKY rats, 14-15 weeks old and weighing approximately 250g, were obtained from Envigo (Indianapolis, Indiana, formerly Harlan Laboratories). Animals treated identically were housed in pairs in standard polypropylene shoebox-type cages (42 x 20.5 x 20 cm) on wood chip bedding throughout the experimental period. Upon arrival, the animals underwent a one-week acclimatization period, during which they were handled daily to minimize any handling-related stress. Throughout the study, except for behavioral tests, the animals had free access to food (Harlan Tech Labs) and water. The room was maintained at 24-26°C and 55-66% relative humidity under a reverse light cycle (lighting from 7:00 PM to 7:00 AM) to allow for convenient evaluation of the animals' behavior during their active hours. The animals were then adapted to a reverse dark cycle over a week, during which the light period was shifted by approximately two hours each day. All behavioral tests and injections were performed between 8:00 AM and 12:00 PM during the animals' active period. All experiments were conducted in accordance with NIH guidelines approved by the Howard University Institutional Animal Care and Use Committee.

[0025] The animals were divided into three groups (n=6 / group) and intramuscularly injected either saline (control) or Hc-TeTx at doses of 20, 40, or 60 μg / kg. The injection was administered into the gastrocnemius muscle. The Hc-TeTx fragment was synthesized as described in detail in a previous report (Herrando-Grabulosa M, Casas C, Aguilera J. The C-terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures. J Neurochem. 2013;124(1):36-44) and as shown below.

[0026] Purification of Hc-TeTx E. coli BL21 cells were transformed with the pQE3 vector (Qiagen, Chatsworth, California, USA) encoding (6×His)-tagged Hc-TeTx, as previously reported (Gil C., Chaib‐Oukadour I. and Aguilera J. (2003) C‐terminal fragment of tetanus toxin heavy chain activates Akt and MEK / ERK signaling pathways in a Trk receptor‐dependent manner in cultured cortical neurons. Biochem. J. 373, 613-620), and grown in Luria Bertani medium containing 100 μg / mL ampicillin. Protein expression was induced by the addition of 0.4 mM isopropyl β-D-thiogalactoside (IPTG). After 3 hours, the cells were pelletized by centrifugation at 4000 g for 20 minutes at 4°C and lysed in lysis buffer (50 mM NaH2PO4). 4、The proteins were resuspended in 300 mM NaCl and 1% Triton-X-100 (pH 8), and subjected to six 30-second sonication cycles on ice. The suspension was centrifuged at 30,000 g for 30 minutes at 4°C. The clear supernatant containing His-tagged proteins was purified by cobalt affinity chromatography. The mixed proteins were injected into a high-performance protein liquid chromatograph (FPLC) containing cobalt-agarose resin (TALON metal affinity resin, Clontech Laboratories, Palo Alto, California, USA) and pre-equilibriumized (50 mM NaH2PO4·H2O and 300 mM NaCl, pH 7). Untagged proteins were eluted by washing the resin with elution buffer (50 mM NaH2PO4·H2O and 300 mM NaCl; pH 7). Hc-TeTx contains six histidine molecules and is held in a resin that forms a Co complex. Hc-TeTx was eluted with elution buffer (50 mM NaH2PO4·H2O, 300 mM NaCl, and 150 mM imidazole, pH 7). The collected fraction was 0.5 mL in volume. The elution process could be tracked using an FPLC system that continuously measured absorbance at 280 nm. The protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained with GelCode blue staining reagent (Pierce Chemical, Rockford, Illinois, USA), and the fractions containing the purified Hc-TeTx protein were dialyzed overnight at 4°C (40 mM Na2HPO4, 10 mM NaH2PO4, and 150 mM NaCl, pH 7.4), and then dialyzed again for 2 hours with fresh buffer. Protein concentrations were determined using a bicinchoninic acid assay (BCA, Pierce Chemical), and the samples were freeze-dried. Hc-TeTx was stored in aliquots at -20°C.

[0027] Furthermore, to determine absorption, uptake, and subsequent arrival at the spinal cord level, Alexa Fluor® 555 was added to Hc-TeTx according to the manufacturer's protocol for the Alexa Fluor® 555 labeling kit (Invitrogen, Carlsbad, California, USA) and stored at -20°C.

[0028] Hc-TeTx solutions were prepared by dissolving 1 mg of lyophilized Hc-TeTx in 1 mL of isotonic saline (0.9% HCl), followed by sequential dilution to obtain final concentrations of 20, 40, or 60 μg / 100 μL. The injection volume was 400 μL / kg. Therefore, each animal was given approximately 100 μL of saline or the drug.

[0029] Approximately 24 hours after the last injection, animals were tested for 5 minutes in an open-field activity monitoring cage (27 × 27 × 20.3 cm, Med Associates, St. Albans, Vermont), and the number of steps, represented by the number of infrared beam interruptions, was recorded. This behavior was evaluated to determine whether the drug treatment affected overall spontaneous motor behavior. This could potentially affect the assessment of immobility in the forced swim test (L. Akinfiresoye, Y. Tizabi, Antidepressant effects of AMPA and ketamine combination: role of hippocampal BDNF, synapsin, and mTOR. Psychopharmacology (Berl), 230 (2013), pp. 291-298; B. Getachew, Y. Tizabi, Both ketamine and NBQX attenuate alcohol-withdrawal induced depression in male rats. J Drug Alc Res, 8 (2019), pp. 1-7).

[0030] Immediately after the open-field activity test, each animal was evaluated for its behavior (immobility) in the forced swimming test (FST). Briefly, each rat was placed in a Pyrex® cylinder pool measuring 17 cm in diameter and 60 cm in height for 5 minutes. The cylinder was filled with 30 cm of water (25 ± 1°C) to prevent the animal from touching the bottom of the container with its hind limbs or tail. FST activity was videotaped for later analysis. After 5 minutes, the rats were removed, dried, and placed in their home cages. Using a time-sampling scoring method, the dominant behavior during each 5-second interval of 300 seconds was recorded. Inactivity (immobility) and activity (swimming) were distinguished as mutually exclusive behavioral states. Swimming behavior was defined as movement (usually horizontal) across the entire cylinder. Immobility was defined as the absence of any additional activity other than that necessary to keep the rat's head above water.

[0031] Since behavioral effects were observed the day after a single Hc-TeTx injection, both OFLA and FST were repeated after a one-week rest period and again after a two-week rest period to determine the sustained effects of a single drug injection on these parameters.

[0032] Another group of rats was treated with a 60 μg / kg dose of Hc-TeTx, as this dose produced the highest behavioral (antidepressant) effect. The animals were euthanized by decapitation approximately 24 hours later, coinciding with the time for behavioral observation. These animals were not subjected to behavioral tests to avoid the potential confounding effect of swimming tests on neurochemical parameters. The brains were promptly removed, frozen on dry ice, and stored at -80°C until excision for BDNF and TNF-alpha measurements. The hippocampus (bilateral) and prefrontal cortex were dissected as described (Tizabi Y, Getachew B, Rezvani AH, Hauser SR, Overstreet DH. Antidepressant-like effects of nicotine and reduced nicotinic receptor binding in the Fawn-Hooded rat, an animal model of co-morbid depression and alcoholism. Prog Neuropsychopharmacol Biol Psychiatry. 2009;33(3):398-402) and as detailed below. The brains were thawed and kept on ice-cold plates. The prefrontal cortex (up to the genu corpus callosum, excluding the olfactory bulb and olfactory tubercle) and hippocampus (bilateral) were removed and stored at -80°C until assay.

[0033] Western blotting was performed as described in detail in the following two documents, the disclosure of which is incorporated herein by reference: B. Getachew, SR Hauser, AB Csoka et al., Role of cortical alpha-2 adrenoceptors in alcohol withdrawal-induced depression and tricyclic antidepressants. Drug Alcohol Depend., 175 (2017), pp. 133-139; and L. Akinfiresoye, Y. Tizabi, Antidepressant effects of AMPA and ketamine combination: role of hippocampal BDNF, synapsin, and mTOR. Psychopharmacology (Berl), 230 (2013), pp. 291-298. In short, homogenates of excised hippocampi (both sides) were prepared in a lysis buffer (10 mM Tris-buffer containing a protease inhibitor (Sigma-Aldrich, St. Louis, Missouri), 5 mM EDTA, 150 mM NaCl, 0.5% TritonX-100 (v / v)). The protein concentration of each sample was determined using the BCA protein assay kit (Pierce Biotechnology, Illinois), and an equal amount of protein (confirmed by β-actin) was loaded into each immunoblot. The proteins were separated using a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membrane was blocked for 1 / 2 hour with a blocking reagent (5% non-fat milk in TBS buffer) and incubated overnight at 4°C with either a primary antibody against BDNF (1:500, Santa Cruz Biotechnology, California) or a primary antibody against TNF-alpha (1:500, Santa Cruz Biotechnology). The membrane was washed with TBST (TBS buffer containing 1% Tween-20) and blocked with the above blocking reagent.Next, the membrane was incubated at room temperature for 1 hour in goat anti-rabbit HRP conjugate secondary antibody (1:3000 in TBS, Bio-Rad Laboratories, California). Then, the membrane was washed with the above TBST washing solution and visualized using an enhanced chemiluminescence kit (Bio-Rad Laboratories, California). The intensity of the protein bands on the gel was quantified using the ChemiDoc XRS system (Bio-Rad Laboratories, California).

[0034] Statistical differences between treatment groups were determined by a one-way analysis of variance (ANOVA) followed by a Newman-Coils post-hoc multiple comparison test to identify which groups were different. A statistical significance level of p<0.05 was set a priori. Data were analyzed using Graphpad Prism6 (Graphpad Software, San Diego, California, USA).

[0035] A single dose of Hc-TeTx resulted in a dose-dependent decrease in FST immobility when tested 24 hours after injection [F(3,28)=6.38, p<0.01]. Thus, compared to the control, the greatest decrease in immobility occurred at a dose of 60 μg / kg (60%, p<0.01), followed by 51% at 40 μg / kg (p<0.01), and 21% at 20 μg / kg (p<0.05) (Figure 1A). Open-field spontaneous motility activity remained unchanged with all treatments (Figure 1B), suggesting that the therapeutic effect of Hc-TeTx on FST is independent of any effect on overall motility.

[0036] Based on these results, the inventors evaluated the neurochemical changes associated with this behavioral effect using the maximum dose of 60 μg / kg.

[0037] One week after the last single injection, the effects of the 40 and 60 μg / kg doses were still evident in the immobility score [F(3,28)=5.96, p<0.01]. Thus, the 40 μg / kg dose reduced immobility by 29% (p<0.05) and the 60 μg / kg dose reduced it by 39% (p<0.01) (Figure 2A). After a two-week rest period, there was no effect of the 40 μg / kg dose (Figure 2B). Although there was still a 13% reduction in the immobility score after administration of the 60 μg / kg dose, this effect was not statistically significant [F(3,28)=0.68, p>0.64].

[0038] Western blot analysis showed that acute treatment with a 60 μg / kg dose of Hc-TeTx resulted in increased BDNF levels in the hippocampus (2.6 times, p<0.01) and prefrontal cortex (2.1 times, p<0.01) 24 hours after a single dose (Figure 3). Regarding TNF-alpha levels, the opposite trend was observed in both regions. Therefore, a 60 μg / kg dose of Hc-TeTx resulted in decreased TNF-alpha levels in the hippocampus (2.5 times, p<0.01) and prefrontal cortex (5 times, p<0.01) 24 hours after a single dose (Figure 4).

[0039] These results suggest an antidepressant-like effect of acute administration of Hc-TeTx in an animal model of treatment-resistant depression. This effect was long-lasting, as behavioral despair, as reflected in the FST immobility score, remained reduced one week after injection. The potential usefulness of Hc-TeTx in movement disorders associated with Parkinson's disease has been demonstrated in numerous preclinical studies (F. Patricio, I. Parra, I. Martinez, et al., Effectiveness of fragment C domain of tetanus toxin and pramipexole in an animal model of Parkinson's disease. Neurotox Res, 35 (2019), pp. 699-710; L. Mendieta, E. Bautista, A. Sanchez, et al., The C-terminal domain of the heavy chain of tetanus toxin given by intramuscular injection causes neuroprotection and improves the motor behavior in rats treated with 6-hydroxydopamine. Neurosci Res, 74 (2012), pp. 156-167), and the comorbidity of depression and PD is well established (RMJ van der Velden, MPG Broen, ML Kuijf, et al., Frequency of mood and anxiety fluctuations in In "Parkinson's disease patients with motor fluctuations: A systematic review. Mov Disord, 33 (2018), pp. 1521-1527," Hc-TeTx can be concluded to be particularly beneficial in such comorbid conditions.This claim is further supported by the finding that neuroprotective drugs generally tend to also have antidepressant effects (Y. Tizabi, Duality of Antidepressants and Neuroprotectants. Neurotox Res, 30 (2016), pp. 1-13).

[0040] These results also suggest a role for the neurotrophic factor BDNF and at least one pro-inflammatory cytokine, TNF-alpha, in the antidepressant effect of Hc-TeTx. This is due to the fact that, accompanied by the observed antidepressant effect, BDNF levels in both the hippocampus and prefrontal cortex increased with Hc-TeTx the day after drug injection, while TNF-alpha levels decreased in both of these areas.

[0041] In summary, adult male Wistar Kyoto rats, a hypothetical animal model of depression, were treated with various doses of Hc-TeTx (0, 20, 40, and 60 μg / kg, intramuscular injection), and performance in open-field spontaneous motility (OFLA) and forced swim test (FST) was evaluated 24 hours, 1 week, and 2 weeks after a single injection. Another group of rats was injected with 60 μg / kg of Hc-TeTx and sacrificed 24 hours later for neurochemical evaluation. Hc-TeTx resulted in a dose-dependent decrease in immobility scores at 24 hours, while OFLA was unaffected. Along with the behavioral effects at 24 hours, BDNF levels in the hippocampus and prefrontal cortex significantly increased, while TNF-alpha levels in both regions significantly decreased. The decrease in immobility scores following high doses of Hc-TeTx was still evident at 1 week but not after 2 weeks of rest. These results demonstrate the long-lasting antidepressant effect of a single dose of Hc-TeTx, suggesting the potential usefulness of Hc-TeTx as a novel intervention for PD-comorbid depression.

[0042] Currently approved antidepressants are primarily based on the monoaminergic hypothesis, which assumes that a decrease in the levels of neurotransmitters such as norepinephrine, dopamine, and serotonin (5HT) in the brain causes mood dysregulation. However, because such antidepressants have a slow onset of action and limited efficacy, attention has shifted to other possible biological substrates. In this regard, the role of neurotrophic factors, particularly BDNF in the hippocampus and prefrontal cortex, and more recently, immune system dysregulation reflected in elevated levels of pro-inflammatory cytokines such as TNF-alpha, have gained substantial traction in this field. Therefore, it is now hypothesized that the slow onset of action of current antidepressants may be due to a slow increase in neurotrophic factors. Furthermore, inhibition of both basal and stimulated serotonin uptake in primary neuron cultures has been demonstrated by Hc-TeTx. Furthermore, in vivo studies have shown that Hc-TeTx increases the activity of tryptophan hydroxylase, a key enzyme in serotonin synthesis, which may lead to the maintenance of high levels of serotonin in the central nervous system.

[0043] While the subject matter disclosed herein has been described in relation to currently accepted practical embodiments, it will be understood that this disclosure is not limited to the disclosed embodiments and includes various variations and equivalent modifications that fall within the spirit and scope set forth in the appended claims.

Claims

1. A method for treating or alleviating depression, comprising administering an effective amount of the C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) to a subject in need thereof.

2. The method according to claim 1, wherein the depression is depression related to Parkinson's disease (PD).

3. The method according to claim 1, wherein the Hc-TeTx is administered by injection.

4. The method according to claim 1, wherein the Hc-TeTx is administered in an amount of 150 μg to 600 μg per dose.

5. The method according to claim 1, wherein the Hc-TeTx is administered in an amount of 180 μg to 540 μg per dose.

6. The method according to claim 1, wherein the Hc-TeTx increases central brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex.

7. The method according to claim 1, wherein the Hc-TeTx reduces tumor necrosis factor (TNF)-alpha (TNF-alpha) in the hippocampus and frontal cortex.

8. A method for treating or alleviating motor impairment associated with Parkinson's disease (PD), comprising administering an effective amount of the C-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) to a subject in need thereof.

9. The method according to claim 8, wherein the Hc-TeTx is administered by injection.

10. The method according to claim 8, wherein the Hc-TeTx is administered in an amount of 150 μg to 600 μg.

11. The method according to claim 8, wherein the Hc-TeTx is administered in an amount of 180 μg to 540 μg.

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