A galantamine–curcumin hybrid lacks the depressant side effect of acetylcholinesterase inhibitors

Abstract

Acetylcholinesterase inhibitors (AChEIs) are drugs that enhance cholinergic neuro-transmission and are used for the treatment of Alzheimer’s disease and myasthenia gravis. Common AChEIs include rivastigmine, donepezil and galantamine (GAL), but they can cause side effects like nausea, vomiting, depression and bradycardia. A potential link exists between AChEIs and increased depression risk. Recently, we have discovered a hybrid compound (4b) combining GAL and curcumin (CCN), which shows improved anticholinesterase activity and neuroprotective effects. It could be developed as a potential multitarget agent for neurodegenerative disorders. Here, we examine the compound’s depressant side effect on mice, comparing it to GAL and CCN. Tests were conducted twice using the tail suspension test (TST) and the forced swim test over an 8-day treatment period. The results showed that 4b lacked the depressant effect of GAL and even displayed a mild antidepressant effect similar to CCN in TST. This suggests that incorporating antidepressant fragments, like CCN, into AChEIs can potentially neutralize their depressive side effects.

Keywords:

• Galantamine
• curcumin
• fluoxetine
• acetylcholinesterase inhibitors
• tail suspension test
• forced swim test

Introduction

Acetylcholinesterase inhibitors (AChEIs) are drugs that enhance cholinergic neurotransmission by inhibiting the enzyme acetylcholinesterase, responsible for the breakdown of acetylcholine. AChEIs are widely used in the treatment of various conditions, including Alzheimer’s disease [1,2] and myasthenia gravis [3]. These drugs increase acetylcholine levels, leading to improved cognitive function and muscle strength. Rivastigmine, donepezil and galantamine (GAL) are commonly prescribed AChEIs. They have demonstrated efficacy in slowing cognitive decline and improving daily functioning in Alzheimer’s patients. However, side effects such as nausea, vomiting, depression and bradycardia may occur [4]. Despite these side effects, AChEIs continue to be an important therapeutic option, providing symptomatic relief and enhancing quality of life for patients. The search for novel AChEIs with multitarget action and fewer side effects continues to be the focus of scientists worldwide.

One of the main and hard side effects of AChEIs is their prodepressant ability. Depression is a mood disorder characterized by persistent feelings of sadness, irritability, poor concentration, feelings of excessive guilt or low self-worth, hopelessness about the future, changes in appetite, weight or sleep and fatigue. It is often treated with antidepressant medications and psychotherapy [5]. Studies have suggested a potential link between AChEIs and an increased risk of depression or worsening of depressive symptoms [6]. The exact mechanisms underlying this association remain unclear.

Recently, a novel hybrid between the AChEI GAL and the antioxidant agent curcumin (CCN), named compound 4b, has been discovered by in silico methods in our lab [7]. The extensive in vitro, in vivo and ex vivo studies demonstrated that the hybrid 4b exhibits enhanced acetylcholinesterase inhibitory activity [8], improved neuroprotective effects against amyloid beta peptide toxicity [9] and scopolamine-induced neurotoxicity [10], and higher antioxidant activity [8,10] compared to the parent compounds alone. These findings highlight the potential of the hybrid 4b as a promising multitarget therapeutic candidate for neurodegenerative disorders.

Here, we examined the prodepressant effect of compound 4b by in vivo tests on mice and compared it with the effects of GAL and CCN. As an AChEI, GAL has a mild prodepressant effect [5], while CCN, on the contrary, improves depressive and anxiety symptoms in people with depression [11]. We studied whether the presence of a CCN fragment in the molecule of the hybrid 4b would counteract the prodepressant effect of GAL.

Materials and methods

Materials

We used the following substances in our study: fluoxetine hydrochloride (Sigma Aldrich, USA, Mw = 345.79 g/mol, purity >98%), galantamine hydrobromide (Sopharma, Sofia, Bulgaria, Mw = 368.3 g/mol, purity >98%), CCN (BioXtract, Les Isnes, Belgium, Mw = 368.4 g/mol, purity >98%). The synthesis of compound 4b is described by Stavrakov et al. [7].

Ethics statement

The experiment was conducted in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and was approved by the Local Animal Care Ethics Committee at the Faculty of Pharmacy, MU-Sofia (No. 329 from 1 June 2022).

Animals

The experiment was conducted on 24 male mice, line ICR, with body weight of 28–32 g. This is a versatile multipurpose line which has been used extensively in pharmacological and toxicological studies. The animals were purchased from the National Breeding Center, Slivnitsa, Bulgaria. A minimum of 7 days of acclimatization was allowed before the start of the study. Standard commercial pelleted mouse food and fresh drinking water were available ad libitum during the experimental period. The animals were housed in Plexiglas cages (4 per cage) in a 12/12 light/dark cycle under standard laboratory conditions (room temperature 20 ± 2 °C and humidity 72 ± 4%), as previously described [10]. The animals were divided into six groups. The tested substances were administered perorally (p.o.) in the following doses:

• Group 1: control group, receiving distilled water only

• Group 2: animals treated with fluoxetine—20 mg/kg

• Group 3: animals treated with GAL—3 mg/kg;

• Group 4: animals treated with CCN—25 mg/kg

• Group 5: animals treated with 4b at low dose—2.5 mg/kg

• Group 6: animals treated with 4b at high dose—5 mg/kg

The substances were ground and the necessary amount of distilled water was added.

Compounds were administered every day for 8 days. To avoid interference between the two tests, they were conducted on different days. The forced swim test (FST) was performed on day 1 and day 7, and the tail suspension test (TST), on day 2 and day 8. On the days of the tests, the substances were administered 60 min before the tests. During this period, animals were observed daily for behavioural changes and signs of toxicity.

Methods

TST

The tail suspension enclosure consisted of plastic enclosures (20 × 25 × 40 cm) separated by an opaque partition which allowed for two animals to be tested at the same time. Following the procedure of Castagné et al. [12] with little modifications, each mouse was suspended by the tail 20 cm above the floor of the enclosure by means of adhesive tape placed <1 cm from the tip of the tail. The behaviour was observed by two investigators for 6 min. The following parameters were recorded: latency to the first immobility and total duration of immobility (in seconds) was recorded from the second minute on. Immobility was defined as hanging passively without any movement of the head or paws. The results of the TSTs were performed on day 2 and day 8, in order to avoid exhaustion in mice.

FST

The FST is a rodent behaviour test used to evaluate the potential antidepressant activity of the tested compounds. Following the procedure of Castagné et al. [12], each of the mice was forced to swim in a narrow plexiglass cylinder with water from which it could not escape. The animals were placed in plexiglass cylinders (45 cm high, 15 cm in diameter, water depth 30 cm, water temperature 23 ± 1 °C) and their behaviour was observed for 6 min. Two animals were observed at the same time with an opaque screen between them. From the second minute onwards, the immobility time (in seconds) of each mouse was recorded. The FSTs were performed on day 1 and day 7.

Data processing and statistics

Statistical processing and graphical presentation of the obtained results were done with the program GraphPad Prism 6.0. The arithmetic mean and the standard errors of the arithmetic mean (SEM) were determined for all data. A statistically significant difference between the compared means was checked using the nonparametric test of Kruskal–Wallis. Differences between the groups were considered statistically significant at a p-value of 0.05 or lower.

Results

The GAL-CU hybrid 4b reduces the immobility time compared with GAL in TST

The TST is an experimental procedure to evaluate depressive-like behaviour and antidepressant efficacy in mice. It involves suspending a mouse by its tail from an elevated support, such as a rod or wire, for a brief period (usually 6 min) [12]. During the test, the mouse is unable to escape or move freely, leading to a state of behavioural despair. This is believed to activate physiological and neurochemical responses associated with depression. Depressed mice exhibit characteristic behaviours such as immobility, reduced struggling, and a ‘frozen’ posture. Antidepressant compounds or treatments are assessed by their ability to decrease the duration of immobility, indicating a potential improvement in mood and motivation.

In the present study, we used 24 mice divided into six groups: control, treated with the antidepressant fluoxetine (positive control), treated with GAL, CCN, 4b low dose (2.5 mg/kg) and 4b high dose (5 mg/kg), respectively. GAL and CCN have intermediate permeability through the blood–brain barrier (BBB), while 4b is highly BBB permeable [8]. Mice were treated for 8 days with the tested compounds. TSTs were performed on day 2 and day 8 from the start of treatment. The average immobility times for the TST are given in Figure 1.

Figure 1. Effect of the administration of the tested substances on the immobility time (seconds) of mice on day 2 and day 8 in the TST. Results are presented as mean values with standard error of the means (±SEM). Fluoxetine (20 mg/kg) was used as a positive control. Differences between groups were statistically non-significant.

Аs expected, fluoxetine reduced the immobility time by 20% as early as day 2, and by day 8 the time was almost half that of the control group. Also expectedly, GAL extended the immobility time by 9% on day 2, and the prolonged treatment did not affect the depressant effect. CCN shortened the immobility time by 11% and 8% on days 2 and 8, respectively. The low dose of 4b had no effect on day 2, but on day 8 a reduction of 13% in the immobility time was observed. Increasing the dose of 4b decreased the depressant effect on the second day of the treatment by 6%. These results suggest that the presence of CCN fragments in the structure of 4b eliminated the depressant effect of GAL.

The latency to the first immobility was also measured. Reduced latency is indicative of depressive-like behaviour. The average latency times are given in Figure 2. Again, as expected, fluoxetine showed a profound antidepressant effect, prolonging the latency times fivefold in comparison to the control group. Both GAL and CCN also increased the latency times (especially on day 2) twofold and threefold, respectively. The low dose of 4b had almost no effect, but the higher dose increased the latency time more than twice after 2 days of treatment. The differences between the groups were not statistically significant.

Figure 2. Latency to the first immobility (seconds) in TST on day 2 and day 8 from the start of the treatment. Results are presented as mean values ± SEM. Fluoxetine (20 mg/kg) was used as a positive control. Differences between groups were statistically non-significant.

The GAL-CU hybrid 4b reduces the immobility time compared with GAL in FST

The FST test is based on the assumption that antidepressant compounds reduce immobility time, whereas the prodepressants increase it. The average immobility times for the FST performed in the present study are given in Figure 3. Here again, fluoxetine and CCN shortened the immobility times by 20% on day 1 and by 10% on day 7, demonstrating antidepressant effects, while GAL prolonged them by 26% and 16% corresponding to its prodepressant effect. The hybrid 4b at both doses showed similar effect to the control group and decreased the immobility time by 17% on day 1 and by 14% on day 7 in comparison to GAL. Once again, the presence of CCN fragments in the molecule of 4b decreased the negative prodepressant effect of AChEIs.

Figure 3. Effect of the administration of the tested substances on the immobility time (seconds) on day 1 and day 7 in the FST. Results are presented as mean values ± SEM. Fluoxetine (20 mg/kg) was used as a positive control. Differences between groups were statistically non-significant.

Discussion

The underlying mechanisms of the depressant effects of AChEIs are not fully understood. It is believed that modulation of cholinergic pathways in the brain, particularly in areas involved in mood regulation such as the basal forebrain and hippocampus, may play a role [13]. The elevated ACh levels in these areas can affect mood and behaviour, leading to depressant effects.

Modelling human depression in laboratory animals is quite a challenging research task due to the complex pathophysiology and multifactorial mechanisms. Many human symptoms of depression, such as recurring thoughts of death or excessive guilt, cannot be replicated in mice. The available models mimic only some of the core aspects of depression. These models include stress paradigms, pharmacological manipulations, lesion studies, environmental manipulations and genetic manipulations [14]. Cryan et al. [14] highlight the importance of complementary approaches and control experiments to validate the results obtained from these models. The more recent genetically altered mice with modified expression of specific proteins offer novel targets for studying antidepressant activity and evaluating the validity of molecular theories of depression [15–17].

Two of the most widely and frequently used experimental procedures in pre-clinical depression research are TST and FST. These models become a standard paradigm to assess ‘depression’ and antidepressant-related phenotypes in wild and/or genetically altered mice [14,18]. They have also been used to detect acute antidepressant effects across different drug classes, making it a valuable tool for rapid screening of novel drugs [19]. Both depression models are based on the observation that, when placed in a stressful situation, animals initially make chaotic movements to escape and then develop an immobile ‘frozen’ posture. In the TST, the stressful situation is hanging by the tail, while in the FST, it is immersion in water. It is recognized that FST and TST may involve different underlying mechanisms for inducing immobility and could yield distinct outcomes following the administration of the same compounds [20].

In our study, we conducted a comparison of the pro- and antidepressant effects of a novel AChEI compound 4b and its parents GAL and CCN in FST and TST. The antidepressant drug fluoxetine was used as a positive control. As an AChEI, GAL has a mild prodepressant effect, while CCN exerts an antidepressant effect. There is no evidence in the literature that the coadministration of both drugs ameliorates the prodepressant effect of GAL.

Arndt et al. [21] reported that fluoxetine (10 and 20 mg/kg, i.p.) generally decreased swimming and increased immobility in male rats across three different environmental conditions. Another study demonstrated that fluoxetine (5–20 mg/kg i.p.) dose-dependently reduced immobility time in the tail-suspension test in both wild-type and 5-HT2A knockout mice, with no significant differences observed between genotypes [22]. These findings could potentially shed light on the divergent results observed in our study with ICR mice, where the immobility time in FST was longer compared to TST following fluoxetine administration.

Several pre-clinical trials have also revealed the antidepressant effects of CCN in rodent models of depression. CCN has been shown to exhibit therapeutic effects comparable to antidepressant drugs such as imipramine and fluoxetine. It has been suggested that CCN can increase serotonin and dopamine levels in the brain at higher doses [23]. Moreover, CCN has the ability to inhibit monoamine oxidase (MAO-A and, to a greater extent, MAO-B), thus regulating the levels of serotonin and dopamine. These mechanisms indicate the potential involvement of the monoaminergic system in the antidepressant effects of CCN. In our study, we observed similar effects of CCN and fluoxetine on immobility time on both days in FST.

A lot of animal trials included acute stress exposures such as the FST, TST, sleep deprivation, immobilization- and cold-induced stress. As recently reviewed [24], chronic stress exposure contained chronic unpredictable stress models (CUMS), restraint stress for different time periods, acute or chronic administration of anxiety, depressive-inducing exogenous agents or some surgical procedures. The narrative review [24] also highlighted that research has mostly focused on the effects of CCN on stress, depression and anxiety induced by these methods, with CCN administration p.o. or by an intraperitoneal injection for different time periods before the stressor. Administered before the stressor, CCN can show antidepressant and anxiolytic effects in animals, evaluated on the basis of endpoints such as changes in behaviour, socialization, appetite and weight [24].

According to the review by Lopresti [24], there are less data from animal studies examining the antidepressant and anxiolytic effects of CCN when it is administered after a stressor. CCN exerted antidepressant and anxiolytic effects when administered after reserpine, pentylenetetrazole and surgical procedures aiming to induce anxious or depressive behaviour. A diet containing CCN consumed before or after fear conditioning could have effects on fear memory consolidation and reconsolidation processes in animals. Accumulating evidence suggests that CCN may be best administered before or during stress exposure in order to manage depression or anxiety. The data from these investigations could have significant influence on the choice of the appropriate time of application of CCN in clinical trials and practice, as it is commonly applied after the first symptoms of depression and stress [24].

The GAL-CCN hybrid 4b, developed as a multitarget agent for treatment of Alzheimer’s disease, was tested here for а depressant side effect inherited from one of its parents GAL. The TST and FST were performed after the mice were treated with two doses of 4b (2.5 and 5.0 mg/kg) for 8 days. The tests showed that this novel agent lacks the depressant effect of GAL, and even in the TST 4b displayed a mild antidepressant effect like its other parent CCN. It is likely that the presence of CCN fragments in the molecule of 4b could eliminate and even reverse the depressant effect of AChEIs. Generally, the inclusion of fragments from antidepressant drugs can be used in the development of novel AChEIs in order to neutralize their ability to cause or trigger depression. This novel approach of including antidepressant fragments in the design of AChEIs offers a promising strategy to mitigate the potential depressant side effects associated with these medications.

Conclusions

This study showed that the GAL-CCN hybrid 4b has the potential to reduce the depressant effect of one of its parents GAL in mice. In TST and FST, the novel agent was associated with a reduced immobility time in comparison to GAL, which indicated that the presence of CCN fragments potentially can reduce the depressant effect of AChEIs. Further research and exploration of this concept are warranted to fully understand the underlying mechanisms and optimize the design of AChEIs with reduced depressive side effects. Such advancements could contribute to the development of safer and more effective treatments for Alzheimer’s disease and other conditions that require AChEI therapy.

Author contributions

Conceptualization, I.K. and I.D.; methodology, I.K.; investigation, I.K., M.A., G.S., I.P. and I.D.; writing—original draft preparation, I.D.; writing—review and editing, I.K., M.A., G.S., I.P. and I.D.; visualization, I.K.; supervision, I.D.; project administration, I.D.; funding acquisition, I.D. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Data availability statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This study was supported by the Science and Education for Smart Growth Operational Program and co-financed by the European Union through the European Structural and Investment funds (Grant No. BG05M2OP001-1.001-0003). The APC was funded by the Bulgarian National Roadmap for Research Infrastructure (Grant No. D01-271/2019).