Main Research Findings

1) Treatment with agomelatine was found to have antidepressant effects on test subjects without eliciting any adverse effects on sexual behavior or development.

2) Agomelatine treatment improved depressive behavior in mice by activating catalase in the hippocampus, increasing superoxide dismutase expression, and reducing malondialdehyde expression and damage related to oxidative stress.

Selected Data

1) The research team of Canpolat et al investigated the effects of agomelatine on sexual response and depressive behavior in male rats. For the purpose of this study, 21 day-old prepubertal Sprague-Dawley rats weighing 40+/- 2 grams were utilized. The animals were weaned from their mothers at 21 days and maintained under a reversed light/dark schedule at a constant temperature and humidity with free access to food and water. Valdoxan tablets containing 25 mg of agomelatine were crushed up everyday and dissolved in saline in order for 10 mg/kg of the nootropic to be administered orally everyday and 9 AM. Agomelatine was administered to the test subjects from day 21 to day 120 in male rats and day 90 in female rats while the control group received a daily 1 mg/kg oral dose of saline.Throughout treatment the daily food and water intake, as well as body weight gain of the animals were carefully monitored. Morphological signs of puberty in both male and female rats were evaluated from day 26 until they reached puberty. Female rats were euthanized after 90 days while males were maintained until 120 days before being euthanized [2].

Prior to euthanasia, sexual behavioral tests were performed between the hours of 1300 hours and 1600 hours in a dark room with a night vision camera system. The male rats were placed in a rectangular testing apparatus where they were allowed to habituate for 15 minutes, followed by the placement of a receptive female into the arena. The female rats were ovariectomized 3 weeks prior but were made sexually receptive through injection of 10 ug of estradiol benzoate dissolved in 0.2 ml of sesame oil 48 hours prior to testing, and 500 ug of progesterone dissolved in 0.2 ml of sesame oil 6 hours prior to testing. Each trial lasted 30 minutes while mount latency of the introduced female rate, intromission latency, ejaculation latency, ejaculation frequency, postejaculatory interval, mount latency prior to first ejaculation, intromission frequency, and copulatory efficiency were measured by the research team [2].

The forced swimming test is typically used to assess potential depressive behavior and antidepressant activity of various drugs. For the purpose of this study a modified version of the forced swimming test was used in order to observe the antidepressant effects of chronic treatment with agomelatine in comparison to treatment with a vehicle. Pretesting sessions began by individually placing the animals into plexiglass cylinders filled with 39 cm of water for 15 minutes. The rats were then removed, dried off, and returned to their cage so the cylinders could be emptied and cleaned. 24 hours after the first official trial of the modified forced swimming test occurred. Each trial was conducted for 4 minutes while a video camera mounted above the apparatus recorded the behavior of the animals while in the apparatus.

The forced swimming test assesses for behavioral despair, represented by the total duration of the animals’ immobile posture while in the water. When becoming immobile the rats float in an upright position only making small movements to keep their head above the water. Additionally, climbing behavior was observed as well and was characterized as struggling movements to get out of the cylinder with the animals’ forepaws placed above the surface of the water. Finally, swimming behavior was recorded and defined as primarily active horizontal swimming motions throughout the cylinder. After the forced swimming tests were completed, female rats were euthanized at day 90 and male rats were euthanized at day 120; blood samples were collected and reproductive organs were dissected and weighed for further analysis [2].

Sperm concentration in the right cauda epididymal tissue was examined using a haemocytometer, while the left cauda epididymal tissue was used to analyze sperm motility. A light microscope with a heated stage was then used to calculate the percentage of sperm motility. Levels of luteinizing hormones and follicle-stimulating hormones were measured in the serum by coating immunoplates with either hormone and transferring the samples to the plates after incubation with primary antibodies. The plates were then washed and the secondary antibody conjugated to streptavidin peroxidase was added into each well while color was developed using tetramethylbenzidine. The plates were read at an absorbance of 450 nm in order to detect levels of luteinizing hormone and follicle-stimulating hormone in the samples [2].

2) The research team of Xu et al examined the effects of agomelatine on depression related to catalase activity, oxidative stress, and inflammation in the body. For the purpose of the study 6-8 week old male, specific pathogen-free C57BL/6 mice were utilized. The animals were maintained under standard laboratory conditions with a standard pellet diet and free access to water. In order to establish a chronic resistant stress (CRS) model, the mice were placed in a cylindrical tube 10 cm long with a diameter of 3 cm in order to restrict head and limb movement from 10:00 hours to 16:00 hours. In order to assure the CRS was sufficiently established, the animals underwent several behavioral performance tests including the sugar-water preference = test, the forced swimming test, and the open field test. The researchers identified a decreased sugar water preference, a prolonged forced swimming rest time, and a decreased central total distance in the open field, as indicators of depression [1].

60 test subjects were randomly divided into 6 groups including: normal control, administration of catalase inhibitor, the CRS model, control rats treated with agomelatine, the CRS model rats treated with the catalase inhibitor, and the CRS model rats administered both agomelatine and the catalase inhibitor. All drugs were administered to the animals for 4 weeks through intragastric administration. The drugs were prepared by dissolving the proper dose in saline; agomelatine was administered in doses of 50 mg/kg when given alone and the catalase inhibitor or a combination treatment of the nootropic and the inhibitor were given in doses of 10 mg/kg.

As it was mentioned the rats underwent three different behavioral tests, the sugar-water preference test, the open field test, and force swimming test, to assess the effects of agomelatine on depressive behavior. The sugar-water preference test is divided into two phases: adaptation and experimental stages. The adaptation stage included a single-cage feeding with two water bottles placed in the cage simultaneously. Within 24 hours both of the bottles were filled with 1% sucrose water. 24 hours after the adaptation stage the experimental stage took place which included giving one bottle of water and one bottle of 1% sucrose water given to the animals and changing their positions after 12 hours. After an additional 24 hours the weight was re-recorded in order to compare how much water versus how much sugar water was drunk. The sugar water preference rate was calculated by dividing the sugar water consumption by the combination of sugar water consumption and pure water consumption [1].

For the open field test, an absent field analysis box was divided into central and peripheral parts. The mice were placed in the central part of the grid in order for their activity to be observed for 5 minutes. The researchers were observing the central crossing time recorded in seconds and the total distance in cm traveled by the rats. The final behavioral test was the forced swimming test where the animals were placed in a circular transparent swimming apparatus filled with water so they could not touch their hind legs to the bottom. The mice were allowed to swim for 1 minute to adapt to their surroundings, followed by a 5 minute trial period where the mice were recorded for 5 minutes in order to record the amount of time they spent stationary [1].

In order to perform ELISA experiments, the mice were anesthetized and blood was collected through a puncture of retrobulbar venous plexus. The blood samples were centrifuged and the resulting serum was stored for further ELISA analysis regarding the levels of catalase, superoxide dismutase, and malondialdehyde levels After behavioral testing was complete and blood samples were collected the mice were perfused with paraformaldehyde and their brains were removed. The hippocampus of each brain was dissected and fixed in paraformaldehyde for 3 days. After fixation the hippocampal tissue was embedded in paraffin and sliced into 10 um sections. The sections were prepared according to procedure and after the final staining with hematoxylin, the researchers used PBS as a primary antibody which was defined as the negative control while the positive control was the normal mucosa [1].

Discussion

1) When looking at the effects of agomelatine on sexual behavior in male rats the results reported that there were no significant differences between the average number of ejaculations recorded in the control group versus the group treatment with agomelatine. However, treatment with agomelatine was also found to decrease the intromission frequencies in both the first and second test sessions that were completed on day 90 and day 105. Over the 30 minute testing period the total intromission frequency was also found to have significantly decreased during the first testing session, in the experimental group treated with agomelatine [2].

In terms of ejaculation latency there was no significant difference between treatment groups during the first session, however, there was a significant decrease in ejaculation latency during the second session in rats treated with agomelatine. Similar results were seen with copulatory efficiency, however, there was a significant decrease in the agomelatine treated group during session 1 while there were no noticeable changes during session 2. In terms of mount latency, intromission latency, postejaculatory intervals, and total copulatory efficiencies, there were no remarkable changes noted between treatment groups.

The results of the forced swimming test reported that administration of agomelatine led to a significant decrease in the duration of immobility while in the water. The rats treated with the nootropic were also found to spend more time swimming around the apparatus in comparison to the animals treated with a vehicle. There were no remarkable changes in the climbing time of the animals treated with agomelatine, however, the research noted that there was a trend in the results exhibiting a tendency for climbing times to be increased in the agomelatine treated animals [2].

Figure 1: Effects of treatment with agomelatine on swimming, climbing, and immobility exhibited by the test subjects during the forced swimming test.

Treatment with agomelatine in male rats was also found to significantly increase pubertal weight in comparison to the control group of rats, however the weight of the reproductive organs including the testicle, epididymis, seminal vesicle, and prostate gland, did not exhibit any noticeable changes. In comparison to the female rats, after administration of agomelatine pubertal weight was found to decrease in comparison to the control rats while both ovarian and uterine weight was shown to significantly increase in response to treatment with agomelatine. Levels of luteinizing hormone and follicle-stimulating hormone did not exhibit any remarkable changes in male or female rats. These findings allowed the research team to conclude that treatment with agomelatine elicits antidepressive effects without causing concurrent sexual and reproductive dysfunction [2].

Figure 2: Changes in pubertal weight of male rats in response to treatment with agomelatine.

Figure 3: Changes in pubertal weight of female rats in response to treatment with agomelatine.

Figure 4: Changes in the levels of luteinizing hormone and follicle stimulating hormone in male rats

Figure 5: Changes in the levels of luteinizing hormone and follicle stimulating hormone in female rats.

2) The research team of Xu et al reported that overall, agomelatine has the potential to alleviate depression in mice. First, the baseline body weight was analyzed across all 6 groups of mice, followed by the analysis of body weight following exposure to chronic restraint stress and drug interventions. The result found that the CRS group of mice lost significantly more weight in comparison to the control group, while treatment with agomelatine and combined treatment with agomelatine and the catalase inhibitor was found to offset the stress-induced weight loss. It was also mentioned by the researchers that the catalase inhibitor was found to weaken the effects of agomelatine and when it was administered by itself there were no significant differences in body weight between the catalase inhibitor group and the CRS group [1].

Figure 6: Changes observed across all experimental treatment groups in the total body weight of the test subjects.

The sugar-water preference test, the open field test, and the forced swimming test were used to observe changes in depressive behavior in the rats. The results found that in the CRS model of animals, the mice showed a lower preference for sucrose-water, a significantly increased rest time during the forced swimming test, and a decrease in the total distance traveled in the central region of the open field test, in comparison to the control group of rats. With administration of agomelatine and combined administration of agomelatine and a catalase inhibitor, the noted changes in the CRS mice were significantly improved. It is important to mention that treatment with a catalase inhibitor alone did not result in any changes in the aforementioned parameters. Additionally, there were no significant differences in the total travel time and central region movement time observed between any of the treatment groups. Based on the findings, the research team was able to conclude that administration of agomelatine has the ability to resolve depressive behavior in rats exposed to chronic resistant stress while only being partially alleviated by administration of a catalase inhibitor [1].

Figure 7. Changes recorded across the different experimental treatment groups in C) weight gain, D) sucrose preference rate in the animals, E) measured rest time during the forced swimming test, F) the total distance traveled by the mice through the central region during the open field test, G) the total time spent by the mice in the central region of the open field test, and H) the total distance traveled by the mice during the open field test

In order to see how treatment with agomelatine affected the hippocampus, hippocampal sections were immunohistochemically stained with catalase. In the CRS group of mice catalase expression was found to significantly decrease in comparison to the control group while treatment with agomelatine improved catalase expression to a level similar to the control group. It is important to mention that treatment with the catalase inhibitor had a clear antagonist effect. Additionally, protein immunoblotting was used to confirm that levels of catalase protein expression was lower in the CRS group in comparison to the control group. That being said, catalase protein expression was restored with agomelatine treatment. These findings allowed the researchers to conclude that catalase expression in rats experiencing depression could be enhanced through treatment with agomelatine [1].

Figure 8: Changes across the different experimental treatment groups in the protein positive rate of catalase.

When evaluating the effects of agomelatine in depression resulting from oxidative stress, an ELISA test was used to measure levels of malondialdehyde and superoxide dismutase in blood samples. The CRS mice were found to have significantly lower levels of malondialdehyde after treatment with agomelatine. However, serum levels of superoxide dismutase increased after treatment with the nootropic. Additionally, the levels of pro-inflammatory cytokines and their downstream regulators such as nuclear factor-KB and IKB-alpha were assessed as an indicator of depression resulting from external stress. Expression of these proteins were analyzed using protein immunoblot while mRNA expression levels of the nuclear factor-KB and IKB-alpha were assessed using quantitative and real-time PCR. Experimental and statistical analysis of the results did not reveal any significant changes in the expression of the proteins across any of the experimental treatment groups [1].

FIgure 9: Changes across the different experimental treatment groups in the levels of superoxide dismutase and malondialdehyde.

FIgure 10: Changes across the different experimental treatment groups in the protein expression of nuclear factor-KB and IKB-alpha.
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Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Xu J, Zhu C, Jin P, Sun W, Yu E. Agomelatine prevented depression in the chronic restraint stress model through enhanced catalase activity and halted oxidative stress. PLoS One. 2024 Feb 9;19(2):e0289248. doi: 10.1371/journal.pone.0289248. PMID: 38335199; PMCID: PMC10857580.

[2] Canpolat S, Ulker N, Yardimci A, Bulmus O, Ozdemir G, Sahin Z, Ercan Z, Serhatlioglu I, Kacar E, Ozcan M, Turk G, Ozkan Y, Atmaca M, Yilmaz B, Kelestimur H. Studies on the reproductive effects of chronic treatment with agomelatine in the rat. Eur J Pharmacol. 2016 Jan 5;770:33-9. doi: 10.1016/j.ejphar.2015.11.054. Epub 2015 Nov 28. PMID: 26643170.

Characteristics of Agomelatine

Agomelatine is a unique antidepressant that has been shown to increase melatonin activity by mimicking the effects of melatonin at the receptor target sites. Melatonin is commonly known as the “sleep hormone” as it affects circadian rhythm and sleep-phase cycles. Important research has concluded that there is a direct link between lack of sleep and depression. That being said, disruption of sleep is considered one of the most prevalent signs of a depressive illness. In animal models, depressed subjects typically exhibit decreases in total sleep time and sleep efficiency, difficulty falling asleep, early wake times, and abnormal instances of rapid eye movement (REM).

Further research solidifies this claim, suggesting that subjects with major depressive order (MDD) or seasonal affective disorder (SAD) have also experienced disruptions in amplitude and rhythm of melatonin secretions as well as circadian rhythm. Because agomelatine has shown promise in treating depression while manipulating melatonin activity, important research is currently being conducted to attempt to understand the mechanism of action of agomelatine (https://pubmed.ncbi.nlm.nih.gov/19454302/).

The compound is considered to be an analog of melatonin due its categorization as an acetamide naphthalene. Research has found that the compound acts as a melatonin agonist with affinity for receptors MT1, MT2, and an antagonist for the 5HT2C (serotonergic) receptor. It’s important to mention that agomelatine acts as a 5HT2C receptor antagonist, considering that activation of this receptor leads to inhibited release of dopamine and norepinephrine. By inhibiting activation of this receptor, agomelatine actually leads to increased levels of norepinephrine and dopamine, explaining its mechanism of action as an antidepressant.

Agomelatine is typically administered orally and is metabolized in the liver by isoenzymes 1A2 and 2C9, as well as cytochrome P450. It’s interesting to note that recent studies have shown that when administering agomelatine in combination with compounds that inhibit the 1A2 isoenzyme pathway, total agomelatine plasma levels increase. Additionally, when compounds that induce activation of the 1A2 isoenzyme platforms are administered, agomelatine plasma levels decrease (https://pubmed.ncbi.nlm.nih.gov/11102739/).

Antidepressant Effects of Agomelatine

Through the use of animal models, researchers have been able to further examine the link between agomelatine’s antidepressant effects and its ability to increase melatonin activity. Because the compound is considered a melatonin agonist, administration of agomelatine typically leads to elicited effects similar to melatonin. For example, studies have found that, like melatonin, agomelatine is able to resynchronize circadian rhythms in animals with induced delayed sleep-phase syndrome. However, unlike melatonin, agomelatine exhibits anxiolytic and antidepressant qualities. Research conducted by Millan et. Al examined the extent of which the anxiolytic effects were expressed in rats. The results reported that when comparing agomelatine to benzodiazepine, clorazepate, and the 5HT2 receptor antagonist, SB243,213, agomelatine yielded the best performance results in tests meant to measure anxiolytic activity.

Many significant studies have found that administration of agomelatine improves performance in terms of measured models of depressions. A study conducted by researchers Bourin et. Al observed the efficacy of agomelatine in treating depression against compounds such as melatonin, fluoxetine, and imipramine. Measuring depression throughout the animal model was based on how the rats performed on a forced swim test (FST). In doses of 4, 16, and 32 mg/kg per day, agomelatine was able to significantly improve FST performance by decreasing immobility duration. This indicates that agomelatine is able to act as an efficient antidepressant in terms of an animal model FST (https://pubmed.ncbi.nlm.nih.gov/15069466/).

A similar study conducted by Norman et. Al assessed the antidepressant effects of agomelatine by measuring depression through locomotor hyperactivity in olfactory bulbectomy rats. Doses of 10 and 50 mg/kg per day of agomelatine were given to male Sprague-Dawley rats over an experimental period of 14 days. Compared to the other compounds administered, both the 10 and 50 mg/kg doses drastically decreased cases of locomotor hyperactivity. This significant reduction in measured behavior supports the claim that agomelatine is an effective antidepressant in this model. It should be noted that administration of melatonin alone led to no significant change in the measurement of either of the previously mentioned models, as it caused no change in the activity of 5HT receptors or the secretion of noradrenaline (https://pubmed.ncbi.nlm.nih.gov/22040921/).

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