Main Research Findings

1) When intravenously administered, Emoxypine succinate was found to evenly distribute over the organs and tissues of the test subjects and effectively penetrate into the cellular and mitochondrial membranes.

2) Administration of Emoxypine succinate has been shown to regulate behavioral and molecular responses to iron overload-induced neuroinflammation by targeting the CDK5/GSK3-beta and NLRP3 inflammatory pathways.

 

Selected Data

1) The research team of Shchulkin et al examined the pharmacokinetics of succinate after intravenous administration of the nootropic, Emoxypine succinate in mature male Wistar rats each weighing 180-200 grams. The protocol followed by the researchers was approved by the Animal Care and Use Committee of the Ryazan State Medical University and included injecting 50 mg/ml of Emoxypine succinate intramuscularly, 100 mg/kg through the caudal vein, or an equivalent volume of saline for the control group of rats. 1, 5, 10, 15, 30, 60, or 90 minutes after the nootropic was administered the animals were euthanized and samples from the liver, myocardium of the left ventricle, and the cerebral cortex were collected for further examination [1].

After the samples were collected from each body region, the blood samples were then prepared by centrifugation for 10 minutes to separate the plasma, while the organ samples were washed in physiological saline and placed in a 0.25 M sucrose solution for homogenization. The homogenates were then centrifuged for an additional 10 minutes to precipitate cells and their nuclei that were incompletely destroyed. Resulting supernatants were centrifuged for 15 minutes to obtain mitochondrial and cytoplasmic cell fractions; the mitochondrial fraction was then resuspended in a medium including 0.1% Triton X-100 to destroy the membranes. 900 uL of methanol plus 100 uL of chloramphenicol, an internal standard, was added to the blood plasma and organ homogenate samples, shaken for 1 minute, and centrifuged for 10 minutes. The supernatant was then separated and stored for further evaluation.

After the samples were prepared accordingly, the concentration of succinate in the organ tissue and blood plasma samples was assessed through the use of HPLC-MS/MS. For this analytic method a mobile phase was used consisting of solvent A containing methanol with 0.1% formic acid, and solvent B containing a formic acid solution of 0.1%, as well as a gradient elution mode of delivery. Additionally, the ionization of succinate and the internal standard, chloramphenicol, were carried out using multiple reaction monitoring and a mode of negative ionization. When setting analytical ranges, the range for succinate was set at 200-20,000 ng/ml for blood plasma samples and 20-500 ng/ml for organ homogenates. It is important to mention that samples were diluted and reanalyzed by the researchers if succinate concentrations measured above the upper limit of quantitation [1].

2) Excessive iron accumulation in the brain is associated with the development of neurodegenerative conditions. The research team of Parab et al examined the effects of Emoxypine succinate as a treatment for neurodegeneration by reducing the negative effects of iron imbalances. For the purpose of this study 90, 6-8 month old, wild type zebrafish were obtained from the Central Institute of Fisheries Education. The subjects were allowed a 2 week acclimatization period to a 30 liter housing tank with continuous or diffused aeration provided through an aeration pump air stone attachment. The tanks were maintained at a constant temperature while the water quality was assessed ensuring a pH between 6.8 and 7.5, ammonia concentrations under 0.02 mg/L, nitrate levels below 50 mg/L, and nitrite levels below 0.1 mg/L. The zebrafish were also kept on a 14 hour light and 10 hour dark cycle with three daily feeding times [2].

After the 2 week acclimatization period, the zebrafish were randomly assigned to 6 groups each including 15 test subjects. The zebrafish were placed in individual 2 liter test tanks either filled with water, to act as a control group, or water containing 1.5 mg/L of ferrous sulfate to induce iron overload neurodegeneration. After 28 in the test tanks the zebrafish were then administered an 8 mg/L dose of deferiprone or Emoxypine succinate in 4 mg/L, 8 mg/L, and 12 mg/L. Both Emoxypine Succinate and deferiprone were administered to the test subjects by dissolving the compound in a 500 ml beaker of water and placing the zebrafish in it for 30 minutes everyday for 14 days. After 14 days of treatment the zebrafish underwent behavioral testing including the Y maze test and the novel tank test.

The Y maze utilized a glass tank apparatus that included three arms each measuring 25 cm in length, 15 cm in height, and 8 cm in width with white floors to allow for video analysis and a contrast in color between the fish and the rest of the maze. Black adhesive plastic sheets were applied to the outer maze walls to obscure the outside while colored paper cues in various geometric shapes were applied to the inner walls of the tank. The research team noted that they excluded the color blue from this experiment due to recent studies suggesting zebrafish have an aversion to the color [2].

The three apparatus arms were the starting arm, the open arm, and the novel arm. The fish were all initially placed in the starting arm at the beginning of both the training and testing trials. The open arm was accessible throughout both the training and testing trials for the zebrafish to explore through the experiment. The novel arm was closed during the initial training sessions but was opened by the research for the purpose of the testing trials. The training trial was performed first, followed by the testing trial that took place 1 hour later; the first training trial lasted 5 minutes and the zebrafish were allowed to explore both the start arm and the open arm. During the second testing trial the novel arm was opened and the zebrafish were allowed to explore all three arms over an experimental period of 5 minutes. The trials were recorded and analyzed in order to determine the total distance each zebrafish traveled and how much time was spent in each arm of the apparatus.

The locomotor activity and anxiety levels of the test subjects were assessed through the novel tank test that took place 24 hours after the Y maze test was completed. The novel tank used was in a trapezoid shape measuring 30 cm in length, 12 cm in width, and 15 cm in height with partitioning separating the tank into upper, middle, and lower sections. The zebrafish were placed into this new tank and their activity was recorded over an experimental period of 5 minutes and analyzed to determine the total distance traveled, the average velocity, the latency to entency the top section of the tank, and the time each zebrafish spent in the top and bottom zones of the tank. Immediately after the behavioral tests were completed the zebrafish were euthanized by immersing them for 10 minutes into cold water maintained at a temperature of 0-4 degrees Celsius. The brains of the test subjects were then dissected, weighed, and cleaned in an isotonic saline solution prior to preservation for further analysis [2].

The brain was homogenized using a 10-fold volume of 0.1 M phosphate buffer solution to assess malondialdehyde levels and perform a catalase test on the resulting homogenates. Additionally, the activity levels of Acetylcholinesterase (AChE) were assessed using a colorimetric AChE assay kit that uses 5,5’-dithiobis-(2-nitrobenzoic acid)) to identify and quantify the production of AChE hydrolytic byproduct, thiocholine. It was also mentioned that an Iron colorimetric Assay Kit was used to quantify the iron content in the brains’ of the zebrafish by isolating ferric ions from within the brain homogenate. Finally, ELISA assay kits were used to evaluate levels of IL-1-beta, TNF-alpha, CDK-5,GSK-3-beta, and NLRP3 in the brain homogenates.

 

Discussion

1) The initial results of the study conducted by Shchulkin et al found that after intravenous administration of Emoxypine succinate into the caudal vein, the concentration of the nootropic in the plasma reached its maximum values 1 minute after administration. Concentrations of the compound rapidly decreased by 90 minutes, however, in comparison to the control group, succinate concentrations were still 2.2 times higher. The pharmacokinetic parameters of succinate were then calculated by the research team by subtracting the levels of endogenous succinate from the concentration of succinate following administration of Emoxypine succinate. The parameters suggest that following intravenous administration of the nootropic, succinate was excreted from the body after being evenly distributed throughout the body and penetrating into the tissues. These effects occurred without excess accumulation of the compound in the body [1].

Figure 1: Concentration of succinate in plasma after an intravenous administration of a 100 mg/kg dose of Emoxypine succinate into the caudal vein of male Wistar rats.

The results reported that after Emoxypine succinate was intravenously administered, there was an increase in succinate levels in cytoplasmic fraction of the myocardial tissues by 76.1% after 10 minutes. The mitochondrial fraction of the myocardial tissue also experienced an increase in succinate levels after 10 minutes. In the cerebral cortex, administration of the nootropic was shown to increase succinate levels by 237.8% in the cytoplasmic fraction after 1 minute. In comparison, succinate levels were detected in the mitochondrial fraction at 10 minutes and at 60 minutes after Emoxypine succinate was administered. Finally, in the liver levels of succinate were found to increase by 114.5% after 90 minutes in the cytoplasmic fraction, and after 5 minutes, 10 minutes, and 60 minutes in the mitochondrial fraction.

The research team noted that after administration of Emoxypine succinate, the differences in succinate concentrations in the three sample tissues is due to the different rates of blood flow and metabolism as well as the differences in overall functioning of the tissues. The rate of blood flow in the rats was highest in the myocardial tissues at 3.27 +/- 0.30 ml/min/g, followed by the cerebral cortex tissues at 1.02 +/- 0.12 ml/min/g, and finally the liver tissues at 0.10 +/- 0.01 ml/min/g. In addition to the highest blood flow rat, the myocardium tissue was also found to have highest metabolic activity at 8986.52 Joules/g, followed by the cerebral cortex tissues at 4901.74 J/g, and the liver tissues at 4084.78 J/g [1].

These findings, along with the understanding of the relationship between blood flow and metabolism and succinate concentrations, allowed the researchers to conclude that succinate levels were the highest in the liver and the lowest in the myocardium. Additionally, due to the high concentration of succinate found in the tissue samples collected from the cerebral cortex, the research team was able to assume that Emoxypine succinate is capable of penetrating the blood-brain-barrier. In regards to the pharmacodynamics of the nootropic, because the compound is so rapidly eliminated from the body, the antihypoxic effects are most likely due to the stimulation of GPR91 receptors and the following biological cascade, rather than maintaining the activity of the respiratory chain [1].

2) Results of the Y maze test were based on the number of entries into each arm of the apparatus and the total distance traveled by each zebrafish. The positive control group was shown to spend a longer amount of time in the novel arm and a longer overall distance traveled compared to the negative control group. The zebrafish included in the negative control group experienced compromised performance shown through decreased time spent in the novel arm and a shorter total distance traveled. This indicates that these subjects had increased anxiety, impaired spatial memory, and reduced exploratory tendencies and locomotor activity. Treatment with all three doses of Emoxypine succinate were found to produce positive outcomes in comparison to the negative control group; these subjects experienced a significant increase in the amount of time spent in the novel arm and the total distance traveled. The findings suggest that the nootropic helps attenuate spatial memory deficits while reducing anxiety and increasing exploratory and locomotor activity [2].

Figure 2: B) The total distance traveled by each of the experimental treatment groups and C) the number of entries in the open arm, starting arm, and novel arm by each of the experimental treatment groups.

The results of the novel tank test were determined based on the total distance traveled, the average velocity, latency to enter the top section of the tank, and the amount of time spent in both the top and bottom sections of the tank. The positive control group exhibited longer exploratory times of the upper section of the tank and less time spent in the lower section, an increased total distance traveled and average velocity, and a shorter latency period to enter the top section in comparison to the negative control group. These positive outcomes were also seen in the zebrafish treated with all three doses of Emoxypine succinate, indicating heightened curiosity and reduced anxiety. Additionally, it was noted that the group of subjects treated with 12 mg/L of the nootropic experienced the highest improvement in spatial memory amongst the experimental treatment groups [2].

Figure 3: B) The total distance traveled by each of the experimental treatment groups; C) the average velocity recorded in each of the treatment groups; D) the latency of each of the treatment groups to enter the top section of the tank; E) the amount of time spent in the top section of the tank by each treatment groups; and F) The amount of time spent in the bottom section of the tank by each of the treatment groups.

Oxidative stress in the zebrafish was assessed by tracking the levels of malondialdehyde (MDA), catalase, superoxide dismutase, and glutathione. MDA was significantly higher in the negative control group, indicating oxidative damage and elevated lipid peroxidation. There was also diminished superoxide dismutase and catalase activity, suggesting impaired antioxidant defense mechanisms, as well as reduced glutathione levels, indicative of poor cellular redox balancing. Treating the zebrafish with Emoxypine succinate led to a dose-dependent reduction in oxidative stress. In all three experimental treatment groups there was a significant decrease in MDA levels, especially in the 8 mg/L and 12 mg/L group, suggesting decreased oxidative damage and lipid peroxidation. Activity levels of catalase, superoxide dismutase, and glutathione were all shown to significantly increase, indicating improvements in antioxidant defense mechanisms, the catalysis of the reactive oxygen species, and cellular redox balance [2].

Figure 4: A) Levels of MDA; B) catalase; C) superoxide dismutase; and D) glutathione (GSH) in the experimental treatment groups.

The negative control group exhibited a significant increase in the activity levels of acetylcholinesterase, suggesting that dysfunction-induced neurodegeneration is associated with impaired cholinergic functioning. However, cholinergic functioning was found to improve following treatment with Emoxypine succinate exhibited by a decrease in AChE activity. These effects were found to be dose-dependent with the 12 mg/L dose of Emoxypine succinate eliciting maximum reduction in the activity of the enzyme. These findings allowed the researchers to infer that the nootropic therapeutically addresses neurodegeneration characterized by cholinergic dysfunction through the restoration of cholinergic neurotransmission [2].

Figure 5: Levels of AChE activity in the different experimental groups.

Additional outcome measures assessed included the accumulation of iron in the brain and levels of the following inflammatory markers: IL-1-beta, TNF-alpha, CDK-5, GSK-3-beta, and NLRP3. In comparison to the negative control group, all three of the experimental groups treated with Emoxypine succinate were found to significantly decrease the susceptibility of iron accumulation, suggesting the potential of the nootropic to regulate brain iron homeostasis and attenuate iron-overload related to neurodegeneration. In regards to the upregulation of inflammatory markers, administration of Emoxypine succinate resulted in significant decreases in IL-1-beta, TNF-alpha, CDK-5, GSK-3-beta, and NLRP3 in comparison to the negative control group. These results indicate that treatment with the nootropic has potential anti-inflammatory effects, as well as the ability to regulate key signaling pathways in order to alleviate neuroinflammation and abnormal signaling related to iron dysregulation in the brain [2].

Figure 6: Levels of iron in the brain in the different experimental treatment groups.

Figure 7: Levels of A) IL-1-beta; B) TNF-alpha; C) CDK-5; D) GSK-3-beta; and E) NLRP3 in the different experimental treatment groups.

 

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] Shchulkin AV, Mylnikov PY, Chernykh IV, Esenina AS, Yakusheva EN. Pharmacokinetics of Succinate in Rats after Intravenous Administration of Mexidol. Bull Exp Biol Med. 2023 May;175(1):54-58. doi: 10.1007/s10517-023-05810-5. Epub 2023 Jun 20. PMID: 37338763.

[2] S. Bagwe Parab, G. Kaur, Emoxypine succinate modulates behavioral and molecular responses in zebrafish model of iron Overload-Induced neuroinflammation via CDK5/GSK3- β and NLRP3 inflammasome pathway, Brain Research (2024), doi: https://doi.org/10.1016/j.brainres.2024.149236

 

 

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