Main Research Findings

1) Selective inhibitors of dopamine, such as S-phenylpiracetam, may have the potential to treat obesity in individuals diagnosed with metabolic syndrome with fewer occurrences of adverse side effects and health consequences.

2) R-phenylpiracetam has been shown to have potential neuroprotective and anti-inflammatory effects indicating that compounds inhibiting the activity of dopamine transporters can be used as effective treatment for cognitive impairments.

 

Selected Data

1) The research team of Zvejniece et al examined the effects of S-phenylpiracetam on weight gain, levels of glucose and leptin, and overall locomotor activity in an animal experimental model. Weight gain is typically caused by excess energy intake and is characterized by increased adipose tissue that results in a higher risk for comorbidities. Previous research has found that dopaminergic pathways play a crucial role in the control of body weight and food consumption, considering that imaging indicates reduced dopamine receptor availability in obese subjects compared to individuals with a healthy BMI [1].

For the in vivo portion of the study, six male Wistar rats were decapitated in order for the brains to be dissected so the researchers could isolate the striatum. Once isolated, the striatum was homogenized in a sucrose-phosphate buffer, followed by centrifugation for 10 minutes. The resulting supernatant was centrifuged again and the pellet obtained from this final process was resuspended in a sodium-phosphate buffer. The binding assay took place using samples consisting of 60 uL of sodium-phosphate buffer, 20 uL of phenylpiracetam, 20 uL of water, 100 uL of membrane aliquots, and 20 uL of [3H}WIN 35,428 [1]. Non-specific binding assays were performed using the same samples with the addition of vanoxerine dihydrochloride and followed by incubation on ice for 2 hours. Finally, in order to measure radioactivity, both free and bound radioligands were separated through the use of rapid filtration achieved by washing of the filters three times with an ice-cold TRIS-HCl buffer. This process allowed for measurement of radioactivity through a liquid scintillation method.

For the in vitro portion of the study sixteen, 6 week old obese male Zucker rats weighing between 130-200 grams were used. These test subjects were compared to eight, age-matched Zucker rats that were leaner weighing from 130-170 grams. The rats were allowed to adapt to their local condition for 7 days prior to the initiation of treatment; the subjects were maintained under standard laboratory conditions with ad libitum access to food and water. The obese Zucker rats were administered a 50 mg/kg dose of S-phenylpiracetam via oral gavage, on a daily basis over a period of 12 weeks. The lean Zucker rats were treated in the same manner, however, instead of S-phenylpiracetam they were administered 50 mg/kg of water [1].

The next in vitro portion of the experiment included 32 male C57BL/6N mice weighing between 21 grams and 25 grams. The mice were allowed to adapt to their local conditions for 7 days prior to the initiation of the experiments; test subjects were maintained under standard laboratory conditions with ad libitum access to food and water. Following the adaptation period the test subjects were randomly assigned to four different treatment groups including: a normal diet, a Western diet, a Western diet with treatment with S-phenylpiracetam, and a Western diet with treatment with R-phenylpiracetam. It is important to mention that the Western diet was characterized as a diet containing 21% fat, 20% protein, and 50% carbohydrates, while the normal diet contained 4.5% fat, 14.5% protein, and 60.1% carbohydrates [1]. The mice receiving active treatment with R- or S-phenylpiracetam were administered 50 mg/kg via oral gavage while the mice on both the Western diet by itself and a normal diet were administered 50 mg/kg of water in the same manner.

The final in vitro experimental group included forty, 16-week old male SW mice weighing from 40-50 grams each. The test subjects were maintained under standard laboratory conditions with ad libitum access to food and water. The mice were randomly assignment to five different groups including: administration of 50 mg/kg of R-phenylpiracetam, administration of 100 mg/kg of R-phenylpiracetam, administration of 50 mg/kg of S-phenylpiracetam, administration of 100 mg/kg of S-phenylpiracetam, and administration of drinking water for the control group [1]. All animals included in the experiment were weighed one time per week in order to make conclusions regarding increases in fat mass by comparing the differences between lean and obese animal mass. The specific formula to calculate fat mass, specifically in the subjects on a Western diet, was as follows: fat mass = average obese animal weight – average lean animal weight. After 12 weeks of treatment and diet manipulation the subjects were placed in individual metabolic cages in order for their food intake to be measured.

Blood glucose tolerance and concentration levels were assessed next. Prior to the glucose tolerance testing the mice and rats were fasted overnight followed by intraperitoneal injection of glucose solution in doses of 1 g/kg for rats and 0.5 g/kg for mice. Blood samples were collected from the tail vein 15, 30, 60, 120, 180, and 240 minutes following administration of the glucose solution. The glucose tolerance test was performed after 8 weeks of treatment for the mice and after 12 weeks of treatment with S-phenylpiracetam for the rats. The same time frames were used when measuring plasma blood glucose concentration, as well as when measuring concentrations of leptin and insulin in the plasma. Glucose concentration values were assessed using a commercially available kit from Instrumentation Laboratory while leptin and insulin was assessed through the use of Mouse Leptin and Rat/Mouse Insulin ELISA kits [1].

Finally, the test subjects underwent an open-field test in order to observe changes in motor activity in the animals following 8 weeks of treatment with S-phenylpiracetam for mice and 12 weeks of treatment for rats. The apparatus used in the test was a square arena where the animals were placed in the center in order for the research team to record the distance moved in cm and the velocity of the movement in cm/seconds, using a video tracking system. The open-field test was performed 60 minutes after administration of the phenylpiracetam enantiomers; the mice were placed in the open field for a 4 minute testing period while the rats were placed in the open field for a 15 minute testing period.

As an additional trial, the research team used the open-field test to examine the motor activity of the rats and mice after a single administration of S- and R-phenylpiracetam, rather than 8-12 weeks of treatment. The test subjects were allowed to explore the apparatus for 10 minutes the day before the experiment followed by return to their original cages. 24 hours after the adaptation period, the experiment took place in two, 12 minute sessions that occurred 30 minutes after the administration of S-phenylpiracetam and 60 minutes after the administration of R-phenylpiracetam. The animals were placed back into the center of the open field so the velocity and the distance traveled could be recorded [1].

2) While the previously mentioned study focused on the efficacy of S-phenylpiracetam, the research team of Zvejniece et al now examined how R-phenylpiracetam changed the anti-inflammatory activity of dopamine transports in inflammatory animal models of study. For the purpose of this experiment, the researchers used 246, 8-10 week old ICR mice each weighing 23-25 grams. The animals were individually housed in a ventilated cage and maintained under standard laboratory conditions and were provided ad libitum access to food and water. From there the mice were randomly assigned into the different treatment groups for the initiation of the experiment and received both an intraperitoneal and peroral dose of 50 mg/kg at 15 minutes, 30 minutes, 1, 2, 4, 6, and 24 hours before the subjects were euthanized in order to collect brain tissue samples [2].

The mice were also injected intraplantarly with carrageenan in order to induce mouse paw oedema. Before the carrageenan was injected, all of the test subjects were perorally administered either 10 mg/kg of saline, 25 or 50 mg/kg of R-phenylpiracetam, or 10 mg/kg of indomethacin, over an experimental period of 7 days. It is important to note that the secondary portion of the carrageenan test compared both the R and S enantiomers by administering a 50 mg/kg peroral dose of each over an experimental period of 7 days. On the last day of treatment the animals received their treatment dose 60 minutes before carrageenan was injected intraplanarly to the right paw; half of the subjects were administered 40 uL of saline or 40 uL of 2% carrageenan. Following the injection the research team monitored changes in the percentage of paw volume related to induced oedema [2].

Related to the carrageenan test, the test subject also underwent an electronic von Frey test in order to examine mechanical allodynia through the withdrawal threshold of the paw injected with carrageenan. 7 hours after the injection an electronic von Frey anesthesiometer was used to apply mechanical stimulus. All of the subjects were placed on a grid floor in individual observation chambers 10 minutes before the trial in order to acclimate to the surroundings. The adaptation period was immediately followed by placement of the von Frey filament to the midplantar surface of the right hind paw while the researchers recorded the force required to cause withdrawal of the paw.

The research team also subjected the mice to a formalin-induced paw-licking test. Prior to testing, the control animals received an administration of saline while animals in the experimental groups received treatment with either 10 or 50 mg/kg of R-phenylpiracetam, or treatment with 50 mg/kg of S-phenylpiracetam over an experimental period of 7 days. On the last day of treatment the animals were administered their final dose 60 minutes before being intraplanarly injected with 30 uL of formalin solution into the right hind paw. Each test subject was then housed in individual observation cages in order for the research team to record the total licking time of the hind paw of each mouse over three separate phases lasting 5-10 minutes a piece [2].

Six hours prior to euthanasia and brain tissue sampling, the mice were given an intraperitoneal injection of 20 mg/kg of LPS in order to obtain data regarding inflammatory gene expression. LPS was injected at the same time as a 50 mg/kg dose of either R-phenylpiracetam or S-phenylpiracetam in experimental animals or an injection of saline in control animals [2]. LPS and the treatment compounds were injected in contralateral sides and the rectal temperature of the test subjects was taken both before the injection and 6 hours after, prior to decapitation and collection of brain and blood samples.

Blood samples collected from the mice were run through centrifugation for 10 minutes in order to isolate the plasma. The brain tissue was divided into right and left hemispheres followed by homogenization, the resulting homogenate was centrifuged again for another 10 minutes in order to leave a supernatant that was homogenized and centrifuged a final time. The resulting pellet was frozen until further analysis took place. Samples of isolated plasma and the final supernatant were prepared through the process of deproteinization, followed by combining 100 uL of sample and 500 uL of 0.1% formic acid solution in acetonitrile. Prepared and deproteinized samples were centrifuged for 20 minutes and transferred to experimental vials so ultra-performance liquid chromatography-tandem mass spectrometry [2].

 

Discussion

1) When looking at the binding assay related to dopamine transportation following treatment with R-phenylpiracetam and S-phenylpiracetam, the results found that both enantiomers of the nootropic competitively bound to the dopamine transport sites, successfully inhibiting the reuptake of dopamine in the synaptic cleft. In addition to the transportation of dopamine, the effects elicited by S-phenylpiracetam on locomotor activity was observed through open-field testing that took place 30 minutes after administration of the nootropic. Treatment with the S enantiomer of phenylpiracetam was shown to increase locomotor activity significantly within the first 4 minutes of the 12 minute trial. From minutes 4-8 and 8-12 locomotion continuously decreased. It is important to note that the 100 mg/kg dose of R-phenylpiracetam was shown to increase locomotor activity to a greater extent than the same dose of S-phenylpiracetam [1].

Figure 1: The recorded locomotor activity levels 30 minutes after administration of either R-phenylpiracetam or S-phenylpiracetam.

When examining the effects of S-phenylpiracetam on weight gain, the researcher utilized two-way ANOVA which revealed a significant correlation between the day of treatment and the experimental group the Zucker rats were included in. The results reported that in comparison to the lean control rats, obese control rats experienced dramatic body weight gain starting at week 2 that persisted until the end of the experiment. The fat mass increase was measured to be 5 grams after the first week of the study; this measurement continued to increase for the remaining experimental time period and was recorded as 173 grams by the end of 12 weeks.

In obese rats treated with S-phenylpiracetam, the rate of body weight gain was significantly decreased in comparison to that of the obese control group. This finding was observed starting at the 4th week of the experiment and persisted until the end of the experiment. After 12 weeks of treatment with the nootropic the researchers recorded a 16% decrease in body weight. In terms of food intake, there were no significant differences observed between lean and obese control rats, however, the groups treated with S-phenylpiracetam exhibited a tendency to eat less food in comparison to the obese control rats [1].

Figure 2: Changes in A) body weight increase, B) fat mass increase, and C) food intake in the lean and obese control groups, as well as the experimental group of obese mice administered S-phenylpiracetam.

Starting from the 2nd week of the experiment, body weight gain was found to be significantly higher in the mice fed with a Western diet rather than the control mice fed a normal diet. As it was previously mentioned, the two-way ANOVA revealed a correlation between day of treatment and experimental group. The related findings of the experiment showed that treatment with R-phenylpiracetam elicited the most significant decrease in body weight during the first two weeks of the study, as well as weeks 6-8. Treatment with S-phenylpiracetam was found to elicit the most significant decrease in body weight starting at week 6 and persisting until the end of the 12 week experimental period.

By week 8, the group administered S-phenylpiracetam experienced a 26% decrease in body weight while the average reduction in body weight in the group administered R-phenylpiracetam was 40%. Additionally, there was a significant increase in fat mass in the mice fed a Western diet compared to the mice fed a normal diet. However, the mice treated with S-phenylpiracetam experienced a 45% decreased in fat mass by the 8th week of treatment, while the mice treated with R-phenylpiracetam experience a 69% decrease in fat mass, in comparison to the Western diet-fed control group [1].

Figure 3: Time dependent changes in A) body weight and B) fat mass in mice fed a normal diet, a Western diet, a Western diet with administration of S-phenylpiracetam, and a Western diet with administration of R-phenylpiracetam.

Finally, the research team examined changes in levels of blood glucose, leptin, and insulin in response to treatment with both S- and R-phenylpiracetam. Baseline levels of the obese and lean control rats were similar; the obese control rats experienced an increase in fasted blood glucose concentration beginning 8 weeks after the initiation of the experiment. After 12 weeks, both the fasted and fed obese control rats were experiencing hyperglycemia, however, the subjects treated with S-phenylpiracetam had a significantly lower fed-state blood glucose concentration when compared to the obese control group. It should be mentioned that treatment with the nootropic did not elicit any changes on the blood glucose concentrations in fasted mice. When observing the data gathered from the glucose tolerance test, the researchers were able to conclude the administration of S-phenylpiracetam improved glucose tolerance when compared to the obese control mice [1].

Figure 4: Changes in A) blood glucose levels and B) glucose tolerance in lean and obese control mice and obese mice treated with S-phenylpiracetam

2) The results of the study performed by Zvejniece et al first focused on the findings observed following the injection of LPS to induce hypothermia and gene overexpression. The initial results found that there was a significant decrease in body temperature in the control animals when temperature was measured 6 hours after the injection of LPS. That being said, pretreatment with both the R and S enantiomers were able to restore the hypothermia caused by LPS. Additionally, injection with LPS was shown to increase gene expression of IL-1 beta by 8-fold, TNF-alpha by 19-fold, and iNOS by 12-fold. Pretreatment with R-phenylpiracetam decreased expression of IL-1 beta by 75%, TNF-alpha by 73%, and iNOS by 65% [2]. On the other hand, pretreatment with S-phenylpiracetam only slightly reduced gene expression of IL-1 beta and TNF-alpha, however, it is important to note that there was a significant difference between the expression of iNOS in subjects treated with R-phenylpiracetam in comparison to S-phenylpiracetam.

Figure 5: Changes in body temperature in response to pretreatment with nootropic enantiomers following injection with LPS

Figure 6: Changes in the gene expression of IL-1 beta, TNF-alpha, and iNOS in response to pretreatment with nootropic enantiomers following injection with LPS.

Carrageenan was injected into the right hind paw of the mice in order to assess the potential of phenylpiracetam to increase inflammation measured by the amount of paw oedema present. Results of this portion of the study revealed that carrageenan increased paw oedema in a dose dependent manner in mice administered saline, rather than an experimental dosage. Pretreatment with 50 mg/kg of R-phenylpiracetam was found to decrease oedema in a time dependent manner. 2 hours post-injection the oedema had reduced by 60%, 4 hours post-injection the oedema had reduced by 40%, and 6 hours post-injection the oedema had reduced by 45% [2]. Lower doses of R-phenylpiracetam did not elicit any significant reduction in paw volume, however, a 10 mg/kg doses of indomethacin significantly reduced paw oedema by 36% at the 4 hour mark and 45% at the 6 hour mark, post-carrageenan injection. Related to the carrageenan injection, the results of the electronic von Frey filament test found that the amount of time it took for withdrawal to occur reduced by 35% when the mice were pretreated with 50 mg/kg of R-phenylpiracetam. Additionally, the amount of time it took for withdrawal to occur reduced by 40% when 10 mg/kg of indomethacin was administered to the test subjects [2].

Figure 7: Changes in A and B) paw volume and C) mechanical withdrawal threshold, in response to administration of R-phenylpiracetam and S-phenylpiracetam

Finally, the pain inhibition effects of the nootropic enantiomers were examined through the formalin-induced paw-licking test. Control mice injected with saline experienced an immediate nociceptive response following injection with formalin. The nociceptive response included shaking and licking of the injected hindpaw that lasted through the entirety of all three phases of the experiment. Treatment with R-phenylpiracetam in doses of 10 and 50 mg/kg was found to reduce the duration of paw licking in a dose dependent manner; the 50 mg/kg dose was shown to reduce paw licking by 50% during the second phase [2].

Figure 8: Changes in the duration of paw licking.

 

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] Zvejniece L, Svalbe B, Vavers E, Makrecka-Kuka M, Makarova E, Liepins V, Kalvinsh I, Liepinsh E, Dambrova M. S-phenylpiracetam, a selective DAT inhibitor, reduces body weight gain without influencing locomotor activity. Pharmacol Biochem Behav. 2017 Sep;160:21-29. doi: 10.1016/j.pbb.2017.07.009. Epub 2017 Jul 22. PMID: 28743458.

[2] Zvejniece L, Zvejniece B, Videja M, Stelfa G, Vavers E, Grinberga S, Svalbe B, Dambrova M. Neuroprotective and anti-inflammatory activity of DAT inhibitor R-phenylpiracetam in experimental models of inflammation in male mice. Inflammopharmacology. 2020 Oct;28(5):1283-1292. doi: 10.1007/s10787-020-00705-7. Epub 2020 Apr 11. PMID: 32279140.

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