Brain
Expert Pharmacologist
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Abstract.
Conducting a study of pro-inflammatory effects of mephedrone and identification of its ethological characteristics are a mandatory stage in researching 4-MMC, which is considered one of the most popular psychoactive substances in terms of recreational use. The administration, transport and concentration of mephedrone in its target tissues depends not only on the administered dose, but also on a variety of other factors - the distribution of the substance, the degree and speed of absorption, the path of metabolism and excretion. Throughout the study, it is important to determine the proportion of the substance distributed in the target tissues or biological fluids which it is transported with, so it can be found out which part of the administered substance undergoes biotransformation, and which part gets into the systemic blood circulation unchanged. Also, at the stage of preclinical testing, in addition to studying the main mechanisms of action, there is a need for a large phenotypic screening, necessary for an in-depth understanding of the pharmacodynamics and side effects of the substance. One of the important aspects of such screening is study on psychoactive properties of the substance.
Study objectives.
1. To determine cognitive motivational and behavioral patterns in rats administered 4-methylmethkathinone.
2. To study endothelial function by conducting vascular pharmacological tests using NO-synthesis blockade under the ultrasound control of blood flow velocity in the rat femoral artery and laser Doppler flowmetry monitoring of general hemodynamics and microcirculation velocity in the medial muscle.
3. To identify and evaluate proinflammatory and immunoregulatory activities of 4-methylmethkathinone using the formalin-induced paw edema model and the NBT test.
Materials and methods.
The study was conducted on 40 male Wistar rats weighing 180-200 g. 4 groups were identified: 1) Control group (intact rats); 2) Rats, which were administered mephedrone intraperitoneally every 48 hours at a dose of 5 mg/kg dissolved in NaCl 0,9 solution (0,5 ml). 3) Rats, which were administered mephedrone orally (through a tube) every 48 hours at a dose of 5 mg/kg dissolved in NaCl 0,9 solution (0,5ml). 4) Rats, which were administered mephedrone intraperitoneally every 48 hours at a dose of 5 mg/kg dissolved in NaCl 0,9 solution (0,5 ml) before administration of formalin for formalin-induced paw edema model.
Study description and discussion.
Determination of cognitive motivational and behavioral patterns in rats administered 4-methylmethkathinone. To achieve this goal, the following objectives were formulated: to assess 4-methylmethkathinone influence on the level of arousal, anxiety and resistance to stress in rats. To determine the psychoemotional state of the studied animals, we used an upgraded Hall test protocol, which involves placing an object in a standardized arena and registering activity indicators using the software and hardware complex PANLAB Harvard Apparatus. Discrete behavioral acts recorded using the software and hardware complex were selected as evaluation units.
The study was carried out on 30 rats (Group 1, 2 and 3 respectively). Before the experiment, the animals were kept in a laboratory vivarium of 10 individuals in a cage, with an unconverted 12-hour regime, at a room temperature of 22-24 °C and humidity of 60%, on a standard diet. The experiments were carried out in the summer. The obtained results were checked for the distribution normality using the Shapiro-Wilk criterion and processed by methods of variational statistics using the Student's t-test and STATISTICA computer software package. The figure schematically shows the design of the experiment. Horizontal activity, grooming reaction, search activity, loco motor activity, urination and defecation were recorded to assess the exploratory behavior. The experiment was carried out in a darkened room, isolated from the extraneous noise and sunlight, in the period from 10 to 14 hours. The open field was a square area of 150x150 cm in size with sides 30 cm high. The temperature in the room was maintained at 23 degrees Celsius. After each animal, the open field was sanitized for deodorization. Before the study of each group, an animal zero was released into the open field.
On day 21, the rats were removed from the experiment. After anesthesia of rats, isolation of the right carotid artery was performed on a fixed platform, the medial thigh muscle was exposed, and the left femoral vein was catheterized. The multicomponent study of hemodynamic parameters was investigated by the method of specialized provocation tests involving the administration of acetylcholine and sodium nitroprusside (NO) into the femoral catheter. Parameters (systolic blood pressure, diastolic blood pressure, heart rate) were recorded using a TDS sensor. The sensor was installed over the middle third of the right femoral artery and the following indicators were recorded: maximum systolic (VS) and diastolic (VD) velocity, average (QAs) and volumetric (QAM) blood flow velocity, pulsation index (PL, Gosling index) and resistivity index (RI, Pourcelot index). To measure the velocity of the blood cells in the vessels of the microcirculatory bed, a needle-shaped probe-sensor was used, which was placed in the lower third of the medial broad thigh muscle. Vasoactive agents (acetylcholine - 40 mcg/kg, sodium nitroprusside - 30 mcg/ kg) were injected through a catheter inserted into the left femoral vein sequentially at intervals of 15 minutes. Thus, there was a possibility of parallel monitoring of hemodynamic and microcirculation parameters in a three-level system (central circulation - elastic vascular system - peripheral vascular system). Statistical processing of the results was performed using the STATISTICA 10 software. Then the coefficient of endothelial dysfunction (QED) was calculated. The statistical analysis was carried out using Spearman's rank test, the reliability calculation between the groups was performed using the Student's parametric t-test.
The diagrams presented show that mephedrone influence on animal behavior corresponds to its main effects, associated with its known mechanism of action. So, diagram 1 illustrates a significant increase in horizontal motor activity compared to the control group. Diagram 2 shows that grooming acts are significantly reduced. This phenomenon is caused by a decrease in motivational influence in regard to the present need. Diagram 3 clearly visualizes an increase in acts of defecation and urination, which is one of frequent effects of 4-mmc intake. An increase in horizontal activity may indicate anxiolytic, anxiogenic, agitation effects or an increase in motor activity without an emotional component (motor arousal). Grooming reactions can also be interpreted ambiguously, since the frequency of grooming acts increases both in a stressful and in a favorable environment. The manifestations of exploratory activity, as well as the frequency of urinations and defecations, can be interpreted most unambiguously. Using this algorithm, it can be concluded that the pharmacodynamic effect regarding higher nervous activity can be interpreted as motor arousal with an emotional component.
The control group demonstrated the same type of responses to the introduction of ACH and NO, which manifested as a drop in systolic, diastolic and mean blood pressure, a decrease in the linear velocity of microcirculation, as well as the diastolic velocity of blood movement in the femoral artery (hereinafter FA), (data indicators - Vd, Vad); 2) an increase in the systolic velocity of blood movement in FA (VS, Vas), the Purcello and Gosling indices. The parameters returned to their original values within 2 minutes.
Further statistical processing was carried out when determining a number of values over time for each sample, which were expressed as a percentage. Spearman's criterion, which was used for statistical analysis, showed the best expression of the necessary difference in statistically important parameters. The greatest degree of correlation (p<0.05) was determined between the index of mean arterial pressure (hereinafter referred to as MAD), linear velocity of microcirculation, systolic velocity and systolic-diastolic velocity difference in the femoral artery. Other sonographic parameters demonstrated a low and less reliable correlation with the dynamics of blood pressure and microcirculation rate. Study of orally administered 4-mmc revealed a statistically significant increase in the indicators of SAD, DAD and the coefficient of endothelial dysfunction (QED) in comparison with the control group. The indicators of maximum systolic (VS) and maximum diastolic velocity (VD) are significantly reduced during provocation tests in rats of Group 3, in which mephedrone was administered orally.
The effectiveness of endothelium-dependent relaxation factors released and activated by the administration of acetylcholine is quantitatively and qualitatively reduced. The NO-mediated effect in response to the ACH provocation test decreases, due to the negative effect of 4-mmc on the production and expression of both this factor and the activity of guanylate cyclase. ACH-induced endothelium-dependent relaxation of the microcirculatory vessels, mediated through muscarinic receptors, is also disrupted due to the degeneration of the receptor activity, and also due to a decrease in the density of receptors on the endotheliocytes surface. It is likely that the mechanism of reducing the ACH-mediated endothelium-dependent reaction in response to mephedrone use is caused by a significant increase in the formation of free radicals (both superoxide and hydroxyl) in the hypoxic state of the cell, which interact with NO2 and NO3.
There was a statistically significant decrease in the metabolic activity of neutrophils by 38.4% (Me = 13.1 with an interquartile range from 10.3 to 15.9 with a difference of 5.3 at p=0.002; z = 3.008) compared with the indicators of the control group. In the study of phagocytosis absorption capacity with the addition of latex, the indicators increased from 13.1 Me to 16.5 Me, with a percentage increase of 18.2% for Group 3 (intraperitoneal administration), however, the percentage difference compared to the control group is 18.0%, which is significant and statistically reliable evidence of immunosuppressive action. Also, a statistically significant decrease in spontaneous phagocytosis in Group 3 was found to be decreased by 25.3% (at p>0.0001, z=2.84) in comparison to intact animals.
Conducting a study of pro-inflammatory effects of mephedrone and identification of its ethological characteristics are a mandatory stage in researching 4-MMC, which is considered one of the most popular psychoactive substances in terms of recreational use. The administration, transport and concentration of mephedrone in its target tissues depends not only on the administered dose, but also on a variety of other factors - the distribution of the substance, the degree and speed of absorption, the path of metabolism and excretion. Throughout the study, it is important to determine the proportion of the substance distributed in the target tissues or biological fluids which it is transported with, so it can be found out which part of the administered substance undergoes biotransformation, and which part gets into the systemic blood circulation unchanged. Also, at the stage of preclinical testing, in addition to studying the main mechanisms of action, there is a need for a large phenotypic screening, necessary for an in-depth understanding of the pharmacodynamics and side effects of the substance. One of the important aspects of such screening is study on psychoactive properties of the substance.
Study objectives.
1. To determine cognitive motivational and behavioral patterns in rats administered 4-methylmethkathinone.
2. To study endothelial function by conducting vascular pharmacological tests using NO-synthesis blockade under the ultrasound control of blood flow velocity in the rat femoral artery and laser Doppler flowmetry monitoring of general hemodynamics and microcirculation velocity in the medial muscle.
3. To identify and evaluate proinflammatory and immunoregulatory activities of 4-methylmethkathinone using the formalin-induced paw edema model and the NBT test.
Materials and methods.
The study was conducted on 40 male Wistar rats weighing 180-200 g. 4 groups were identified: 1) Control group (intact rats); 2) Rats, which were administered mephedrone intraperitoneally every 48 hours at a dose of 5 mg/kg dissolved in NaCl 0,9 solution (0,5 ml). 3) Rats, which were administered mephedrone orally (through a tube) every 48 hours at a dose of 5 mg/kg dissolved in NaCl 0,9 solution (0,5ml). 4) Rats, which were administered mephedrone intraperitoneally every 48 hours at a dose of 5 mg/kg dissolved in NaCl 0,9 solution (0,5 ml) before administration of formalin for formalin-induced paw edema model.
Study description and discussion.
Determination of cognitive motivational and behavioral patterns in rats administered 4-methylmethkathinone. To achieve this goal, the following objectives were formulated: to assess 4-methylmethkathinone influence on the level of arousal, anxiety and resistance to stress in rats. To determine the psychoemotional state of the studied animals, we used an upgraded Hall test protocol, which involves placing an object in a standardized arena and registering activity indicators using the software and hardware complex PANLAB Harvard Apparatus. Discrete behavioral acts recorded using the software and hardware complex were selected as evaluation units.
The study was carried out on 30 rats (Group 1, 2 and 3 respectively). Before the experiment, the animals were kept in a laboratory vivarium of 10 individuals in a cage, with an unconverted 12-hour regime, at a room temperature of 22-24 °C and humidity of 60%, on a standard diet. The experiments were carried out in the summer. The obtained results were checked for the distribution normality using the Shapiro-Wilk criterion and processed by methods of variational statistics using the Student's t-test and STATISTICA computer software package. The figure schematically shows the design of the experiment. Horizontal activity, grooming reaction, search activity, loco motor activity, urination and defecation were recorded to assess the exploratory behavior. The experiment was carried out in a darkened room, isolated from the extraneous noise and sunlight, in the period from 10 to 14 hours. The open field was a square area of 150x150 cm in size with sides 30 cm high. The temperature in the room was maintained at 23 degrees Celsius. After each animal, the open field was sanitized for deodorization. Before the study of each group, an animal zero was released into the open field.
On day 21, the rats were removed from the experiment. After anesthesia of rats, isolation of the right carotid artery was performed on a fixed platform, the medial thigh muscle was exposed, and the left femoral vein was catheterized. The multicomponent study of hemodynamic parameters was investigated by the method of specialized provocation tests involving the administration of acetylcholine and sodium nitroprusside (NO) into the femoral catheter. Parameters (systolic blood pressure, diastolic blood pressure, heart rate) were recorded using a TDS sensor. The sensor was installed over the middle third of the right femoral artery and the following indicators were recorded: maximum systolic (VS) and diastolic (VD) velocity, average (QAs) and volumetric (QAM) blood flow velocity, pulsation index (PL, Gosling index) and resistivity index (RI, Pourcelot index). To measure the velocity of the blood cells in the vessels of the microcirculatory bed, a needle-shaped probe-sensor was used, which was placed in the lower third of the medial broad thigh muscle. Vasoactive agents (acetylcholine - 40 mcg/kg, sodium nitroprusside - 30 mcg/ kg) were injected through a catheter inserted into the left femoral vein sequentially at intervals of 15 minutes. Thus, there was a possibility of parallel monitoring of hemodynamic and microcirculation parameters in a three-level system (central circulation - elastic vascular system - peripheral vascular system). Statistical processing of the results was performed using the STATISTICA 10 software. Then the coefficient of endothelial dysfunction (QED) was calculated. The statistical analysis was carried out using Spearman's rank test, the reliability calculation between the groups was performed using the Student's parametric t-test.
The diagrams presented show that mephedrone influence on animal behavior corresponds to its main effects, associated with its known mechanism of action. So, diagram 1 illustrates a significant increase in horizontal motor activity compared to the control group. Diagram 2 shows that grooming acts are significantly reduced. This phenomenon is caused by a decrease in motivational influence in regard to the present need. Diagram 3 clearly visualizes an increase in acts of defecation and urination, which is one of frequent effects of 4-mmc intake. An increase in horizontal activity may indicate anxiolytic, anxiogenic, agitation effects or an increase in motor activity without an emotional component (motor arousal). Grooming reactions can also be interpreted ambiguously, since the frequency of grooming acts increases both in a stressful and in a favorable environment. The manifestations of exploratory activity, as well as the frequency of urinations and defecations, can be interpreted most unambiguously. Using this algorithm, it can be concluded that the pharmacodynamic effect regarding higher nervous activity can be interpreted as motor arousal with an emotional component.
The control group demonstrated the same type of responses to the introduction of ACH and NO, which manifested as a drop in systolic, diastolic and mean blood pressure, a decrease in the linear velocity of microcirculation, as well as the diastolic velocity of blood movement in the femoral artery (hereinafter FA), (data indicators - Vd, Vad); 2) an increase in the systolic velocity of blood movement in FA (VS, Vas), the Purcello and Gosling indices. The parameters returned to their original values within 2 minutes.
Further statistical processing was carried out when determining a number of values over time for each sample, which were expressed as a percentage. Spearman's criterion, which was used for statistical analysis, showed the best expression of the necessary difference in statistically important parameters. The greatest degree of correlation (p<0.05) was determined between the index of mean arterial pressure (hereinafter referred to as MAD), linear velocity of microcirculation, systolic velocity and systolic-diastolic velocity difference in the femoral artery. Other sonographic parameters demonstrated a low and less reliable correlation with the dynamics of blood pressure and microcirculation rate. Study of orally administered 4-mmc revealed a statistically significant increase in the indicators of SAD, DAD and the coefficient of endothelial dysfunction (QED) in comparison with the control group. The indicators of maximum systolic (VS) and maximum diastolic velocity (VD) are significantly reduced during provocation tests in rats of Group 3, in which mephedrone was administered orally.
The effectiveness of endothelium-dependent relaxation factors released and activated by the administration of acetylcholine is quantitatively and qualitatively reduced. The NO-mediated effect in response to the ACH provocation test decreases, due to the negative effect of 4-mmc on the production and expression of both this factor and the activity of guanylate cyclase. ACH-induced endothelium-dependent relaxation of the microcirculatory vessels, mediated through muscarinic receptors, is also disrupted due to the degeneration of the receptor activity, and also due to a decrease in the density of receptors on the endotheliocytes surface. It is likely that the mechanism of reducing the ACH-mediated endothelium-dependent reaction in response to mephedrone use is caused by a significant increase in the formation of free radicals (both superoxide and hydroxyl) in the hypoxic state of the cell, which interact with NO2 and NO3.
There was a statistically significant decrease in the metabolic activity of neutrophils by 38.4% (Me = 13.1 with an interquartile range from 10.3 to 15.9 with a difference of 5.3 at p=0.002; z = 3.008) compared with the indicators of the control group. In the study of phagocytosis absorption capacity with the addition of latex, the indicators increased from 13.1 Me to 16.5 Me, with a percentage increase of 18.2% for Group 3 (intraperitoneal administration), however, the percentage difference compared to the control group is 18.0%, which is significant and statistically reliable evidence of immunosuppressive action. Also, a statistically significant decrease in spontaneous phagocytosis in Group 3 was found to be decreased by 25.3% (at p>0.0001, z=2.84) in comparison to intact animals.
- When mephedrone was administered to experimental animals, a statistically significant change in the analyzed behavioral acts was recorded. This fact indicates the presence of specific psychotropic activity of this compound associated with excessively elevated levels of arousal and anxiety, however, with reduced resistance to stress. Thus, the study results at this stage expanded the information about the profile of the compound action. The observed psychotropic effects of 4-mmc deepen the understanding of its pharmacological activity and should be the subject of further study.
- Persistent elevated blood pressure parameters in the initial states of Group 3 rats (mephedrone), as well as their relative indicators after provocation tests, are caused by a disruption not only in the expression of NO (one of the main vasodilating factors regulating the work of microcirculatory vessels), but also prostacyclines and endothelial hyperpolarization factors (EDHF). The study results at this stage confirmed the theoretical assumption about the excessive negative effect of mephedrone on both the endothelium of the microcirculatory vessels and on the viability of the cardiovascular system as a whole. The data obtained can be used in planning preclinical studies to expand the spectrum of complications associated with 4-mmc use, as well as to build an algorithm of clinical recommendations for pharmacological correction of these complicated conditions.
- Taking into consideration that mephedrone likely reduces the perfusion permeability of the cell wall and the transport function of proteins, affects both passive and facilitated diffusion and active transport, affects cognitive patterns in a certain way (including delayed phenomena), and also has the ability to influence the coagulation component of hemostasis positively (hypercoagulation), it can be assumed that COX-2 activity is predominant as the main enzyme playing a role in the induction of the alternative phase of inflammation. Thus, the use of selective COX-2 inhibitors for treatment of inflammatory diseases in patients using 4-mmc is the most preferable and justified. However, for a precise definition of pharmacological treatment aimed at relieving the inflammatory processes associated with 4-mmc use, additional preclinical studies are necessary.
- The results at third stage of the study proved a decrease in the metabolic and absorption activity of neutrophils in rats belonging to Group 3 (intraperitoneal administration of 4-mmc) compared to the indicators of control group animals. This defines mephedrone as a substance that has immunosuppressive effect. This conclusion is the basis for further studies on pathogenetic corrective immunotherapy.
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