DOI 10.1007/s10517-015-2929-2 Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 PHARMACOLOGY AND TOXICOLOGY
229
Pharmacokinetic Properties of a New Glutamic Acid Derivative Glutaron L. A. Smirnova, A. F. Ryabukha, K. A. Kuznetsov, E. A. Suchkov, V. N. Perfilova, and I. N. Tyurenkov Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 2, pp. 188-191, February, 2015 Original article submitted January 9, 2014 Inhomogenous distribution of glutaron in organs and tissues was found after intravenous and peroral administration: the agent demonstrated high affinity to organs with high degree of vascularization (lungs and heart) and elimination (kidney). Glutaron easily penetrates through the blood-brain barrier, which is consistent with its concentration in the adipose tissue. Key Words: derivatives of glutamic acid; pharmacokinetics; tissue bioavailability; glutaron An important stage of preclinical testing of new drugs is the analysis of their properties pharmacokinetic, i.e. absorption, distribution, metabolism, and excretion of the substances from the body. Knowledge on distribution processes enable estimation of organs and tissues with the highest drug concentration or retention level, which helps to understand the mechanisms of its action [1,2,5]. Here we studied the distribution and tissue bioavailability of a new structural analogue of glutamic acid glutaron, the drug exhibiting antidepressant, anxiolytic, and neuroprotective properties [3,6].
MATERIALS AND METHODS Quantitative analysis was performed on a Shimadzu liquid chromatograph with a SUPELCOSIL LC-18 column (5 μm, 150×4.6 mm). Detection was conducted at λex=215 nm and λem=285 nm. Mobile phase included acetonitrile (UV 210) and buffer system containing 50 mM monobasic potassium phosphate (pH 2.1) and 0.06% heptanesulfonic acid [7,8]; acetonitrile:buffer ratio 10:90% (v/v). Extraction of glutaron and simultaneous protein precipitation from biological samples of rat plasma Laboratory of Pharmacological Kinetics, Department of Pharmacology and Biopharmaceutics of the Faculty of Continuing Medical Education, Volgograd State Medical University, Volgograd, Russia. Address for correspondence: [email protected]. A. F. Ryabukha
was performed using 10% trichloracetic acid (1:0.2). The level of glutaron extraction was not less than 90%. Method sensitivity was 0.5 μg/ml. Glutaron pharmacokinetics was studied on male rats (n=100) weighing 180-220 g. The experiments were conducted with strict adherence to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (1997). Glutaron was administered intravenously and orally in therapeutic dose of 26 mg/kg. The animals were decapitated. Samples of the blood and organs were taken 5, 15, 30 min, and 1, 2, 4, 8, 12, and 24 h after intravenous injection and 15, 30 min, 1, 2, 4, 8, 12, and 24 h after oral administration. The intensity of penetration of the substance into tissues was estimated by the index of tissue availability (ft) calculated as a ratio between AUC in the tissue (area under pharmacokinetic curve) and AUC in the blood. Model-independent pharmacokinetic parameters were calculated using a method of statistical moments for time interval from 0 to ∞ for both administration routes [4]. Experimental data were analyzed by Microsoft Excel software.
RESULTS Averaged pharmacokinetic profiles of substance concentration in rat blood plasma as a function of time
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Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 PHARMACOLOGY AND TOXICOLOGY
Fig. 1. Concentration of glutaron in rat plasma after intravenous administration in the dose of 26 mg/kg.
Fig. 2. Concentration of glutaron in rat plasma after oral administration in the dose of 26 mg/kg.
after its intravenous administration were obtained (Fig. 1). The concentration of glutaron (13.99 μg/ml) attained maximum in 5 min after injection and then decreased. The decrease was biexponential: fast distribution phase was followed by slow elimination phase. Elimination phase predominated up to 12 h, and only traces of glutaron were found in the plasma and organs 25 h after administration. The main pharmacokinetic parameters calculated from the concentration curve (Table 1) showed relatively low elimination half-time and mean retention time of one molecule of the agent in the body. Systemic clearance was rather low. The total distribution volume after intravenous injection surpassed the volume of extracellular fluid in the body by 3.5 times, which reflects intensive penetration of the substance into organs and tissue. After oral administration, glutaron was detected in blood plasma in 15 min. Pharmacokinetic profile had an absorption phase (concentration increases to 1.82 μg/ml in 30 min after administration; Fig. 2) and elimination phase (the agent was detected in the plasma over 24 h). Second concentration peak in 12 h after treatment can be related to hepatoduodenal circulation of glutaron. Pharmacokinetic parameters after oral administration of glutaron slightly differed from the corresponding values after intravenous injection (Table 1). Elimination half-time, mean retention time of one molecule, and distribution volume were higher after oral treatment. Inhomogeneity in glutaron distribution in organs and tissues was found after different administration routes. Glutaron crossed blood-brain barrier reaching maximum concentration (10.27 μg/g) in the brain in 5 min after administration, then this level decreased, and the second peak was observed 1 h after admin-
istration (7.46 μg/g). Then, this parameter gradually decreased over 12 h. Tissue bioavailability was higher after intravenous injection (Tables 2 and 3). Two concentration peaks were found in the heart after intravenous injection: one peak (28.44 μg/g) was detected 5 min after administration, the second peak (18.73 μg/g) was detected 40 min after treatment, then the concentration decreased, but was higher than detection limit over 12 h. Tissue availability was 2.31 after intravenous administration (Table 2), but was lower after oral treatment (Table 3). Similar curve was obtained for the spleen (Table 2). The level of glutaron was high in the lungs, maximum concentration was observed 10 min after administration (62.68 μg/g). Tissue availability was 2.95 after intravenous administration (Table 2). Glutaron concentration in muscles was similar to that in the blood: peaked in 5 min after administration and remained above the detection limit TABLE 1. Pharmacokinetic Parameters of Glutaron in Rat Blood Plasma after Intravenous and Oral Administration in a Dose of 26 mg/kg Parameter
AUC, μg×h/ml Kel, h
–1
Intravenous administration
Oral administration
41.18
34.52
0.185
0.051
Т1/2, h
3.75
13.61
MRT, h
5.77
15.37
Cl, liter/(h/kg)
0.63
0.75
Vd, liter/kg
3.42
14.79
Note. T1/2, elimination half-time; MRT, retention time for one molecule; Cl, systemic clearance; Vd, total distribution volume; Kel, elimination constant.
L. A. Smirnova, A. F. Ryabukha, et al.
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TABLE 2. Pharmacokinetic Parameters of Glutaron Distribution in Organs and Tissues after Intravenous Administration in a Dose of 26 mg/kg Organ
AUC, μg×h/ml
Tissue availability (ft)
Brain
38.75
0.94
Heart
95.23
2.31
Spleen
23.07
0.56
Lungs
121.53
2.95
Muscles
47.09
1.14
Kidneys
197.89
4.81
Liver
49.47
1.2
Omentum
109.29
2.65
TABLE 3. Pharmacokinetic Parameters of Glutaron Distribution in Organs and Tissues after Oral Administration in a Dose of 26 mg/kg Organ
AUC, μg×h/ml
Tissue availability (ft)
Brain
13.58
0.28
Heart
16.94
0.34
Kidneys
20.4
0.41
Liver
66.03
1.34
over 8 h. Tissue availability was 1.14 after intravenous administration (Table 2). In the omentum, glutaron concentration increased gradually, and was maximum (22.61 μg/g) 4 h after administration. Tissue availability was 2.65 (Table 2). After oral administration, glutaron concentrations in the spleen, lungs, muscles, and omentum were below the detection limit. In the kidneys and liver, the drug concentration was high, and its maximum in the kidneys (145.57 μg/g) was detected in 5 min after treatment. Tissue availability was 4.81 after intravenous injection and 0.41 after oral administration. The
maximum concentration in the liver (32.97 μg/g) was detected also in 5 min after treatment. Tissue availability differed after intravenous and oral administration (Tables 2, 3). The distribution of glutaron in organs and tissues correlated with previous data on substance excretion. Glutaron was present in the urine for >72 h. Maximum excretion was found on day 2 after administration. Renal clearance was 123 ml/h, nonrenal clearance was 34.84 ml/h. Thus, glutaron was detected in all organs and tissues over at least 12 h after administration. The distribution of the agent was inhomogenous. Pharmacokinetic profiles of glutaron in organs and tissues and blood plasma were similar. The substance has maximum affinity to the kidneys and high affinity to the lungs and heart. It can be suggested that the pattern of distribution of the test agent mostly depended on the level of vascularization of organs and was determined by physical and chemical properties of the substance. The greater part of unchanged glutaron was excreted by the kidneys.
REFERENCES 1. V. P. Zherdev, S. S. Boiko, N. V. Mesonzhnik, et al., Eksp. Klin. Farmakol., 72, No. 3, 16-21 (2009). 2. M. V. Karlina, O. N. Pozharitskaya, V. M. Kosman, et al., Eksp. Klin. Farmakol., 73, No. 8, 46-48 (2010). 3. V. I. Petrov, I. N. Tyurenkov, V. V. Bagmetova, et al., Patent RF No. 2429834, Substance with Antidepressive, Anxiolytic, Neuroprotective, and Immunostimulating Effects, Byull., No. 27, September 27, 2011. 4. Manual for Performance of Preclinical Studies [in Russian], Ed. A. N. Mironov, Tula (2012). 5. A. A. Spasov, L. A. Smirnova, I. N. Iyozhitsa, et al., Vopr. Med. Khimii, 48, No. 3, 233-258 (2002). 6. I. N. Tyurenkov, V. V. Bagmetova, Yu. V. Chernysheva, et al., Vestn. Vol. Gos. Med. Univer., No. 3, 21-24 (2012). 7. I. N. Tyurenkov, V. N. Perfilova, L. A. Smirnova, et al., Khim. Farm. Zh., 44, No. 12, 53-56 (2010). 8. I. N. Tyurenkov, V. N. Perfilova, L. A. Smirnova, et al., Khim. Farm. Zh., 47, No. 3, 55-56 (2013).
229
Pharmacokinetic Properties of a New Glutamic Acid Derivative Glutaron L. A. Smirnova, A. F. Ryabukha, K. A. Kuznetsov, E. A. Suchkov, V. N. Perfilova, and I. N. Tyurenkov Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 159, No. 2, pp. 188-191, February, 2015 Original article submitted January 9, 2014 Inhomogenous distribution of glutaron in organs and tissues was found after intravenous and peroral administration: the agent demonstrated high affinity to organs with high degree of vascularization (lungs and heart) and elimination (kidney). Glutaron easily penetrates through the blood-brain barrier, which is consistent with its concentration in the adipose tissue. Key Words: derivatives of glutamic acid; pharmacokinetics; tissue bioavailability; glutaron An important stage of preclinical testing of new drugs is the analysis of their properties pharmacokinetic, i.e. absorption, distribution, metabolism, and excretion of the substances from the body. Knowledge on distribution processes enable estimation of organs and tissues with the highest drug concentration or retention level, which helps to understand the mechanisms of its action [1,2,5]. Here we studied the distribution and tissue bioavailability of a new structural analogue of glutamic acid glutaron, the drug exhibiting antidepressant, anxiolytic, and neuroprotective properties [3,6].
MATERIALS AND METHODS Quantitative analysis was performed on a Shimadzu liquid chromatograph with a SUPELCOSIL LC-18 column (5 μm, 150×4.6 mm). Detection was conducted at λex=215 nm and λem=285 nm. Mobile phase included acetonitrile (UV 210) and buffer system containing 50 mM monobasic potassium phosphate (pH 2.1) and 0.06% heptanesulfonic acid [7,8]; acetonitrile:buffer ratio 10:90% (v/v). Extraction of glutaron and simultaneous protein precipitation from biological samples of rat plasma Laboratory of Pharmacological Kinetics, Department of Pharmacology and Biopharmaceutics of the Faculty of Continuing Medical Education, Volgograd State Medical University, Volgograd, Russia. Address for correspondence: [email protected]. A. F. Ryabukha
was performed using 10% trichloracetic acid (1:0.2). The level of glutaron extraction was not less than 90%. Method sensitivity was 0.5 μg/ml. Glutaron pharmacokinetics was studied on male rats (n=100) weighing 180-220 g. The experiments were conducted with strict adherence to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (1997). Glutaron was administered intravenously and orally in therapeutic dose of 26 mg/kg. The animals were decapitated. Samples of the blood and organs were taken 5, 15, 30 min, and 1, 2, 4, 8, 12, and 24 h after intravenous injection and 15, 30 min, 1, 2, 4, 8, 12, and 24 h after oral administration. The intensity of penetration of the substance into tissues was estimated by the index of tissue availability (ft) calculated as a ratio between AUC in the tissue (area under pharmacokinetic curve) and AUC in the blood. Model-independent pharmacokinetic parameters were calculated using a method of statistical moments for time interval from 0 to ∞ for both administration routes [4]. Experimental data were analyzed by Microsoft Excel software.
RESULTS Averaged pharmacokinetic profiles of substance concentration in rat blood plasma as a function of time
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Bulletin of Experimental Biology and Medicine, Vol. 159, No. 2, June, 2015 PHARMACOLOGY AND TOXICOLOGY
Fig. 1. Concentration of glutaron in rat plasma after intravenous administration in the dose of 26 mg/kg.
Fig. 2. Concentration of glutaron in rat plasma after oral administration in the dose of 26 mg/kg.
after its intravenous administration were obtained (Fig. 1). The concentration of glutaron (13.99 μg/ml) attained maximum in 5 min after injection and then decreased. The decrease was biexponential: fast distribution phase was followed by slow elimination phase. Elimination phase predominated up to 12 h, and only traces of glutaron were found in the plasma and organs 25 h after administration. The main pharmacokinetic parameters calculated from the concentration curve (Table 1) showed relatively low elimination half-time and mean retention time of one molecule of the agent in the body. Systemic clearance was rather low. The total distribution volume after intravenous injection surpassed the volume of extracellular fluid in the body by 3.5 times, which reflects intensive penetration of the substance into organs and tissue. After oral administration, glutaron was detected in blood plasma in 15 min. Pharmacokinetic profile had an absorption phase (concentration increases to 1.82 μg/ml in 30 min after administration; Fig. 2) and elimination phase (the agent was detected in the plasma over 24 h). Second concentration peak in 12 h after treatment can be related to hepatoduodenal circulation of glutaron. Pharmacokinetic parameters after oral administration of glutaron slightly differed from the corresponding values after intravenous injection (Table 1). Elimination half-time, mean retention time of one molecule, and distribution volume were higher after oral treatment. Inhomogeneity in glutaron distribution in organs and tissues was found after different administration routes. Glutaron crossed blood-brain barrier reaching maximum concentration (10.27 μg/g) in the brain in 5 min after administration, then this level decreased, and the second peak was observed 1 h after admin-
istration (7.46 μg/g). Then, this parameter gradually decreased over 12 h. Tissue bioavailability was higher after intravenous injection (Tables 2 and 3). Two concentration peaks were found in the heart after intravenous injection: one peak (28.44 μg/g) was detected 5 min after administration, the second peak (18.73 μg/g) was detected 40 min after treatment, then the concentration decreased, but was higher than detection limit over 12 h. Tissue availability was 2.31 after intravenous administration (Table 2), but was lower after oral treatment (Table 3). Similar curve was obtained for the spleen (Table 2). The level of glutaron was high in the lungs, maximum concentration was observed 10 min after administration (62.68 μg/g). Tissue availability was 2.95 after intravenous administration (Table 2). Glutaron concentration in muscles was similar to that in the blood: peaked in 5 min after administration and remained above the detection limit TABLE 1. Pharmacokinetic Parameters of Glutaron in Rat Blood Plasma after Intravenous and Oral Administration in a Dose of 26 mg/kg Parameter
AUC, μg×h/ml Kel, h
–1
Intravenous administration
Oral administration
41.18
34.52
0.185
0.051
Т1/2, h
3.75
13.61
MRT, h
5.77
15.37
Cl, liter/(h/kg)
0.63
0.75
Vd, liter/kg
3.42
14.79
Note. T1/2, elimination half-time; MRT, retention time for one molecule; Cl, systemic clearance; Vd, total distribution volume; Kel, elimination constant.
L. A. Smirnova, A. F. Ryabukha, et al.
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TABLE 2. Pharmacokinetic Parameters of Glutaron Distribution in Organs and Tissues after Intravenous Administration in a Dose of 26 mg/kg Organ
AUC, μg×h/ml
Tissue availability (ft)
Brain
38.75
0.94
Heart
95.23
2.31
Spleen
23.07
0.56
Lungs
121.53
2.95
Muscles
47.09
1.14
Kidneys
197.89
4.81
Liver
49.47
1.2
Omentum
109.29
2.65
TABLE 3. Pharmacokinetic Parameters of Glutaron Distribution in Organs and Tissues after Oral Administration in a Dose of 26 mg/kg Organ
AUC, μg×h/ml
Tissue availability (ft)
Brain
13.58
0.28
Heart
16.94
0.34
Kidneys
20.4
0.41
Liver
66.03
1.34
over 8 h. Tissue availability was 1.14 after intravenous administration (Table 2). In the omentum, glutaron concentration increased gradually, and was maximum (22.61 μg/g) 4 h after administration. Tissue availability was 2.65 (Table 2). After oral administration, glutaron concentrations in the spleen, lungs, muscles, and omentum were below the detection limit. In the kidneys and liver, the drug concentration was high, and its maximum in the kidneys (145.57 μg/g) was detected in 5 min after treatment. Tissue availability was 4.81 after intravenous injection and 0.41 after oral administration. The
maximum concentration in the liver (32.97 μg/g) was detected also in 5 min after treatment. Tissue availability differed after intravenous and oral administration (Tables 2, 3). The distribution of glutaron in organs and tissues correlated with previous data on substance excretion. Glutaron was present in the urine for >72 h. Maximum excretion was found on day 2 after administration. Renal clearance was 123 ml/h, nonrenal clearance was 34.84 ml/h. Thus, glutaron was detected in all organs and tissues over at least 12 h after administration. The distribution of the agent was inhomogenous. Pharmacokinetic profiles of glutaron in organs and tissues and blood plasma were similar. The substance has maximum affinity to the kidneys and high affinity to the lungs and heart. It can be suggested that the pattern of distribution of the test agent mostly depended on the level of vascularization of organs and was determined by physical and chemical properties of the substance. The greater part of unchanged glutaron was excreted by the kidneys.
REFERENCES 1. V. P. Zherdev, S. S. Boiko, N. V. Mesonzhnik, et al., Eksp. Klin. Farmakol., 72, No. 3, 16-21 (2009). 2. M. V. Karlina, O. N. Pozharitskaya, V. M. Kosman, et al., Eksp. Klin. Farmakol., 73, No. 8, 46-48 (2010). 3. V. I. Petrov, I. N. Tyurenkov, V. V. Bagmetova, et al., Patent RF No. 2429834, Substance with Antidepressive, Anxiolytic, Neuroprotective, and Immunostimulating Effects, Byull., No. 27, September 27, 2011. 4. Manual for Performance of Preclinical Studies [in Russian], Ed. A. N. Mironov, Tula (2012). 5. A. A. Spasov, L. A. Smirnova, I. N. Iyozhitsa, et al., Vopr. Med. Khimii, 48, No. 3, 233-258 (2002). 6. I. N. Tyurenkov, V. V. Bagmetova, Yu. V. Chernysheva, et al., Vestn. Vol. Gos. Med. Univer., No. 3, 21-24 (2012). 7. I. N. Tyurenkov, V. N. Perfilova, L. A. Smirnova, et al., Khim. Farm. Zh., 44, No. 12, 53-56 (2010). 8. I. N. Tyurenkov, V. N. Perfilova, L. A. Smirnova, et al., Khim. Farm. Zh., 47, No. 3, 55-56 (2013).
