DOI 10.1007/s10517-014-2655-1 Bulletin of Experimental Biology and Medicine, Vol. 157, No. 6, October, 2014 PHARMACOLOGY AND TOXICOLOGY
735
Comparison of Pharmacokinetics and Relative Bioavailability of Tablets and Substance of New Dipeptide Neuroleptic Dilept R. V. Shevchenko, A. A. Litvin, G. B. Kolyvanov, V. P. Zherdev, and V. V. Smirnov Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 6, pp. 704-706, June, 2014 Original article submitted July 10, 2013 We studied pharmacokinetics of dilept after single cross-administration of tablets and substance of dilept in a dose of 40 mg to rabbits. The following pharmacokinetic parameters were calculated: maximum plasma concentration of dilept, time to maximum observed concentration, area under the pharmacokinetic curve, elimination half-life, and relative bioavailability. Dilept concentration in blood plasma was estimated using ultra-fast liquid chromatography with mass spectrometry detection. Relative bioavailability of dilept tablets was 93.46±28.91% of that for the substance. Key Words: dilept; short peptides; pharmacokinetics; ultra-fast liquid chromatography
Dilept is an original agent with antipsychotic properties developed in V. V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences. Dilpet, N-caproyl-L-prolyl-L-tyrosine methyl ether, is a modified dipeptide mimicking the structure of atypical neuroleptic sulpiride and the head of β-reverse structure of neurotensin (HT8-13) [2]. Here we studied relative bioavailability of dilept in rabbits after administration of tablets and substance of this agent in a single dose of 40 mg.
MATERIALS AND METHODS Pharmacokinetic studies were performed on 6 male Chinchilla rabbits (Stolbovaya Breeding Center, Russian Academy of Medical Sciences, Moscow Region) weighting 3.34±0.18 kg. The animals were kept under standard vivarium conditions at 12-h light/dark regimen and deprived of food 12 h before the experiment. Each experimental animal had its own experimental number. The animals randomly received 2 tablets of Laboratory of Pharmacokinetics, V. V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia. Address for correspondence: [email protected]. R. V. Shevchenko
dilept (2×20=40 mg) or the substance (40 mg, suspension containing 1% starch gel) through a tube. After 10 days (wash-out period), the order of administration was changed. Blood samples (1.5 ml) were taken from the auricular vein with needles and transferred to conic polyethylene tubes preliminary treated with K3-EDTA. Blood samples for measuring of agent concentration in blood plasma were taken in 0, 0.25, 0.5, 0.45, 1.0, 2.0, 4.0, and 6.0 h of the experiment. The samples were centrifuged for 15 min at 3000g and 4ºC. Blood plasma was placed into sterile plastic tubes and stored at -24ºC. During the preparation of biological samples, hydrophobic substances of blood plasma (lipids) were removed by hexane extraction. To this end, 0.5 ml hexane was added to the plasma (1:2 v/v), vortexed for 5 min, and centrifuged for layer separation. Hexane layer was wasted and water layer was used for further experiments. Acetic acid (0.3 ml, 0.7%) was added to water phase for making of optimal extraction pH. Dilept was extracted with 3.0 ml chloroform during vortexing for 15 min. After centrifugation (8000g, 15 min, 20ºC), the organic layer was removed and exsiccated under nitrogen at 60ºC [1]. Dry residue was dissolved in 200 μl acetonitrile and analyzed us-
0007-4888/14/15760735 © 2014 Springer Science+Business Media New York
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 6, October, 2014 PHARMACOLOGY AND TOXICOLOGY
736
ing ultra-fast liquid chromatography (UFLC) with tandem mass spectrometry detection. Chromatography was performed using Zorbax XDB-C18 analytical column (4.6×50 mm; particle size of sorbent 5 μ; Agilent Technologies). Mobile phase consisted of an acetonitrile:deionized water (300:200) mixture. UFLC analysis was performed by isocratic elution. Flow rate of mobile phase was 0.5 ml/min, volume of injected sample was 10 μl. Statistical analysis of the obtained data was performed. Non-compartmental model (R software) was used for calculations of pharmacokinetic parameters.
RESULTS Mean pharmacokinetic parameters of dilept in rabbit blood plasma after single administration of tablets and substance are presented in Figure 1. The concentration of the test agent significantly varied (CV%, up to 65%). However, the concentration of unchanged substance was measured in all animals and complete individual pharmacokinetic profiles and pharmacokinetic parameters were determined. Dilept was assayed in rabbit blood plasma over 4 h after administration of the tablets or substance. Pharmacokinetic parameters of dilept after single administration of tablets or substance to rabbits are presented in Table 1.
Fig. 1. Mean pharmacokinetics profiles of dilept in rabbit blood plasma after oral administration of tablets (1) or substance (2) in a single dose of 40 mg. n=6.
Analysis of absorption phase showed that mean time to maximum concentration (tmax) in rabbit blood plasma was 0.75±0.16 h after tablet administration and 0.79±0.10 h after substance administration. Maximum concentration (Cmax) was 3.59±1.94 and 3.10±1.21 ng/ml, respectively. tmax and Cmax of tablets and substance of dilept did not significantly differ. Dosage form also did not affect the elimination kinetics of dilept. The mean elimination rate constant (kel) of
TABLE 1. Pharmacokinetic Parameters of Dilept after Administration of Tablets or Substance of Dilept in a Single Dose of 40 mg to Rabbits AUC0-t, ng/ml×h
AUC0-∞, ng/ml×h
tmax, h
Cmax, ng/ml
kel, h–1
T1/2el, h
MRT, h
Tablets
4.04
4.41
0.75
3.59
0.692
1.06
1.46
Substance
4.41
4.96
0.79
3.10
0.621
1.13
1.50
Dilept form
TABLE 2. Relative Bioavailability of Dilept Tablets in Rabbits Animal number
AUC0-t tablets, ng/ml×h
AUC0-t substance, ng/ml×h
f, %
AUC0-∞ tablets, ng/ml×h
AUC0-∞ substance, ng/ml×h
f, %
1
2.32
2.23
104.04
2.54
2.61
97.32
2
3.22
2.91
110.65
3.86
3.20
120.63
3
7.31
5.35
136.64
7.51
5.89
127.50
4
3.84
5.04
76.19
4.25
6.02
70.60
5
5.3
7.00
75.71
5.69
7.64
74.48
6
2.25
3.91
57.54
2.62
4.40
59.55
X
4.04
4.41
93.46
4.41
4.96
91.68
SD
1.96
1.75
28.90
1.91
1.90
28.02
CV%
48.45
39.62
30.92
43.34
38.36
30.57
R. V. Shevchenko, A. A. Litvin, et al.
dilept was 0.692±0.188 h–1 in rabbits receiving tablets and 0.621±0.070 h–1 in rabbits receiving the substance. Half-elimination period (T1/2el) of dilept from the plasma was 1.06±0.26 and 1.13±0.12 h after administration of tablets and substance, respectively. Mean retention time (MRT) of dilept in rabbit organism was 1.46±0.26 (tablets) and 1.50±0.13 h (substance). High total clearance (CL=9.26 liter/h) reflects the significant contribution of biotransformation into the process of dilept elimination from the body. Similar data on dilept pharmacokinetics were observed in healthy volunteers [3]. Comparison of relative bioavailability of tablets and substance of dilept showed that relative bioavailability (f) of dilept tablets was 93.46±28.91% (calculated using the area under curve “dilept concentration
737 in blood plasma – time (0-6h)”, AUC0-t; Table 2). This parameter (f’) was 91.86±28.02% if the calculations were performed using AUC0-∞ (area under curve from 0 to infinity). Thus, the obtained data suggest that dilept tablets can be recommended for the second stage of clinical studies.
REFERENCES 1. N. V: Arkhipenko, S. A. Appolonova, T. G. Sobolevskii, et al., Khim.-Farm. Zh., 43, No. 5, 53-56 (2009). 2. S. B. Seredenin, T. A. Voronina, T. A. Gudasheva, et al., Patent RF No. 2091390, Substituted Prolyl-Tyrosine with Psychotropic Activity, Byull. No. 27. 3. R. V. Shevchenko, A. A. Litvin, G. B. Kolyvanov, et al., Eksp. Klin. Farmakol., 76, No. 6, 34-37 (2013).
735
Comparison of Pharmacokinetics and Relative Bioavailability of Tablets and Substance of New Dipeptide Neuroleptic Dilept R. V. Shevchenko, A. A. Litvin, G. B. Kolyvanov, V. P. Zherdev, and V. V. Smirnov Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 157, No. 6, pp. 704-706, June, 2014 Original article submitted July 10, 2013 We studied pharmacokinetics of dilept after single cross-administration of tablets and substance of dilept in a dose of 40 mg to rabbits. The following pharmacokinetic parameters were calculated: maximum plasma concentration of dilept, time to maximum observed concentration, area under the pharmacokinetic curve, elimination half-life, and relative bioavailability. Dilept concentration in blood plasma was estimated using ultra-fast liquid chromatography with mass spectrometry detection. Relative bioavailability of dilept tablets was 93.46±28.91% of that for the substance. Key Words: dilept; short peptides; pharmacokinetics; ultra-fast liquid chromatography
Dilept is an original agent with antipsychotic properties developed in V. V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences. Dilpet, N-caproyl-L-prolyl-L-tyrosine methyl ether, is a modified dipeptide mimicking the structure of atypical neuroleptic sulpiride and the head of β-reverse structure of neurotensin (HT8-13) [2]. Here we studied relative bioavailability of dilept in rabbits after administration of tablets and substance of this agent in a single dose of 40 mg.
MATERIALS AND METHODS Pharmacokinetic studies were performed on 6 male Chinchilla rabbits (Stolbovaya Breeding Center, Russian Academy of Medical Sciences, Moscow Region) weighting 3.34±0.18 kg. The animals were kept under standard vivarium conditions at 12-h light/dark regimen and deprived of food 12 h before the experiment. Each experimental animal had its own experimental number. The animals randomly received 2 tablets of Laboratory of Pharmacokinetics, V. V. Zakusov Research Institute of Pharmacology, Russian Academy of Medical Sciences, Moscow, Russia. Address for correspondence: [email protected]. R. V. Shevchenko
dilept (2×20=40 mg) or the substance (40 mg, suspension containing 1% starch gel) through a tube. After 10 days (wash-out period), the order of administration was changed. Blood samples (1.5 ml) were taken from the auricular vein with needles and transferred to conic polyethylene tubes preliminary treated with K3-EDTA. Blood samples for measuring of agent concentration in blood plasma were taken in 0, 0.25, 0.5, 0.45, 1.0, 2.0, 4.0, and 6.0 h of the experiment. The samples were centrifuged for 15 min at 3000g and 4ºC. Blood plasma was placed into sterile plastic tubes and stored at -24ºC. During the preparation of biological samples, hydrophobic substances of blood plasma (lipids) were removed by hexane extraction. To this end, 0.5 ml hexane was added to the plasma (1:2 v/v), vortexed for 5 min, and centrifuged for layer separation. Hexane layer was wasted and water layer was used for further experiments. Acetic acid (0.3 ml, 0.7%) was added to water phase for making of optimal extraction pH. Dilept was extracted with 3.0 ml chloroform during vortexing for 15 min. After centrifugation (8000g, 15 min, 20ºC), the organic layer was removed and exsiccated under nitrogen at 60ºC [1]. Dry residue was dissolved in 200 μl acetonitrile and analyzed us-
0007-4888/14/15760735 © 2014 Springer Science+Business Media New York
Bulletin of Experimental Biology and Medicine, Vol. 157, No. 6, October, 2014 PHARMACOLOGY AND TOXICOLOGY
736
ing ultra-fast liquid chromatography (UFLC) with tandem mass spectrometry detection. Chromatography was performed using Zorbax XDB-C18 analytical column (4.6×50 mm; particle size of sorbent 5 μ; Agilent Technologies). Mobile phase consisted of an acetonitrile:deionized water (300:200) mixture. UFLC analysis was performed by isocratic elution. Flow rate of mobile phase was 0.5 ml/min, volume of injected sample was 10 μl. Statistical analysis of the obtained data was performed. Non-compartmental model (R software) was used for calculations of pharmacokinetic parameters.
RESULTS Mean pharmacokinetic parameters of dilept in rabbit blood plasma after single administration of tablets and substance are presented in Figure 1. The concentration of the test agent significantly varied (CV%, up to 65%). However, the concentration of unchanged substance was measured in all animals and complete individual pharmacokinetic profiles and pharmacokinetic parameters were determined. Dilept was assayed in rabbit blood plasma over 4 h after administration of the tablets or substance. Pharmacokinetic parameters of dilept after single administration of tablets or substance to rabbits are presented in Table 1.
Fig. 1. Mean pharmacokinetics profiles of dilept in rabbit blood plasma after oral administration of tablets (1) or substance (2) in a single dose of 40 mg. n=6.
Analysis of absorption phase showed that mean time to maximum concentration (tmax) in rabbit blood plasma was 0.75±0.16 h after tablet administration and 0.79±0.10 h after substance administration. Maximum concentration (Cmax) was 3.59±1.94 and 3.10±1.21 ng/ml, respectively. tmax and Cmax of tablets and substance of dilept did not significantly differ. Dosage form also did not affect the elimination kinetics of dilept. The mean elimination rate constant (kel) of
TABLE 1. Pharmacokinetic Parameters of Dilept after Administration of Tablets or Substance of Dilept in a Single Dose of 40 mg to Rabbits AUC0-t, ng/ml×h
AUC0-∞, ng/ml×h
tmax, h
Cmax, ng/ml
kel, h–1
T1/2el, h
MRT, h
Tablets
4.04
4.41
0.75
3.59
0.692
1.06
1.46
Substance
4.41
4.96
0.79
3.10
0.621
1.13
1.50
Dilept form
TABLE 2. Relative Bioavailability of Dilept Tablets in Rabbits Animal number
AUC0-t tablets, ng/ml×h
AUC0-t substance, ng/ml×h
f, %
AUC0-∞ tablets, ng/ml×h
AUC0-∞ substance, ng/ml×h
f, %
1
2.32
2.23
104.04
2.54
2.61
97.32
2
3.22
2.91
110.65
3.86
3.20
120.63
3
7.31
5.35
136.64
7.51
5.89
127.50
4
3.84
5.04
76.19
4.25
6.02
70.60
5
5.3
7.00
75.71
5.69
7.64
74.48
6
2.25
3.91
57.54
2.62
4.40
59.55
X
4.04
4.41
93.46
4.41
4.96
91.68
SD
1.96
1.75
28.90
1.91
1.90
28.02
CV%
48.45
39.62
30.92
43.34
38.36
30.57
R. V. Shevchenko, A. A. Litvin, et al.
dilept was 0.692±0.188 h–1 in rabbits receiving tablets and 0.621±0.070 h–1 in rabbits receiving the substance. Half-elimination period (T1/2el) of dilept from the plasma was 1.06±0.26 and 1.13±0.12 h after administration of tablets and substance, respectively. Mean retention time (MRT) of dilept in rabbit organism was 1.46±0.26 (tablets) and 1.50±0.13 h (substance). High total clearance (CL=9.26 liter/h) reflects the significant contribution of biotransformation into the process of dilept elimination from the body. Similar data on dilept pharmacokinetics were observed in healthy volunteers [3]. Comparison of relative bioavailability of tablets and substance of dilept showed that relative bioavailability (f) of dilept tablets was 93.46±28.91% (calculated using the area under curve “dilept concentration
737 in blood plasma – time (0-6h)”, AUC0-t; Table 2). This parameter (f’) was 91.86±28.02% if the calculations were performed using AUC0-∞ (area under curve from 0 to infinity). Thus, the obtained data suggest that dilept tablets can be recommended for the second stage of clinical studies.
REFERENCES 1. N. V: Arkhipenko, S. A. Appolonova, T. G. Sobolevskii, et al., Khim.-Farm. Zh., 43, No. 5, 53-56 (2009). 2. S. B. Seredenin, T. A. Voronina, T. A. Gudasheva, et al., Patent RF No. 2091390, Substituted Prolyl-Tyrosine with Psychotropic Activity, Byull. No. 27. 3. R. V. Shevchenko, A. A. Litvin, G. B. Kolyvanov, et al., Eksp. Klin. Farmakol., 76, No. 6, 34-37 (2013).
