European Journal of Clinical Pharmacology
Europ. J. din. Pharmacol. 12, 355-357 (1977)
© by Springer:Verlag 1977
Transfer of Nitrazepam Across the Human Placenta L. Kangas, J. Kanto, and R. Erkkola Department of Pharmacology, University of Turku and Department of Obstetrics and Gyneacology, University Central Hospital, Turku, Finland
Summary. Six women from 14 to 17 weeks pregnant, and 12 woman from 36 to 40 weeks pregnant, were given nitrazepam 5 mg orally about 12 h before legal abortion by hysterotomy in the former group and elective caesarean section in the latter group. The concentration of nitrazepam was determined by gas-liquid chromatography. Binding to plasma proteins was evaluated by separation of the protein-free fraction by ultracentrifugation. In the first group (early pregnancy) the level of nitrazepam was found to be lower in the fetal than in the maternal drculation. The concentration in amniotic fluid was still lower. In the latter group (late pregnancy) the concentration both of unbound and total nitrazepam in maternal and fetal plasma were in equilibrium, which indicated an increase in transplacental transfer in late pregnancy. The percentage of unbound nitrazepam in both cases was 12%.
Key words: Nitrazepam, placental transfer, pharmacokinetics, plasma levels, protein binding
Nitrazepam is one of the most widely used hypnotics in Europe. Its toxicity has been proved to be low and it has a wide dose range for safe use. Nitrazepam is often administered during pregnancy for sleep disturbances. As far as is known, no clinical studies of its placental transfer in humans have been made. The aim of the present work was to investigate the fetal and maternal plasma concentration of nitrazepam in early and late pregnancy. Special interest was taken in the concentrations of free, protein unbound drug.
Subjects and Methods Six women, 14-17 weeks pregnant, were admitted to hospital for legal abortion. Their mean age was 22.0_+3.4 years and weight 59_+10.5kg (mean + S.D.). In the evening prior to hysterotomy they were given nitrazepam 5 mg orally (Mogadon ®, Hoffmann la Roche, Basle, Switzerland). As premedication they received atropine 0.5mg and pethidine 50 mg i. m. Anaesthesia was induced with i. v. thiopentone (dose up to 350 mg) and was maintained with 70/30 N20/02 mixture. Relaxation was obtained with a 50-100 mg bolus of succinylcholine and was maintained with an infusion of succinylcholine in glucose 5%. During the hysterotomy a sample of amniotic fluid was obtained (AF, n = 5). Blood samples from the maternal cubital vein (MV 1) and umbilical cord (UC) were taken after the fetus had been removed. Elective caesarean section was done in twelve women, aged 28.8_+7.5 years, weighing 74.1_+ 13.0 kg (mean _+ S. D.), who had been pregnant for 36 to 40 weeks. Each received an oral dose of nitrazepam 5 mg (Mogadon ®) in the evening prior to the operation. In the course of the operation blood samples were collected simultaneously from the maternal cubital vein (MV 2), fetal umbilical vein (UV) and from the umbilical artery (UA). All the women were informed about the study and they gave their written consent to it. Blood samples were immediately centrifuged and the plasma separated. The erythrocytes were lysed with distilled water according to Zingales (1969). Protein bound and free drug were separated by ultracentrifugation of 10 ml plasma at 407000 × g, at 37°C, according to Kanto et al. (1975). The concentration of nitrazepam was determined by
356
L Kangas et al.: Nitrazepam Transfer Across the Placenta
Table 1. Concentration of nitrazepam (ng/ml) in maternal plasma (MV 1), the umbilical circulation (UC) and amniotic fluid (AF) in early pregnancy Subject
MV 1
UC
AF
SO LL LR KR LV SB
23 28 34 30 12 38
15 11 26 18 10 11
12 6 16 6 4 - (no sample)
mean S.D.
27.5 9.2
15.2 6.1
8.8 5.0
Table 2. Concentration of nitrazepam (ng/ml) in maternal plasma (MV 2), umbilical vein (UV) and umbilical artery (UA) in late pregnancy, and the protein unbound fraction of nitrazepam (ng/ml and percentage of total) in plasma from a maternal and the umbilical vein Subject
Nitrazepam (total) in plasma (ng/ml)
Protein unbound nitrazepam (ng/ml)
Protein unbound nitrazepam (percentage of total) MV2
UV
13.2 15.6 14.1 11.0 10.6 9.6 9.6 12.5 12.8 9.6
11.7 10.7 12.4 8.6 13,5 14.1 10.8 13.2 13.9 10.8
11.9 2.1
12.0 1,8
MV2 UV
UA
MV2 UV
SU SA HE AN LU LA OL VE RA HA RE HL
34 31 22 16 17 21 36 28 26 28 25 20
26 22 23 14 21 21 31 17 24 28 23 24
37 22 19 18 23 24 33 16 26 25 19 18
. . . . . . 2.9 2.7 2.5 1.5 2.4 2.6 2.3 1.8 3.8 4.2 2.7 2.4 2.5 2.6 3.5 3.7 3.2 3.2 2.5 2.6
mean S.D.
25.3 6.4
22.8 4.5
23.3 6.3
2.8 0.51
2.7 0.81
. .
63Ni-EC gas-liquid chromatography (Kangas, 1977). Before analysis nitrazepam was hydrolyzed with 6 N H2SO 4 to the corresponding benzophenone in order to attain high sensitivity in the protein binding studies. The recovery of the method was 96.8+3.3% (mean + S.D.), the sensitivity 0.5 ng/ml and the coefficient of variation of interday analyses was 6.3%. The sensitivity in the red cell samples was 2 ng/ml, due to the high frequency of impurities found in them. Plasma 0.5 ml, washed red cells 1.5 ml and protein free supernatant 1.0 ml was used for each determination. Statistical analysis was performed by Student's t-test.
Results
In the six cases of early pregnancy (14-17 weeks) the concentration of nitrazepam in MV 1 was 27.5+9.2 ng/ml (mean _+ S. D.), which was significantly higher than in UC (15.2 _+6.1 ng/ml, p
Europ. J. din. Pharmacol. 12, 355-357 (1977)
© by Springer:Verlag 1977
Transfer of Nitrazepam Across the Human Placenta L. Kangas, J. Kanto, and R. Erkkola Department of Pharmacology, University of Turku and Department of Obstetrics and Gyneacology, University Central Hospital, Turku, Finland
Summary. Six women from 14 to 17 weeks pregnant, and 12 woman from 36 to 40 weeks pregnant, were given nitrazepam 5 mg orally about 12 h before legal abortion by hysterotomy in the former group and elective caesarean section in the latter group. The concentration of nitrazepam was determined by gas-liquid chromatography. Binding to plasma proteins was evaluated by separation of the protein-free fraction by ultracentrifugation. In the first group (early pregnancy) the level of nitrazepam was found to be lower in the fetal than in the maternal drculation. The concentration in amniotic fluid was still lower. In the latter group (late pregnancy) the concentration both of unbound and total nitrazepam in maternal and fetal plasma were in equilibrium, which indicated an increase in transplacental transfer in late pregnancy. The percentage of unbound nitrazepam in both cases was 12%.
Key words: Nitrazepam, placental transfer, pharmacokinetics, plasma levels, protein binding
Nitrazepam is one of the most widely used hypnotics in Europe. Its toxicity has been proved to be low and it has a wide dose range for safe use. Nitrazepam is often administered during pregnancy for sleep disturbances. As far as is known, no clinical studies of its placental transfer in humans have been made. The aim of the present work was to investigate the fetal and maternal plasma concentration of nitrazepam in early and late pregnancy. Special interest was taken in the concentrations of free, protein unbound drug.
Subjects and Methods Six women, 14-17 weeks pregnant, were admitted to hospital for legal abortion. Their mean age was 22.0_+3.4 years and weight 59_+10.5kg (mean + S.D.). In the evening prior to hysterotomy they were given nitrazepam 5 mg orally (Mogadon ®, Hoffmann la Roche, Basle, Switzerland). As premedication they received atropine 0.5mg and pethidine 50 mg i. m. Anaesthesia was induced with i. v. thiopentone (dose up to 350 mg) and was maintained with 70/30 N20/02 mixture. Relaxation was obtained with a 50-100 mg bolus of succinylcholine and was maintained with an infusion of succinylcholine in glucose 5%. During the hysterotomy a sample of amniotic fluid was obtained (AF, n = 5). Blood samples from the maternal cubital vein (MV 1) and umbilical cord (UC) were taken after the fetus had been removed. Elective caesarean section was done in twelve women, aged 28.8_+7.5 years, weighing 74.1_+ 13.0 kg (mean _+ S. D.), who had been pregnant for 36 to 40 weeks. Each received an oral dose of nitrazepam 5 mg (Mogadon ®) in the evening prior to the operation. In the course of the operation blood samples were collected simultaneously from the maternal cubital vein (MV 2), fetal umbilical vein (UV) and from the umbilical artery (UA). All the women were informed about the study and they gave their written consent to it. Blood samples were immediately centrifuged and the plasma separated. The erythrocytes were lysed with distilled water according to Zingales (1969). Protein bound and free drug were separated by ultracentrifugation of 10 ml plasma at 407000 × g, at 37°C, according to Kanto et al. (1975). The concentration of nitrazepam was determined by
356
L Kangas et al.: Nitrazepam Transfer Across the Placenta
Table 1. Concentration of nitrazepam (ng/ml) in maternal plasma (MV 1), the umbilical circulation (UC) and amniotic fluid (AF) in early pregnancy Subject
MV 1
UC
AF
SO LL LR KR LV SB
23 28 34 30 12 38
15 11 26 18 10 11
12 6 16 6 4 - (no sample)
mean S.D.
27.5 9.2
15.2 6.1
8.8 5.0
Table 2. Concentration of nitrazepam (ng/ml) in maternal plasma (MV 2), umbilical vein (UV) and umbilical artery (UA) in late pregnancy, and the protein unbound fraction of nitrazepam (ng/ml and percentage of total) in plasma from a maternal and the umbilical vein Subject
Nitrazepam (total) in plasma (ng/ml)
Protein unbound nitrazepam (ng/ml)
Protein unbound nitrazepam (percentage of total) MV2
UV
13.2 15.6 14.1 11.0 10.6 9.6 9.6 12.5 12.8 9.6
11.7 10.7 12.4 8.6 13,5 14.1 10.8 13.2 13.9 10.8
11.9 2.1
12.0 1,8
MV2 UV
UA
MV2 UV
SU SA HE AN LU LA OL VE RA HA RE HL
34 31 22 16 17 21 36 28 26 28 25 20
26 22 23 14 21 21 31 17 24 28 23 24
37 22 19 18 23 24 33 16 26 25 19 18
. . . . . . 2.9 2.7 2.5 1.5 2.4 2.6 2.3 1.8 3.8 4.2 2.7 2.4 2.5 2.6 3.5 3.7 3.2 3.2 2.5 2.6
mean S.D.
25.3 6.4
22.8 4.5
23.3 6.3
2.8 0.51
2.7 0.81
. .
63Ni-EC gas-liquid chromatography (Kangas, 1977). Before analysis nitrazepam was hydrolyzed with 6 N H2SO 4 to the corresponding benzophenone in order to attain high sensitivity in the protein binding studies. The recovery of the method was 96.8+3.3% (mean + S.D.), the sensitivity 0.5 ng/ml and the coefficient of variation of interday analyses was 6.3%. The sensitivity in the red cell samples was 2 ng/ml, due to the high frequency of impurities found in them. Plasma 0.5 ml, washed red cells 1.5 ml and protein free supernatant 1.0 ml was used for each determination. Statistical analysis was performed by Student's t-test.
Results
In the six cases of early pregnancy (14-17 weeks) the concentration of nitrazepam in MV 1 was 27.5+9.2 ng/ml (mean _+ S. D.), which was significantly higher than in UC (15.2 _+6.1 ng/ml, p
