Abstract
AIM
To investigate the influence of gestational diabetes mellitus (GDM) on the kinetic disposition and transplacental and amniotic fluid distribution of metoprolol and its metabolites O-desmethylmetoproloic acid and α-hydroxymetoprolol stereoisomers in hypertensive parturients receiving a single dose of the racemic drug.
METHODS
The study was conducted on hypertensive parturients with well-controlled GDM (n = 11) and non-diabetic hypertensive parturients (n = 24), all receiving a single 100 mg oral dose of racemic metoprolol tartrate before delivery. Serial maternal blood samples (0–24 h) and umbilical blood and amniotic fluid samples were collected for the quantitation of metoprolol and its metabolite stereoisomers using LC-MS/MS or fluorescence detection.
RESULTS
The kinetic disposition of metoprolol and its metabolites was stereoselective in the diabetic and control groups. Well-controlled GDM prolonged tmax for both enantiomers of metoprolol (1.5 vs. 2.5 h R-(+)-MET; 1.5 vs. 2.75 h S-(−)-MET) and O-desmethylmetoproloic acid (2.0 vs. 3.5 h R-(+)-AOMD; 2.0 vs. 3.0 h S-(−)-OAMD), and for the four stereoisomers of α-hydroxymetoprolol (2.0 vs. 3.0 h for 1′S,2R-, 1′R,2R- and 1′R,2S-OHM; 2.0 vs. 3.5 h for 1′S,2S-OHM) and reduced the transplacental distribution of 1′S,2S-, 1′R,2R-, and 1′R,2S-OHM by approximately 20%.
CONCLUSIONS
The kinetic disposition of metoprolol was enantioselective, with plasma accumulation of the S-(−)-MET eutomer. Well-controlled GDM prolonged the tmax of metoprolol and O-desmethylmetoproloic acid enantiomers and the α-hydroxymetoprolol stereoisomers and reduced by about 20% the transplacental distribution of 1′S,2S-, 1′R,2R-, and 1′R,2S-OHM. Thus, well-controlled GDM did not change the activity of CYP2D6 and CYP3A involved in metoprolol metabolism.
Keywords: arterial hypertension, enantiomers, gestational diabetes mellitus, metoprolol, parturients, pharmacokinetics
WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT
Metoprolol, a drug clinically available as a racemic compound, is a drug indicated for the treatment of hypertension during pregnancy because of its low fetal and neonatal risk, although S-(−)-MET is considered to be the eutomer in terms of blockade of the β1-adrenergic receptor. Metoprolol is eliminated by metabolism dependent on CYP2D6 (α-hydroxymetoprolol) and CYP3A (O-desmethylmetoproloic acid).
WHAT THIS STUDY ADDS
The higher plasma concentrations of S-(−)-metoprolol in the parturients with or without diabetes is explained by the favoured formation of the enantiomer R-(+)-O-desmethylmetoproloic acid. Well-controlled gestational diabetes mellitus (GDM) prolonged the tmax of both metoprolol enantiomers as well as the tmax for the formation of all stereoisomers of the metabolites O-desmethylmetoproloic acid (AODM) and α-hydroxymetoprolol (OHM). Well-controlled GDM showed plasma accumulation of both AODM enantiomers and reduced by about 20% the transplacental distribution of 1′S,2S-, 1′R,2R-, and 1′R,2S-OHM.
Introduction
Gestational diabetes mellitus (GDM) is one of the most frequent obstetrical dysfunctions occurring during pregnancy, affecting about 90% of all pregnancies complicated by diabetes. Its prevalence is estimated at 15 to 20% depending on the population studied and it is related to increased perinatal morbidity and mortality [1–3].
Metoprolol (MET) is a selective antagonist of the β-adrenergic receptor used for the treatment of hypertension during pregnancy because of its low fetal and neonatal risk [4–6]. MET has an asymmetrical centre and is available for clinical use in the form of a racemic compound with the S-(−)- and R-(+)-MET enantiomers [7,8]. The S-(−)-MET enantiomer expresses a 500 times higher affinity for the β1-adrenergic receptor compared with the R-(+)-MET enantiomer [9].
Metoprolol is mainly eliminated as inactive O-desmethylmetoproloic acid (AODM – 65% of the dose) and as active α-hydroxymetoprolol (OHM) (10% of the dose) [10,11]. CYP2D6 and CYP3A, in addition to other CYP isoforms, participate in the O-demethylation of metoprolol [12,13]. The oxidative metabolism of metoprolol represents 85% of its elimination, CYP2D6 being responsible for approximately 70% of this metabolism, whereas a small proportion is dependent on CYP3A4 activity [10,12,13]. The formation of α-hydroxymetoprolol depends on the genetically determined activity of CYP2D6 [14]. In vivo and in vitro studies with human liver microsomes have suggested that α-hydroxylation and O-demethylation preponderantly occur for the R-(+)-MET enantiomer [15].
Pregnancy is considered to be a physiological condition associated with changes in the kinetic disposition and metabolism of drugs. CYP3A4, CYP2D6, CYP2C9 and uridine diphosphate glucuronosyltransferase (UGT1A4 and UGT2B7) activities increase during the gestational period, whereas there are reports of reduced metabolism of drugs dependent on CYP1A2 and CYP2C19 [16].
Diabetes mellitus can also change the kinetic disposition and metabolism of clinically used drugs depending on the type and time of diagnosis of the disease, as well as the substrate investigated [17]. Clinical and experimental studies are demonstrating that diabetes mellitus can alter the activity of various enzymes, such as those of the CYP gene family, leading to differentiated modifications of the expression of their isoforms [18]. Clinical studies developed by our group have suggested that type 2 diabetes [19] and gestational diabetes [20] inhibit CYP3A and/or CYP1A2, with the occurrence of a probable induction of UGT1A and UGT2B7 in parturients with gestational diabetes [20].
In view of the ability of diabetes to modify the activity of enzyme systems involved in drug metabolism [18,21], the objective of the present study was to assess for the first time the influence of well-controlled gestational diabetes mellitus on the kinetic disposition, metabolism and distribution in the placenta and amniotic fluid of the enantiomers of metoprolol and O-desmethylmetoproloic (AODM) acid and the stereoisomers of the α-hydroxymetoprolol (OHM) in hypertensive parturients treated with a single oral dose of the racemic drug. Considering that hypertension and GDM are the most frequent diseases manifested during pregnancy [2], this study is relevant in the treatment choice during this period not only for metoprolol but also for other drugs with metabolism dependent on CYP3A and CYP2D6.
Methods
Clinical protocol
This investigation was conducted as an open, randomized, monocentric and single dose study on hypertensive parturients seen at the Obstetrical Centre of the University Hospital, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil (HCFMRP-USP).
Sample size for the pharmacokinetics study was calculated using the Power and Sample Size software, version 2.1.31 (Vanderbilt, USA), considering the variability of the pharmacokinetics of metoprolol enantiomers in healthy volunteers treated with a single dose of the racemic drug [12] and using the data of the isomer with greater inter-individual variability R-(+)-MET. We considered a power of 80%, a type I standard error of 5%, the mean AUC value (468.60 ng ml−1 h) and the standard deviation (288.98 ng ml−1 h) for the R-(+)-MET isomer and a difference of at least 50% between the control and diabetes group [12]. The study was approved by the Research Ethics Committee of HCFMRP-USP, Protocol HCRP 3974/2008, and all subjects gave written informed consent to participate.
In the clinical protocol were included 35 hypertensive parturients aged between 21 to 45 years old, gestational weight of 58.5 to 145.8 kg and gestational body mass index (BMI) of 26 to 51.7 kg m−2) at a gestational age of 35 to 42 weeks. Parturients with a singleton pregnancy, absence of fetal intercurrences, liver and kidney function within normal limits and classified as extensive metabolizers of metoprolol (CYP2D6) were included. Parturients could be receiving treatment with other antihypertensive drugs, but those treated with insulin, oral hypoglycaemic drugs, CYP inducers or CYP inhibitors 1 month before or during the period of the study were excluded.
During the study period, the haemodynamic parameters of the hypertensive parturients were monitored by recording systolic and diastolic arterial pressure and heart rate, with the results showing haemodynamic stability. Arterial hypertension was diagnosed according to the criteria of the National High Blood Pressure Education Program (NHBEP) [22], including parturients with chronic hypertension or gestational hypertension. The values recorded before and 2 h after drug administration are listed in Table 1. The newborns of the hypertensive parturients did not show any disease at birth. Median birth weight and length values are listed in Table 1.
Table 1.
Demographic data of the parturients investigated and their newborns (median and 95% CI)
| Control group (n = 24) | Diabetes group (n = 11) | |||
|---|---|---|---|---|
| Parameters | PK + TD (n = 13) | TD (n = 11) | PK + TD (n = 10) | TD (n = 1) |
| Age (years) | 29.23 (25.96, 32.50) | 30.50 (27.23, 35.20) | 33.00 (28.41, 37.57) | 36.00 |
| Weight (kg) | 107.00 (88.81, 111.76) | 101.70 (91.60, 119.64) | 100.15 (92.08, 106.71) | 102.80 |
| BMI (kg m−2) | 39.91 (34.37, 41.85) | 39.51 (35.52, 43.62) | 38.42 (36.14, 41.79) | 42.30 |
| Gestational age (days) | 275.00 (267.66, 283.42) | 268.00 (261.80, 277.47) | 270.50 (268.06, 284.54) | 264.00 |
| Fasting glycaemia (mmol l−1) | 4.33 (3.67, 4.73) | 4.14 (3.27, 4.79) | 5.05*(4.38, 5.50) | 3.55 |
| 2 h glycaemia (OGTT 75-mmol l−1) | – | – | 7.83 (5.64, 10.19) | 6.33 |
| Glycated haemoglobin (%) | – | – | 5.20 (4.59, 6.11) | 5.30 |
| Systolic arterial pressure (mmHg)† | 140.00 (129.99, 148.78) | 140.00 (125.90, 157.19) | 140.00 (122.64, 161.58) | 135.00 |
| Systolic arterial pressure (mmHg)‡ | 135.50 (127.38, 150.78) | 124.00 (110.42, 134.15) | 120.00* (111.63, 123.7) | 130.00 |
| Diastolic arterial pressure (mmHg)† | 80.00 (58.90, 91.86) | 90.00 (80.33, 102.94) | 83.00 (69.19, 98.41) | 90.00 |
| Diastolic arterial pressure (mmHg)‡ | 81.00 (68.91, 92.59) | 68.00 (58.27, 72.87) | 55.00* (49.11, 76.00) | 90.00 |
| Heart rate (beats min−1)† | 76.00 (71.95, 87.69) | 82.00 (73.01, 92.99) | 84.00 (79.75, 88.91) | 80.00 |
| Heart rate (beats min−1)‡ | 80.00 (76.04, 84.84) | 70.00 (63.36, 76.07) | 77.00 (66.83, 83.61) | 72.00 |
| Associated pharmaceuticals | 1;2;3;4;5;6;7;8;9;10 | 1;2;3;4;5;6;8;10;11 | 1;2;3;4;5;6;8;10;12 | 4;5;6;8 |
| Newborn weight (g) | 3170.0 (2783.2, 3313.0) | 3345.0 (3096.5, 3986.0) | 3647.5*(3441.5, 3974.5) | 2780 |
| Newborn length (cm) | 48.50 (46.84, 50.09) | 49.00 (48.07, 50.83) | 50.50* (49.44, 51.66) | 46.50 |
Haemodynamic parameters measured before metoprolol administration;
Haemodynamic parameters measured 2 h after metoprolol administration; PK + TD = parturients included in the pharmacokinetics and transplacental distribution study; TD = parturients included only in the transplacental distribution study; Associated pharmaceuticals: 1 methyldopa, 2 nifedipine, 3 hydralazine, 4 dipyrone, 5 oxytocin, 6 cefazolin, 7 dimethicone, 8 ferrous sulphate, 9 metronidazole, 10 misoprostol, 11 betamethasone, 12 furosemide; BMI: Body mass index;
Mann–Whitney test, P < 0.05 (PK + TD Control group vs. PK + TD diabetes group).
The parturients were divided into two groups, the control group consisting of 24 hypertensive parturients without diabetes mellitus (13 parturients for plasma pharmacokinetics including transplacental distribution and 11 parturients only for the transplacental distribution study) and the diabetes group consisting of 11 hypertensive parturients with well-controlled gestational diabetes mellitus (10 parturients for pharmacokinetics and transplacental distribution studies and one only for the transplacental distribution study). Well-controlled gestational diabetes mellitus diagnosed according to the oral glucose tolerance test (OGTT) with a 75 g overload of anhydrous glucose (OGTT-75), was considered to be present in situations of fasting glycaemia of 7.1 mmol l−1 or more and/or 2 h glycaemia of 7.8 mmol l−1 or more and glycated haemoglobin below 6.5% [23]. The diabetes control was done only with diet.
The median weight of the newborns of the diabetic parturients was significantly greater than that of the newborns of control parturients, a result compatible with the maternal glycaemic fluctuation observed in patients with gestational diabetes [24].
Only five newborns in the control group and one newborn in the diabetes group showed a first minute Apgar score of less than 7 and all had fifth minute scores higher than 7, showing a favourable post-natal course.
The hypertensive parturients received a single 100 mg dose of racemic metoprolol tartrate (Lopressor®, Novartis, Brazil) with 20 ml of water. Meals were provided at standardized times. Maternal blood samples (5 ml) were collected through an intravenous catheter into heparinized syringes (Liquemine® 5000IU, Roche) at times, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16, 20 and 24 h after metoprolol administration. Maternal and umbilical cord blood samples were collected at delivery in order to determine the rate of placental transfer of the drug. For the parturients with resolution of pregnancy by the abdominal route (n = 18), 5 ml aliquots of amniotic fluid were also collected for the determination of drug distribution in this compartment. Deliveries were performed in a median time of 3.5 h (range 1.0–6.75 h) after metoprolol administration for the control group and 2.5 h (range 0.5–4.5 h) for the diabetes group. The plasma aliquots obtained after blood centrifugation at 850 g for 20 min and the amniotic fluid samples were stored at −70°C until analysis.
Analytical assays
Analysis of the metoprolol enantiomers and the α-hydroxymetoprolol stereoisomers in plasma
The enantiomers of metoprolol and the stereoisomers of α-hydroxymetoprolol were separated as previously described by our group [25]. The plasma concentrations of the metoprolol enantiomers and α-hydroxymetoprolol stereoisomers were determinated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with a chiral phase Chiralpak® AD column using hexane : ethanol : isopropanol : diethylamine (88:10.2:1.8:0.2, v/v/v/v) as the mobile phase. The calibration curves constructed using the height vs. concentration ratios of metoprolol enantiomers (0.2–250 ng of each enantiomer ml−1 plasma) and α-hydroxymetoprolol stereoisomers (0.1–125 ng of each stereoisomer ml−1 plasma) showed linearity during the intervals mentioned, with correlation coefficients higher than 0.99. The limits of quantitation were 0.2 ng ml−1 plasma for metoprolol enantiomers and 0.1 ng ml−1 plasma for α-hydroxymetoprolol stereoisomers. The coefficients of variation obtained in the inter- and intra-assay precision and percent inaccuracy studies were lower than 15%, ensuring the reproducibility and repeatability of the results.
Analysis of the O-desmethylmetoproloic acid enantiomers in plasma
Analysis of the O-desmethylmetoproloic acid enantiomers in plasma was carried out using a procedure developed and validated in a previous study by our group [26]. The O-desmethylmetoproloic acid enantiomers were separated by high performance liquid chromatography (h.p.l.c.) with ultraviolet detection, on a Chiracel OD-R column with a mixture of 0.5 m sodium perchlorate/perchloric acid (pH 3.0) : acetonitrile (85:15, v/v) as mobile phase. The method was linear in the 17–3300 ng ml−1 plasma range for each enantiomer of O-desmethylmetoproloic acid, with correlation coefficients higher than 0.99. The quantitation limit detected was 17 ng ml−1 for both enantiomers. The coefficients of variation obtained in the inter- and intra-assay precision and percent inaccuracy studies were lower than 15%, ensuring the reproducibility and repeatability of the results.
Analysis of the metoprolol and O-desmethylmetoproloic acid enantiomers and the α-hydroxymetoprolol stereoisomers in amniotic fluid
The enantiomers of metoprolol and O-desmethylmetoproloic acid and α-hydroxy metoprolol stereoisomers were analyzed in amniotic fluid as described in previous studies by our group using plasma as matrix [25,26]. The concentration of the metoprolol enantiomers and α-hydroxymetoprolol stereoisomers were determinated in amniotic fluid by LC-MS/MS coupled with a chiral phase Chiralpak® AD column using hexane : ethanol : isopropanol : diethylamine (88:10.2:1.8:0.2, v/v/v/v) as the mobile phase. The O-desmethylmetoproloic acid enantiomers were analyzed by h.p.l.c. with ultraviolet detection, on a Chiracel® OD-R column with a mixture of 0.5 m sodium perchlorate/perchloric acid (pH 3.0) : acetonitrile (85:15, v/v) as the mobile phase. The calibration curves constructed using the height vs. concentration ratios of the enantiomers of metoprolol (0.2–250 ng of each enantiomer ml−1 amniotic fluid) and O-desmethylmetoproloic acid (13.75–1375.00 ng of each enantiomer ml−1 amniotic fluid) and the stereoisomer of α-hydroxymetoprolol (0.1–125 ng of each stereoisomer ml−1 amniotic fluid) showed linearity during the intervals mentioned, with correlation coefficients higher than 0.99. The coefficients of variation obtained in the inter- and intra-assay precision and percent inaccuracy studies were lower than 15%, ensuring the reproducibility and repeatability of the results.
Oxidative phenotype of the metoprolol type
The metoprolol : α-hydroxymetoprolol (MET : OHM) metabolic ratio was calculated by dividing the metoprolol concentration by the α-hydroxymetoprolol concentration in plasma after a single oral dose of 100 mg racemic metoprolol tartrate [27,28] and the log of metabolic ratio MET : OHM was then calculated. Subjects whose metabolic ratios were lower than 1.5 were considered to be poor metabolizers (PM) and subjects whose values were higher than 1.5 were considered to be extensive metabolizers (EM) [28]. The calculations were performed using the stereoisomer mixture of the plasma concentrations. This method presented good correlation with the MET : OHM metabolic ratio of the AUC(0,12 h) [27,29] and reasonable correlation with the 0–8 h urine MET : OHM metabolic ratio, suggesting that the metabolic ratio from one point obtained 3 h after the administration of metoprolol was also useful for the oxidative phenotyping of this drug [28].
Pharmacokinetic analysis
Pharmacokinetic analysis was performed using the WinNonlin software, version 4.0 (Pharsight Corp, Moutain View, CA, USA). The pharmacokinetic parameters were calculated based on the plasma concentrations obtained experimentally using a non-compartmental model.
Statistical analysis
The median, mean and 95% confidence interval (95% CI) of the data were calculated with the aid of GraphPad Instat software. The two-tailed Wilcoxon test for paired data was used to determine the enantiomer ratios differing from unity for metoprolol and O-desmethylmetoproloic acid, and for the multiple comparisons the non-parametric Kruskal−Wallis test was used for the stereoisomers of α-hydroxymetoprolol. The two-tailed Mann−Whitney test for unpaired data was used to compare the pharmacokinetic parameters between the control and diabetic groups. The level of significance was set at 5% in all statistical analysis.
Results
The kinetic disposition of metoprolol and its metabolites O-desmethylmetoproloic acid and the α-hydroxymetoprolol was enantioselective in both groups (Figure 1 and Tables 2 and 3). The data show higher AUC and lower CL/F values for S-(−)-MET compared with its antipode in both groups.
Figure 1.
Plasma concentration vs. time curves for the metoprolol (MET - I), α-hydroxymetoprolol (α-OHM - II) and O-desmethylmetoproloic acid (AODM - III) isomers in parturients of the control group, n = 13 (A–C), and of the diabetes group, n = 10 (D–F), treated with a single oral dose of racemic metoprolol tartrate. Data are reported as mean and standard error of the mean. *Mann–Whitney test, P < 0.05 (Control group vs. diabetes group). 
, R-(+) MET; 
, S-(–) MET; 
, 1′S,2R-OHM; 
, 1′S,2S-OHM; 
, 1′R,2R-OHM; 
, 1′R,2S-OHM; 
, R-(+) AODM; 
, S-(–) AODM
Table 2.
Pharmacokinetics of the metoprolol (MET) isomers in the control (n = 13) and diabetes (n = 10) groups (median and 95% CI)
| Parameter | R-(+)-MET | S-(−)-MET | ||
|---|---|---|---|---|
| Control group | Diabetes group | Control group | Diabetes group | |
| Cmax (ng ml−1) | 22.89 (20.56, 64.26) | 18.98 (12.53, 42.14) | 41.42† (30.16, 67.51) | 22.90 (12.97, 49.54) |
| tmax (h) | 1.50 (1.16, 2.15) | 2.50* (1.55, 3.94) | 1.50 (1.19, 2.15) | 2.75* (1.94, 4.35) |
| AUC(0,∞) (ng ml−1 h) | 62.65 (58.39, 225.03) | 84.63 (41.48, 196.92) | 113.42† (89.73, 266.58) | 125.32† (62.81, 238.70) |
| MRT (h) | 6.71 (5.65, 10.14) | 6.54 (4.78, 8.00) | 5.87† (4.85, 8.28) | 6.37† (4.66, 9.01) |
| t1/2 (h) | 7.74 (6.38, 11.24) | 3.57* (2.60, 7.38) | 7.01 (5.26, 9.77) | 4.38 (3.08, 7.13) |
| Kel (h−1) | 0.09 (0.06, 0.13) | 0.19* (0.12, 0.26) | 0.10 (0.08, 0.15) | 0.16 (0.11, 0.24) |
| V/F (l kg−1) | 35.38 (23.81, 87.46) | 25.60 (15.95, 66.19) | 26.87† (21.03, 62.98) | 20.02† (14.58, 39.98) |
| CL/F (l h−1 kg−1) | 5.29 (3.19, 7.48) | 4.85 (1.97, 12.62) | 3.19† (2.29, 4.95) | 3.05† (1.88, 7.68) |
Mann−Whitney test, P < 0.05 (Control group vs. diabetes group).
Wilcoxon test for paired data, P < 0.05 (R-(+)-MET vs. S-(–)-MET). AUC(0,∞), area under the curve for plasma concentration vs. time; CL/F, total apparent clearance; Cmax, maximum plasma concentration; Kel, elimination rate constant; MRT, mean residence time; t1/2, elimination half-life; tmax, time needed to reach Cmax; V/F, apparent distribution volume.
Table 3.
Kinetic disposition of the α-hydroxymetoprolol (OHM) and O-demethylmetoproloic acid (AODM) isomers in the control (n = 13) and diabetes (n = 10) groups (median and 95% CI)
| Isomer | Cmax (ng ml−1) | tmax (h) | AUC(0,∞) (ng ml−1 h) | t1/2 (h) | Kel (h−1) | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Control group | Diabetes group | Control group | Diabetes group | Control group | Diabetes group | Control group | Diabetes group | Control group | Diabetes group | |
| 1′S,2R-OHM | 9.64#♦ (7.34, 11.35) | 6.48#♦ (4.42, 11.51) | 2.00 (1.24, 2.37) | 3.00* (2.30, 5.04) | 57.27#♦ (48.37, 66.38) | 54.29#♦ (42.16, 71.49) | 5.87 (4.95, 7.50) | 5.36 (4.09, 8.56) | 0.11 (0.10, 0.15) | 0.13 (0.10, 0.15) |
| 1′S,2S-OHM | 11.31♣⊗ (8.41, 13.52) | 7.25♣⊗ (4.93, 12.39) | 2.00 (1.25, 2.52) | 2.75* (1.92, 4.38) | 79.76♣⊗ (60.48, 88.39) | 59.84♣⊗ (46.26, 81.79) | 6.31 (5.50, 7.50) | 6.20 (4.82, 8.47) | 0.11 (0.10, 0.13) | 0.11 (0.10, 0.15) |
| 1′R,2R-OHM | 26.14 (19.50, 30.24) | 18.56 (14.93, 31.05) | 2.00 (1.21, 2.49) | 3.00* (2.36, 5.04) | 168.52 (136.69, 183.94) | 171.37 (140.63, 202.29) | 6.48 (5.32, 6.77) | 5.53 (4.72, 7.57) | 0.11 (0, 0.13) | 0.12 (0.10, 0.15) |
| 1′R,2S-OHM | 33.68 (25.08, 40.39) | 28.45 (19.62, 42.51) | 2.00 (1.27, 2.50) | 3.00* (2.25, 5.05) | 223.99 (179.47, 254.32) | 248.09 (175.26, 316.34) | 5.95 (5.16, 6.55) | 5.74 (4.11, 8.13) | 0.12 (0.11, 0.13) | 0.12 (0.11, 0.17) |
| R-(+)-AODM | 523.63 (430.52, 610.87) | 456.86 (384.48, 640.91) | 2.00 (1.95, 3.12) | 3.50* (2.83, 5.17) | 2769.70 (2429.10, 3795.20) | 3678.80* (3375.00, 4604.80) | 4.59 (3.35, 6.52) | 5.85* (5.10, 7.89) | 3.62 (2.95, 4.62) | 5.42* (4.93, 5.98) |
| S-(–)-AODM | 505.34▪ (402.84, 564.65) | 459.96 (367.73, 635.23) | 2.00 (1.90, 3.10) | 3.00* (2.64, 4.96) | 2660.40▪ (2213.30, 3482.40) | 3414.90*▪ (3018.40, 4180.10) | 0.15 (0.12, 0.21) | 0.12 (0.09, 0.14) | 0.18 (0.15–0.23) | 0.13* (0.11–0.14) |
Kruskal-Wallis test for paired data, P < 0.05: #1′S.2R vs. 1′R.2R; ♣1′S.2S vs. 1′R.2R; ♦1′S.2R vs. 1′R.2S; ⊗1′S.2S vs. 1′R.2S. ▪Wilcoxon test for paired data, P < 0.05 (R-(+)-AODM vs. S-(–)-AODM) *Mann–Whitney test, P < 0.05 (Control group vs. diabetes group). AUC(0,∞), area under the curve for plasma concentration vs. time; Cmax, maximum plasma concentration; Kel, elimination rate constant; t1/2, elimination half-life; tmax, time needed to reach Cmax.
The pharmacokinetic data of the diabetic group showed a prolonged time to reach Cmax (tmax) compared with control group (Tables 2 and 3) for metoprolol, AODM and OHM stereoisomers.
The values listed in Table 4 represent the umbilical cord: maternal plasma concentration ratios close to 1 for the metoprolol enantiomers and OHM stereoisomers and close to 0.8 for both AODM enantiomers (control group = 22). The transplacental distribution of the enantiomers of metoprolol and AODM in the diabetes group can be considered to be similar to that obtained for the control group. However, the transplacental distribution of the 1′S,2S, 1′R,2R and 1′R,2S-OHM stereoisomers was approximately 20% lower compared with the control group (Table 4).
Table 4.
Ratios of the umbilical cord : maternal plasma and amniotic fluid : maternal plasma concentrations of the metoprolol (MET), α-hydroxymetoprolol (OHM) and O-desmethylmetoproloic acid (AODM) isomers in the parturients of the control and diabetic groups (median and 95% CI)
| Isomer | Umbilical cord/maternal plasma | Amniotic fluid/maternal plasma | ||
|---|---|---|---|---|
| Control group (n = 22) | Diabetes group (n = 10) | Control group (n = 13) | Diabetes group (n = 5) | |
| R-(+)-MET | 1.04 (0.91, 1.70) | 1.06 (0.63, 1.42) | 2.99 (2.09, 6.89) | 0.40 (0.01, 2.18) |
| S-(–)-MET | 1.15 (1.06, 1.41) | 1.08 (0.64, 1.32) | 3.23 (2.27, 6.96) | 0.51 (0.39, 2.12) |
| 1′S,2R-OHM | 1.10 (1.01, 1.51) | 1.06 (0.63, 1.42) | 5.06 (2.28, 10.02) | 0.44 (0.03, 0.99) |
| 1′S,2S-OHM | 1.09 (1.00, 1.39) | 0.79* (0.42, 1.06) | 3.95 (1.74, 9.58) | 0.39 (0.072, 0.66) |
| 1′R,2R-OHM | 0.95 (0.83, 1.28) | 0.73* (0.39, 1.01) | 1.63 (0.34, 11.90) | 0.20 (0.01, 3.76) |
| 1′R,2S-OHM | 0.92 (0.83, 1.28) | 0.66* (0.38, 1.03) | 1.45 (0.24, 9.28) | 0.29 (0.01, 3.36) |
| S-(-)-AODM | 0.83 (0.70, 0.94) | 0.65 (0.51, 0.76) | 0.83 (0.67, 0.92) | 0.63 (0.45, 0.73) |
| R-(+)-AODM | 0.29 (0.02, 2.50) | 0.13 (0.01, 0.44) | 0.37 (0.05, 3.22) | 0.12 (0.10, 0.61) |
Kruskal–Wallis test for paired data, P < 0.05: #1′S.2R vs. 1′R.2R; ♣1′S.2S vs. 1′R.2R; ♦1′S.2R vs. 1′R.2S; ⊗1′S.2S vs. 1′R.2S. ▪Wilcoxon test for paired data, P < 0.05 (R-(+) vs. S-(–)).
Mann–Whitney test, P < 0.05 (Control group vs. diabetes group).
Related to the amniotic fluid : maternal plasma concentration ratios we observed ratios of 2.99 for R-(+)-MET and 3.23 for S-(−)-MET. The amniotic fluid : maternal plasma ratios of α-hydroxymetoprolol were 5.06 for the 1′S,2R, 3.95 for 1′S,2S, 1.63 for 1′R,2R and 1.45 for the1′R,2S α-hydroxymetoprolol stereoisomer (Table 4), showing that the 1′S,2R and 1′S,2S stereoisomers of α-hydroxymetoprolol accumulate in amniotic fluid to a greater extent than the metoprolol enantiomers. Both AODM enantiomers reached lower concentrations in the amniotic fluid than in the plasma of the parturients in the control group (Table 4). The amniotic fluid : maternal plasma concentration ratios were lower than those observed for metoprolol and α-hydroxymetoprolol (0.29 and 0.37 for R-(+)- and S-(−)-AODM, respectively).
Figure 2 shows the amniotic fluid : maternal plasma concentration ratios of control parturients as a function of time between metoprolol administration and sample collection.
Figure 2.
Concentration ratios of amniotic fluid : maternal plasma related to the isomers of metoprolol (MET), α-hydroxymetoprolol (OHM) and O-desmethylmetoproloic acid (AODM) as a function of time between metoprolol administration and sample collection in hypertensive parturients (control group; n = 11) treated with a single oral dose of 100 mg racemic metoprolol tartrate. Each point on the curve corresponds to the sample from one patient. 
, R-(+)-MET; 
, S-(–)-MET; 
, 1′S,2R-OHM; 
, 1′S,2S-OHM; 
, 1′R,2R-OHM; 
, 1′R,2S-OHM; 
, R-(+)-AODM; 
, S-(–)-AODM
Discussion
The present study investigated the influence of well-controlled gestational diabetes mellitus on the kinetic disposition and the metabolism of the metoprolol and O-desmethylmetoproloic acid enantiomers and the α-hydroxymetoprolol stereoisomers in hypertensive parturients.
The metoprolol hepatic extraction ratio is medium to high in patients phenotyped as CYP2D6 EM and consequently metoprolol clearance depends on both hepatic blood flow and enzymatic activity [30]. The log of the metabolic ratios metoprolol : α-hydroxymetoprolol as a stereoisomeric mixture were less than 1.5 for all patients investigated, designating the EM phenotype [27].
The kinetic disposition of metoprolol was enantioselective in the control patients (Figure 1A, Table 2) with lower CL/F values for S-(−)-MET (3.19 vs. 5.29 l h−1 kg−1) (Table 2). The CL/F values of both metoprolol enantiomers obtained for the parturients investigated were higher than those reported by Cerqueira et al. [13] in a study of hypertensive patients (men and non-pregnant women) (1.26 vs. 1.53 l h−1 kg−1) and by Boralli [12] in a study of non-pregnant healthy volunteers (1.66 vs. 2.31 l h−1 kg−1). AUC(0,∞) S-(−) : R-(+) enantiomeric ratios of 1.39 were previously reported by Johnson & Burlew [31] who investigated non-pregnant healthy volunteers treated with a single dose of racemic metoprolol. The approximately two-fold increase of CYP2D6 activity during the third trimester of pregnancy, determined as the reduction of the AUC values of metoprolol as an enantiomer mixture, agrees with previous reports [32–34]. During pregnancy, there is an increase in extracellular fluid, reduction in plasma total protein concentration, increase in the concentration of free fatty acids, increase in the glomerular filtration rate and the state of enzyme induction, which results in plasma concentrations of unchanged drug smaller than those observed in non-pregnant patients [16]. Moreover, according to work reported by Nakai et al. [35], the total liver blood flow increases significantly (2.98 ± 1.13 l min−1) after 28 weeks gestation when compared with non-pregnant women (1.82 ± 0.63 l min−1). As metoprolol is a medium to high extraction ratio drug, there is some evidence that increased total liver blood flow, in addition to decreased protein binding, may result in increased clearance [16].
The V/F parameter also exhibited enantioselectivity in the control group, with V/F values for R-(+)-MET being higher than for S-(−)-MET (35.38 vs. 26.87 l kg−1; Table 2). These differences in V/F values cannot be explained as a function of probable changes in binding to plasma proteins in view of the low binding of metoprolol to plasma proteins (approximately 11%). Thus, differences in oral bioavailability between the R-(+)- and S-(−)-MET enantiomers may have contributed to the observation of enantioselectivity in the V/F parameter. Boralli [12] also reported a higher V/F value for the R-(+)-MET enantiomer in healthy volunteers treated with a single dose of the racemic drug (5.7 vs. 4.0 l kg−1). The higher V/F values for both metoprolol enantiomers obtained for the present hypertensive parturients may be explained by the increased extracellular fluid volume during pregnancy [36].
The formation of the α-hydroxymetoprolol metabolite, which is active in the blockade of the β1-adrenergic receptor [11] and which contributes to the elimination of 10% of the metoprolol dose [10], proved to be stereoselective for the control parturients, favouring the formation of the new chiral centre 1′R (Figure 1B). A statistically significant difference in Cmax and AUC between the 1′S and 1′R stereoisomers was observed (Table 3). The favoured formation of a new 1′R chiral centre agrees with literature studies employing human liver microsomes [10]. AUC 1′R : 1′S ratios of 2.84 for the present hypertensive parturients agree with the values reported by Cerqueira et al. [13] in a study of hypertensive patients (AUC 1′R : 1′S = 2.87) and with the values reported by Boralli [12] in a study of healthy volunteers (AUC 1′R : 1′S = 3.07).
The formation of the AODM metabolite, responsible for the elimination of 65% of the metoprolol dose [10], also showed enantioselectivity (Table 3, Figure 1C), with the observation of higher AUC values for the R-(+)-AODM isomer (2.77 vs. 2.66 μg ml−1 h). Mistry et al. [37] reported that healthy volunteers treated with an oral solution of racemic metoprolol exhibit a favoured formation of R-(+)-AODM (AUC S-(−) : R-(+) = 0.85).. Boralli [12] and Cerqueira et al. [13] also reported higher AUC values for the R-(+)-AODM enantiomer in their respective investigations of hypertensive patients and healthy volunteers. The favoured formation of the R-(+)-AODM enantiomer explains the plasma accumulation of S-(−)-MET, if we consider that O-desmethylation contributes to the elimination of approximately 65% of the metoprolol dose in patients who are EM for CYP2D6 [32].
Diabetes mellitus can alter the pharmacokinetics by various mechanisms, including a change in the intestinal absorption, distribution and elimination of clinically used drugs [21,38,39]. Hyperglycaemia is considered the cause of tissue damage in diabetes due to alterations in cellular metabolism of glucose accumulation or long term glycated biomolecules and advanced glycation of the final products. The glycation processes affecting the movement of proteins such as serum albumin, insulin, haemoglobin and glycoproteins, while the formation of advanced glycation of the final products, represented by a heterogeneous group of chemicals resulting from non-enzymatic reaction between sugars and proteins, lipids and/or nucleic acids, involves reactive intermediates and result in oxidative stress among other complications [40]. Oxidative stress associated with the molecular mechanism of decrease in the biosynthesis and secretion of insulin is the main aetiology of glucose toxicity, responsible for complications caused by diabetes mellitus. The extent of these complications depends on the time since diagnosis of the disease and on the control of glycaemia levels [41].
In the present study we investigated hypertensive parturients with gestational diabetes mellitus who did not take insulin or hypoglycaemic agents and who presented with good control of glycaemia levels (glycated haemoglobin <6.5%, Table 1). The enantioselectivity in the kinetic disposition of metoprolol and its metabolites in the diabetic parturients was similar to that observed in the controls.
The pharmacokinetic data of the diabetic group showed a prolonged time to reach Cmax (tmax) compared with the control group (Tables 2 and 3) for all metoprolol and AODM enantiomers and OHM stereoisomers. The delayed rate of absorption of both metoprolol enantiomers in the diabetic group may be explained as a function of motor abnormalities caused by diabetes in the stomach and small bowel [38]. Hyperglycaemia has a strong impact on gastrointestinal motility and also affects gastric emptying time by different mechanisms. In addition, marked hyperglycaemia seems to affect all regions of the gastrointestinal tract [21,42–44]. Although there are no data related to gastrointestinal motility and well-controlled gestational diabetes, according to the glucose concentrations below the reference limits in the investigated patients shown in Table 1, there is probably a metabolic effect of mild hyperglycemia on gastrointestinal motility.
Regarding AODM, there was also a statistically significant difference between the control and diabetes groups in the AUC parameters. The data presented in Table 3 show that the diabetic parturients had a plasma accumulation of both AODM enantiomers (3.68 vs. 2.77 μg ml−1 h for R-(+)- and 3.41 vs. 2.66 μg ml−1 h for S-(−)-AODM). AODM formation depends on CYP3A and probably on other CYP isoforms and represents the main pathway of metoprolol elimination in patients who are EM for CYP2D6 (65% of the dose) [10,31,45]. Previous studies by our group have shown reduced CYP3A activity in patients with poorly controlled type 2 diabetes mellitus or in patients with poorly controlled gestational diabetes mellitus [19,20,46]. Dostalek et al. [21] also reported a significant reduction of CYP3A activity in hepatic microsome fractions of diabetic patients. Thus, the reduction of CYP3A activity in diabetes does not explain the plasma accumulation of both AODM enantiomers in the diabetic group. Changes in CYP2D6 activity in diabetes also do not contribute to the explanation of the plasma accumulation of this metabolite if we consider that, in the present study, the kinetic disposition of α-hydroxymetoprolol depending on CYP2D6 activity was not altered. Thus, the induced activity of other CYP isoforms probably involved in AODM formation, as well as the possible changes in the capacity of elimination of the metabolite, may explain the plasma accumulation of both AODM enantiomers observed in the diabetic group.
Borbás et al. [47] showed significant correlations between blood glucose concentrations in rats with streptozotocin-induced diabetes and the activity of total CYP, CYP1A, CYP3A and flavin-containing monooxygenase (FMO) in the liver and the activity of intestinal CYP3A. Clinical studies by our group have also indicated changes in CYP activity only in patients with poorly controlled diabetes. Boralli [12] reported that well-controlled type 2 diabetes mellitus does not change the enantioselective kinetic disposition or metabolism of metoprolol.
Like the liver, placenta is also involved in drug metabolism, although the mRNAs of CYP1A1, CYP2E1, CYP2F1, CYP3A3, CYP3A4, CYP3A5 and CYP4B1 are expressed at much lower levels in the term placenta than in the liver. However, gestational diabetes mellitus does not seem to change the activity of CYP enzymes in the placenta [48].
Transplacental drug distribution usually occurs by passive diffusion, a process depending on the degree of ionization, lipophilicity, protein binding and molecular weight along with blood flow. However, efflux transporters such as P-glycoprotein (P-gp) limit distribution of drugs to the fetus [49].
The transplacental distribution of metoprolol and its metabolite, α-hydroxymetoprolol, as an enantiomeric mixture is extensive, with umbilical cord : maternal plasma concentration ratios close to 1 [50].
The data presented in Table 4 show absence of enantioselectivity in the transplacental distribution of metoprolol and its metabolites, which can be explained by the fact that the drug is not a P-gp substrate and it does not have any action on the expression of this transporter [51]. When the diabetes group was compared with the control group, we observed that the transplacental distribution of the 1′S,2S, 1′R,2R and 1′R,2S-OHM stereoisomers in the diabetes group was approximately 20% lower compared with the control group (Table 4). The present results cannot be explained as a function of metabolism in the placenta since the formation of OHM depends on CYP2D6, an isoform that is not expressed in the term placenta [48].
Amniotic fluid is the product of numerous exchanges with the fetus. Exposure of the fetus to amniotic fluid occurs by inhalation through the respiratory tract, deglutition through the gastrointestinal tract, and also by possible transdermal absorption. A fetus at term swallows 210–760 ml day−1 amniotic fluid and produces 600–800 ml urine day−1 independently of amniotic fluid volume. Thus, amniotic fluid may represent an important source of fetal exposure to clinically used drugs [50,52]. Metoprolol and its metabolites are also distributed in amniotic fluid [50], with S-(−)-MET concentrations being higher than R-(+)-MET concentrations in the control group. The amniotic fluid : maternal plasma ratios presented in Table 4 show that the 1′S,2R and 1′S,2S stereoisomers of α-hydroxymetoprolol accumulate in amniotic fluid to a greater extent than the metoprolol stereoisomers. It should be pointed out that α-hydroxymetoprolol is an active metabolite with a potency of approximately 1/10 of metoprolol in terms of reduction of heart rate [12]. Both AODM enantiomers reached lower concentrations in amniotic fluid than in plasma of the parturients of the control group (Table 4). The amniotic fluid/maternal plasma concentration ratios were lower than those observed for metoprolol and α-hydroxymetoprolol (0.29 and 0.37 for R-(+)- and S-(−)-AODM, respectively).
The concentrations of metoprolol and AODM enantiomers and OHM stereoisomers in amniotic fluid are influenced by the time between drug administration and amniotic fluid collection as a function of the dynamics of the processes involved in the formation of amniotic fluid. The data presented in Figure 2 show the influence of time between metoprolol administration and amniotic fluid collection on the stereoisomeric ratios of the amniotic fluid: maternal plasma concentrations for the control patients. The data permit us to infer that, even though the tmax values for maternal plasma were 1.5 to 2 h for metoprolol and its metabolites (Tables 2 and 3), the higher amniotic fluid: maternal plasma ratios for both the metoprolol enantiomers and the α-hydroxymetoprolol stereoisomers were observed starting 4 h after the oral administration of metoprolol and remained stable until approximately 8 h after administration. The amniotic fluid: maternal plasma ratios of the AODM enantiomers remained low up to 8 h after metoprolol administration. Previous studies have also reported higher metoprolol and α-hydroxymetoprolol concentrations as stereoisomeric mixtures in the amniotic fluid than in the maternal plasma of hypertensive parturients treated with multiple doses of racemic metoprolol. It has been reported that the highest amniotic fluid : maternal plasma ratios for both metoprolol and α-hydroxymetoprolol were observed 5 h after metoprolol administration [50].
The group of parturients with well-controlled gestational diabetes mellitus consisted of only five women for the investigation of the distribution of metoprolol and its metabolites in amniotic fluid, with the time between metoprolol administration and amniotic fluid collection ranging from 1.5 to 3.5 h. Thus, no statistical analyses were carried out to assess the influence of gestational diabetes mellitus on the distribution of the pharmaceuticals in amniotic fluid. The data presented in Table 4 show that there was no accumulation of metoprolol or its metabolites in the amniotic fluid, probably due to the fact that the sample was collected before the time needed for the equilibrium of drug distribution (4–8 h for the parturients of the control group).
In conclusion, the present data suggest that the kinetic disposition of metoprolol is enantioselective, with plasma accumulation of the S-(−)-MET eutomer in all parturients investigated, with or without gestational diabetes mellitus. The plasma accumulation of S-(−)-MET can be explained by the preferential metabolism of R-(+)-MET to R-(+)-AODM. Well-controlled gestational diabetes mellitus prolonged the tmax of all metoprolol and AODM enantiomers and α-hydroxymetoprolol stereoisomers. The 1′S,2R and 1′S,2S stereoisomers of α-hydroxymetoprolol were present in amniotic fluid to a greater extent than the metoprolol enantiomers, whereas the distribution of AODM in this compartment was not significant. The transplacental distribution of metoprolol and its AODM metabolite enantiomers was not influenced by gestational diabetes mellitus. However, the disease reduced by approximately 20% the transplacental distribution of the 1′S,2S, 1′R,2R and 1′R,2S-OHM stereoisomers.
Acknowledgments
The authors wish to thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support and for granting a research fellowship, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (FAEPA) for financial support.
Competing Interests
All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work.
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