The hypothesis that variability in metoprolol metabolism stereoselectivity is ... metoprolol was also stereoselective but to the same extent in both EM and PM.
Differential stereoselective metabolism of metoprolol in extensive and poor debrisoquin metabolizers The hypothesis that variability in metoprolol metabolism stereoselectivity is related to debrisoquin oxidation phenotype was tested in six extensive (EM) and six poor (PM) debrisoquin
metabolizers. In EM, plasma AUCs for (S)-metoprolol were 35% higher than for (R)-metoprolol, whereas in PM, AUCs for (S)-metoprolol were lower than for (R)-metoprolol. AUCs for total metoprolol correlated with the ratio of (S)- to (R)-metoprolol AUG. The renal clearance of metoprolol was also stereoselective but to the same extent in both EM and PM. Findings suggest that the enzyme system responsible for polymorphic oxidation of the debrisoquin-type is stereoselective. The relation between log total metoprolol plasma concentration and response (/3-blockade) was shifted to the right in PM relative to EM, which is compatible with a difference in pharmacologic activity of metoprolol enantiomers. Kinetic predictions based on total drug measurements will tend to overestimate dynamic differences between EM and PM, but the magnitude of the error is relatively small, and, in absolute terms, there is a large difference in pharmacologic activity between the phenotypes (/3-blockade at 24 hr: EM PM = 18.9 ± 3.8%).
=-
5.3 ± 5.6%;
M. S. Lennard, Ph.D., G. T. Tucker, Ph.D., J. H. Silas, M.D.,*
S. Freestone, M.B.Ch.B., L. E. Ramsay, M.B.Ch.B., and H. F. Woods, D. Phil.
Sheffield, England University Department of Therapeutics, The Royal Hallamshire Hospital
Metoprolol is used as a racemic mixture of its enantiomers, (S)- and (R)-metoprolol. Although we know of no information in the literature on the relative pharmacologic activity of these forms, by analogy with propranolol and other 0-adrenoceptor antagonists, it seems likely that most of the 0-blocking activity resides in the
Presented at the British Pharmacological Society Meeting, Jan, 5-7, London, 1983. Received for publication April 15, 1983; accepted June 6, 1983. Reprint requests to: Dr. M. S. Lennard, University Department of Therapeutics, The Royal Hallamshire Hospital, Sheffield, SIO 2JF, England. *Present address: The Hypertension Unit, Clatterbridge Hospital, Bebington, Wirral, Merseyside, L63 4JY, England.
732
(S)-enantiomer.' Differences in the kinetics of the two isomers have been reported by Hermansson and von Bahr,7 who also noted marked between-subject variability in stereoselective metabolism. We demonstrated a large genetic influence on the metabolism of metoprolol with debrisoquin phenotyping as an index of polymorphic oxidation.' After a 200-mg oral dose, plasma metoprolol concentrations were six times as high and elimination t1/2s were three times as long in the poor (PM) than in the extensive (EM) debrisoquin metabolizers In the present study the hypothesis was tested that variability in the stereoselective metabolism of metoprolol is related to oxidation phenotype. .
Volume 34
Number
Stereoselective metabolism of metoprolol
6
EM
subject
PM
733
subject
300
Plasma concentration (ng/ml)
100
(S)-M
10
(R)-M
8
12
0
6
12
24
Time (h)
Fig. 1. Log plasma concentration-time curves for (S)-metoprolol [(S)-M] and (R)-metoprolol [(R)-M] after 200 mg metoprolol by mouth to one EM and to one PM of debrisoquin.
Methods Our subjects were six EM and six PM of debrisoquin. There were four hypertensive men in each group, matched for age (EM, 56 SD yr; PM, 58 ± 11 SD yr). The remaining subjects were healthy men aged 28 to 35 yr. The groups were matched for alcohol and tobacco use. All hypertensive subjects were taking atenolol, which they discontinued 72 hr before the study. Hepatic and renal function were within normal limits in all subjects. Metoprolol tartrate (200 mg) was given orally 1.5 hr after a light breakfast. Venous blood was drawn before (0) and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hr after dosing, and a 24-hr urine collection was made. Subjects exercised on a bicycle ergometer for 4 min at a work load sufficient to achieve a heart rate of over 130 bpm in those over 60 yr and 140 bpm in younger subjects. This test was performed before and at 2, 12, and 24 hr after metoprolol dosing. After exercise, heart rate was measured from an ECG with the mean of the first four beats after exercise. /3-Blockade was expressed as percentage reduction of control heart rate. Debrisoquin oxidation phenotype was determined by measuring concentrations of debrisoquin and 4-hydroxydebrisoquin in an 8-hr 1
urine sample collected after 10 mg debrisoquin hemisulfate by mouth." Subjects with debrisoquin/4-hydroxydebrisoquin ratios above 12.6 were designated PM and those with ratios below 12.6, EM.4 Plasma and urine concentrations of total metoprolol were measured by HPLC.9 The ratios of metoprolol enantiomer concentrations were determined by a modification of the HPLC method of Hermansson and von Bahr,' involving separation of the (R)- and (S)-isomers as their diastereoisomeric L-leucine derivatives as follows. Plasma or urine (0.5 to 1 ml) and sodium carbonate solution (1 ml of 0.5M) were shaken gently with dichloromethane (5 ml for 10 min). After centrifugation and removal of the upper aqueous layer, the organic layer was evaporated to dryness at 30° in a Buchler Vortex Evaporator. The residue was dissolved in dichloromethane (250 ,u1) containing triethylamine (35.6 ,umol) and a tenfold dilution of butoxycarbonyl-L-leucine anhydride reagent' (200 fil) and left at room temperature for 30 min. After evaporation of the reaction mixture to dryness, trifluoroacetic acid (250 jul) was incubated with the residue (0° for 10 min) to remove the butoxycarbonyl-protecting group. Excess acid was neutralized with NaOH solution (2 ml of 2M) and the free diastereoisomers were
Clin. Pharmacol. Ther.
734 Lennard et al.
December 1983
Table I. AUCs and elimination t1/2s of (S)-metoprolol [(S)-M] and (R)-metoprolol [(R)-M] in EM and PM of debrisoquin after a 200-mg oral dose of metoprolol. ml
AUC (ng (S)-M
(R)-M
188 159
4
349 269 723 553
582 468
5
1336
1261
6
844
742
Mean SD PM
679 388
408 409
3920 3779 4565 4127 2657
Subject
-1
hr)
t1/2
(S)-M
(hr)
Ratio (R)-M
(S)-M
(R)-M
1.86 1.69 1.24 1.18 1.09 1.14
1.96 1.60 2.66 2.81
2.07
1.37
2.87
2.47 2.73 4.48 3.22 2.76
0.32
1.09
1.01
0.87 0.94 0.85
6.09 6.39 7.84 9.98 5.82 7.13
6.46 6.60 9.35
EM 1
2 3
3
3414 3563 3862
4
4121
5
2314 3312
1
7
6
Mean SD
3431
623
1.00
0.87 0.88 0.90 0.06
3761
3802 635
Statistics: AUC EM: (S)-M:(R)-M; P < 0.05. AUC PM: (S)-M:(R)-M; P 0.05. t1/2 PM: (S)-M:(R)-M; P < 0.05.
P>
extracted into diethyl ether (5 ml for 10 min) and then back extracted into dilute orthophosphoric acid (100 ttl of 0.1M). An aliquot of the acid phase (5 to 90 jul) was injected into the chromatograph, which consisted of a Waters 6000A pump, a Rheodyne 7125 injector (0.5 ml loop), a column (10 cm x 5 mm) packed with Hypersil 5-0DS, and a Schoeffel 970FS fluorimetric detector. A water-acetonitrile mixture (70:30) containing 1% triethylamine and adjusted to pH3 with orthophosphoric acid was used as the mobile phase. Chromatography was performed isocratically at a flow rate of 2 ml/min and at ambient temperature. The detector was operated at an excitation wavelength of 193 nm without a cutoff emission filter. The diastereoisomers were identified by analysis of reference standards of (R)- and (S)-metoprolol. Less than 3% isomeric contamination was present in each enantiomer. Plasma drug AUCs were calculated by the trapezoidal rule and extrapolated to time infinity. Renal clearance of the enantiomers was cal-
< 0.05.
1.58
4.56 3.61
6.30 7.19 7.69
7.21 1.54
(S)-M AUC (R)-M AUC EM :PM; P
< 0.001.
1.68 t1/2
EM: (S)-M:(R)-M;
culated by dividing the amount excreted in the 24-hr urine sample by the plasma AUC time curve up to 24 hr. Statistical methods used were the Wilcoxon test for paired data, the MannWhitney U test for unpaired data and Spearman 's rank correlation coefficient (rs). Differences in plasma concentration-response relationships between phenotypes were assessed by an analysis of covariance test after angular transformation of the 0-blockade data to make their distribution normal. Results
Plasma concentrations and AUCs for (S)metoprolol were higher than for the (R)-form in EM, whereas the opposite was true in PM (mean ± SD ratio (S)-metoprolol AUC/(R)metoprolol AUC: EM, 1.37 ± 0.32; PM, 0.9 ± 0.06) (Fig. 1, Table I). AUCs for total metoprolol were inversely related to the ratio of (S)metoprolol AUC/(R)-metoprolol AUC (Fig. 2). Elimination tihs of (R)-metoprolol were longer than those of (S)-metoprolol in PM (Table I), but
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Stereoselective metabolism of metoprolol
Number 6
Table II. Renal clearance values of (S)-M and (R)-M in EM and PM of debrisoquin. Data were available from only five EM owing to technical difficulties in the urine analysis in one case Renal clearance (ml
20. EM AUC(Fo-m
10
Subject
Ratio
(R)-M
70.6 96.5 82.5 37.8 60.9 69.7 22.2
77.6
0.91
101.1
0.95 0.94 0.86 0.94 0.92 0.04
3
4 5
Mean SD 1
4 5 6
Mean SD
cto)
0
-o
05
87.6 42.7 64.9 74.8 22.3
5.000
A UC TOTAL
65.5 83.6 32.5 80.6 55.2 21.4 56.5 25.3
68.9 92.4 37.8 84.2 63.2 25.9 62.1
25.9
0.95 0.90 0.86 0.96 0.87 0.83 0.90 0.05
Statistics: (S)-M: EM:PM; P > 0.05. (R)-M: EM:PM; 0.05. (S)-M < (R)-M; P < 0.01.
10,000
Ing/m1.111
Fig. 2. Relationship between the ratio of (5)-metoprolol:(R)-metoprolol AUC (AUC/s)-m/AUCao-m) and total [(5)- + (R)-] metoprolol AUC (AUG..) after 200 mg metoprolol by mouth. The dashed line indicates absence of stereoselectivity.
PM 2 3
-
(R)-1
EM 2
rs.- 0-87, p
