single oral doses of amitriptyline and nortriptyline on different occasions. The urinary D/4-hydroxy-D ratio correlated significantly (P < 0.01) with all three ...
Amitriptyline metabolism: Association with debrisoquin hydroxylation in nonsmokers Eleven healthy nonsmokers with wide variation in the ability to hydroxylate debrisoquin (D) were given single oral doses of amitriptyline and nortriptyline on different occasions. The urinary D/4-hydroxy-D ratio correlated significantly (P < 0.01) with all three parameters of amitryptyline disposition measured (total plasma clearance, clearance by demethylation, and clearance by pathways other than demethylation), with r, = 0.89, 0.78, and 0.83, respectively. In contrast, we failed to demonstrate such correlations in a previous sample of smokers. Our data suggest that there may be a common regulation of the hydroxylation of D and the oxidative metabolism of amitriptyline in nonsmokers. It is hypothesized that an additional demethylase/hydroxylase is induced in smokers that is not involved in D hydroxylation. (CLIN PHARMACOL THER 1986;39:369-71.)
Britt Mellstrom, Ph.D., Juliette Sawe, M.D., Leif Bertilsson, Ph.D., and Folke Sjoqvist, M.D. Huddinge, Sweden Amitriptyline (AT) is mainly metabolized by demethylation to nortriptyline (NT).1 Both AT and NT are 10-hydroxylated, and it has been shown in healthy subjects and patients that these hydroxylations covary with that of debrisoquin (D).2-5 In a previous study in nine healthy subjects' we failed to demonstrate a significant relationship between the demethylation of AT and the D hydroxylation phenotype. We observed, however, that there was a significant relationship between these reactions in five nonsmokers, of which four were women. We speculated that AT demethylation is mediated by at least two isozymes of the cytochrome P-450 family, one of which was induced in smokers and the other of which was regulated in manner similar to that of the D hydroxylase. We have now extended our study by investigating the relationship between AT metabolism and D hydroxylation in six nonsmoking men.
METHODS Eleven nonsmoking subjects were investigated and the results from five of these have been reported earlier.' Subjects were selected from healthy individuals whose From the Department of Clinical Pharmacology at the Karolinska Institute, Huddinge Hospital. Supported by grants from the Swedish Medical Research Council (No. 3902) and the Karolinska Institute. Received for publication Sept. 26, 1985; accepted Nov. 25, 1985. Reprint requests to: Dr. Leif Bertilsson, Department of Clinical Pharmacology, Huddinge Hospital, S-141 86 Huddinge, Sweden.
D hydroxylation phenotype' had been determined after a single oral dose of 10 mg D (Declinax; Hoffman-La Roche). The ratio D/4-hydroxy-D was measured in the urine collected for 6 hours after the dose. D and its metabolite were determined by gas chromatography ac-
cording to Lennard et al.' Single 28.5 mg oral doses of NT HCI and 50 mg AT HC1 were given to subjects after an overnight fast, with a 2-week washout between drugs. Food was allowed after 1 hour. Nine venous blood samples over 192 hours were drawn into Venoject tubes (Terumo Medical Corp.). After centrifugation the plasma was collected and stored at 20° C. Plasma concentrations of AT were determined by HPLC' and NT was determined by GC/MS.9 The fraction of an oral dose of AT that is demethylated to NT f-NT(AT) was estimated from the equation: fNT(AT) = (AUCNT(AT)/CIOSeAT)/(AUCNT/CIOSeNT), where AUCNT(AT) is the plasma AUC of NT formed from oral AT and AUCNT is the AUC of oral NT.' The AUC was estimated by the trapezoidal rule, and the AUC from the last measurable concentration (C) to infinity (AUC(t-00)) was estimated by the relationship: AUC(t-00) = C/13, where 13 is the constant during the elimination phase. It was assumed that the administered dose of NT and the NT formed from AT are handled in the same way by the liver. Plasma clearance (CL) after oral dosing was estimated as: CL = Dose/AUC, with the assumption of complete absorption. The clearance of AT by demethylation was calculated by the
369
CLIN PHARMACOL THER APRIL 1986
370 Mellstrom et al.
2.0
0.8-
1.0
0.8 0.6
1.5
7.; 0.6 0.0
1.0
2..7c
o.s
.=
0.2
0.2
03
10
100
0.3
10
1
MataboliO ratio debrisoquin
0.83
-0.78
r,
100
0.3
10
1
100
4-0H-debrisoquin
Fig. 1. Relationship between the D metabolic ratio and amitriptyline kinetics in smokers. All relationships are significant (P < 0.01).
healthy non-
11
Table I. Plasma kinetics in eleven healthy subjects based on plasma concentrations after single doses of AT and NT Sex B. H. F. S. T. W. T. T.
6.
E. L. P. B. I. L. 0. E.* E. H.* A. M.* I. B.*
Age (yr)
Body weight (kg)
27 50 28 33 38 26 25
67 80
Urinary ratio D14-0H-D 112 47
61
8.2
0.9 0.8 0.6
29
65 69 74 53 72 58
36
65
49
52
31
60 7.0 3.0
0.7 0.4
CL, (LIhrlkg)
finy,,
0.44 0.60 0.92 0.88
0.64
1.63 1.41
0.85 0.80 1.07 1.23 1.74
0.71
0.59 0.46 0.58 0.55 0.62 0.54 0.58 0.71
0.56
f(AT) CLAT (LIhrlkg)
0.28 0.43 0.54 0.40 0.95 0.77 0.53 0.43 0.62 0.87 0.97
M = Male; F = female. *Data from these subjects have been reported.6
multiplication of f(AT) by the total body clearance of
fNT(AT), CL
AT (CLAT).
routes other than demethylation (II = -0.83; P < 0.01; Fig. 1).
RESULTS In the 11 healthy nonsmokers CLAT varied between 0.44 and 1.74 L/hr/kg, and the fraction of the dose that was demethylated varied between 0.46 and 0.71 (Table I). The relationship between fNT(AT) CLAT and the metabolic ratio D/4-0H-D in the six "new" subjects (r, = -0.77) was similar to that in the five subjects who had been previously investigated (r, = -0.90; P = 0.05).6 The relationship was highly significant for the entire group of 11 nonsmokers (r, = -0.78; < 0.01; Fig. 1). There were even stronger correlations between the D metabolic ratio and the CLAT (r, = -0.89; P < 0.01) and the clearance of AT by
DISCUSSION Our previous study6 of the demethylation of AT indicated a relationship with D hydroxylation in the five nonsmoking subjects. Because cigarette smoking is known to induce N-demethylations of several drugs such as antipyrine," imipramine,I2 and theophylline,13'14 it seems plausible to assume that this is also true for AT. Men might be slower demethylators of AT than women,' and four of the five nonsmokers in our previous study were women. We could therefore not exclude that sex is as important as smoking habits for the activity of the enzymes that demethylate AT. Our present extended study in six nonsmoking men suggests that there may be a common regulation of AT
1
VOLUME 39 NUMBER 4
demethylation and D hydroxylation. As this seems to be true both in men and women, smoking habits in addition to genetic factors are important determinants of the activity of AT demethylation. The N-demethylation of amiflamine also covaries with D hydroxylation,' while the N-demethylations of antipyrine,'" diazepam (unpublished results), and theophylline" do not. Obviously there are several cytochrome P-450 isozymes that catalyze N-demethylations of drugs. In a recent study' in human liver microsomes it was shown that the N-demethylation of imipramine was impaired in a patient who was a poor hydroxylator of D as shown both in vivo and in vitro. This gives further evidence that the N-demethylation of tertiary amine tricyclics and the hydroxylation of D are coregulated. In the present study there was a strong correlation between the clearance of AT by metabolic routes other than demethylation and D hydroxylation (rs = -0.83; n = 11; P < 0.01). As 10-hydroxylation in addition to demethylation is a major metabolic reaction of AT, further evidence is given to the suggestion of BalantGorgia et al.2 that the AT and D hydroxylations covary. The fact that the rate of demethylation of AT, and most probably also of other tertiary amine tricyclics like chlorimipramine," might vary with the D hydroxylation phenotype has clinical implications. We recently described an extremely rapid hydroxylator of D who developed very low levels of both NT and AT while following conventional dosage schedules:9
References Rollins DE, Alvan G, Bertilsson L, et al. Interindividual differences in amitriptyline demethylation. CLIN PHARMACOL THER 1980;28:121-9. Balant-Gorgia AE, Schulz P. Dayer P, et al. Role of oxidation polymorphism on blood and urine concentrations of amitriptyline and its metabolites in man. Arch Psychiatr Nervenkr 1982;232:215-22. Mellstrom B, Bertilsson L, Sawe J, Schulz H-U, Sjoqvist F. E- and Z-10-hydroxylation of nortriptyline: Relationship to polymorphic debrisoquine hydroxylation. CLIN PHARMACOL THER 1981;30:189-93. Nordin C, Siwers B, Benitez J, Bertilsson L. Plasma concentrations of nortriptyline and its 10-hydroxy metabolite in depressed patients-Relationship to the debrisoquine hydroxylation metabolic ratio. Br J Clin Pharmacol 1985;19:832-5. Woolhouse NM, Adjepon-Yamoah KK, Mellstrom B, Hedman A, Bertilsson L, Sjoqvist F. Nortriptyline and debrisoquine hydroxylations in Ghanaian and Swedish subjects. CLIN PHARMACOL THER 1984;36:374-8. Mellstrom B, Bertilsson L, Lou Y-C, Sainte J, Sjoqvist F. Amitriptyline metabolism: Relationship to poly-
Amitriptyline and debris-oquin metabolism
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morphic debrisoquine hydroxylation. CLIN PHARMACOL THER 1983;34:516-20. Mahgoub A, Idle JR, Dring LG, Lancaster R, Smith RL. Polymorphic hydroxylation of debrisoquine in man. Lancet 1977;2:584-6. Lennard MS, Silas JH, Smith AJ, Tucker GT. Determination of debrisoquine and its 4-hydroxy metabolite in biological fluids by gas chromatography with flameionization and nitrogen-sensitive detection. J Chromatogr 1977;133:161-6. Borga 0, Palmer L, Sjoqvist F, Holmstedt B. Mass fragmentography used in quantitative analysis of drugs and endogenous compounds in biological fluids. Pharmacology and the future of man. vol III. In: Acheson GH, ed. Proceedings of the Fifth International Congress on Pharmacology. Basel: S Karger AG, 1972:56-68. Eichelbaum M, Bertilsson L, Sawe J. Antipyrine metabolism in relation to polymorphic oxidations of sparteine and debrisoquine. Br J Clin Pharnaacol 1983;15:317-21. Vestal RE, Norris AH, Tobin JO, Cohen BH, Shock NW, Anders R. Antipyrine metabolism in man: Influence of age, alcohol, caffeine and smoking. CLIN PHARMACOL THER 1975;18:425-32. Perel JM, Shostak M, Gann E, Kantor SJ, Glassman AH. Pharmacodynamics of imipramine and clinical outcome in depressed patients. In: Gottshalk LA, Merlis S. eds. Pharmacokinetics of psychoactive drugs. Blood levels and clinical response. New York: Spectrum, 1976:229-41. Dahlqvist R, Bertilsson L, Birkett DJ, Eichelbaum M, Sawe J, Sjoqvist F. Theophylline metabolism in relation to antipyrine, debrisoquine, and sparteine metabolism. CLIN PHARMACOL THER 1984;35:815-21. Grygiel JJ, Birkett DJ. Cigarette smoking and theophylline clearance and metabolism. CLIN PHARMACOL THER 1981;30:491-6. Edelbroek PM, deWolff FA. Amitriptyline: looking through the therapeutic window. Lancet 1984;1:1123. Alvan G, Grind M, Graffner C, Sjoqvist F. Relationship of N-demethylation of amiflamine and its metabolite to debrisoquine hydroxylation polymorphism. CLIN PHARMACOL THER 1984;36:515-9. von Bahr C, Birgersson C, Morgan ET, et al. Oxidation of tricyclic antidepressant drugs, debrisoquine and 7ethoxyresorufin by human liver preparation. Xenobiotica. (in press) Mellstrom B, Bertilsson L, Traskman L, Rollins D, Asberg M, Sjoqvist F. Intraindividual similarity in the metabolism of amitriptyline and chlorimipramine in depressed patients. Pharmacology 1979;19:282-7. Bertilsson L, Aberg-Wistedt A, Gustafsson L, Nordin C. Extremely rapid hydroxylation of debrisoquine-A case report with implication for treatment with nortriptyline and other tricyclic antidepressants. Ther Drug Monit 1985;7:478-80.
