Br. J. clin. Pharmac. (1990), 29, 248-253
Quinidine kinetics after a single oral dose in relation to the sparteine oxidation polymorphism in man K. BR0SEN, F. DAVIDSEN & L. F. GRAM Department of Clinical Pharmacology, Odense University. J.B. Winsl0ws Vej 19, DK-5000 Odense C, Denmark
The kinetics at a single oral dose (400 mg) of quinidine were studied in four extensive metabolizers (EM) and four poor metabolizers (PM) of sparteine. The clearance of quinidine by 3-hydroxylation was significantly lower in PM than in EM, but the difference was small (25-30%). This finding suggests that 3-hydroxylation, in part, is catalyzed by the same isoenzyme of cytochrome P450, P450dbl which oxidizes sparteine. Otherwise, no significant phenotypic differences in total or metabolic clearance were found and it is concluded that the metabolism of quinidine is largely carried out by P450 isoenzymes different from P450dbl. A biexponential decline in the log plasma quinidine concentration vs time curves was observed in all subjects, and the mean elimination half-life was 11-12 h. This is about twice as long as generally reported in the literature.
Keywords quinidine sparteine genetic polymorphism metabolism pharmacokinetics Introduction The oxidation of the pharmacogenetic probe drugs sparteine and debrisoquine reflects the activity of a distinct isoenzyme of cytochrome P450, P450dbl (Gonzalez et al., 1988). The oxidations exhibit genetic polymorphism in vivo (Eichelbaum et al., 1979; Evans et al., 1980) and in Caucasians two phenotypes are clearly distinguished: about 7% are poor metabolizers (PM) and more than 90% are extensive metabolizers (EM). In the livers of PM, different forms of incorrectly spliced mRNA for P450dbl have been identified (Gonzalez et al., 1988), and this may explain why P450dbl is absent in the PM (Zanger et al., 1988). The elimination of more than 20 clinically used drugs is influenced by the sparteine/debrisoquine oxidation polymorphism (Br0sen & Gram, 1989). In preparations of human liver microsomes, quinidine has been shown to be a very potent competitive inhibitor of sparteine and debrisoquine oxidation (Guengerich et al., 1986; Inaba
et al., 1985; Otton et al., 1986). In accordance with this, clinical studies have shown that therapeutic doses of quinidine almost completely abolish the oxidation of sparteine and debrisoquine in EM subjects (Brinn et al., 1986; Br0sen et al., 1987; Leemann et al., 1986; Speirs et al., 1986). Many in vitro inhibitors of sparteine/debrisoquine oxidation, such as tricyclic antidepressants and beta-adrenoceptor antagonists have been shown to be themselves substrates for P450dbl (Inaba et al., 1985; Otton et al., 1983, 1984). Therefore, it could be expected that quinidine also is oxidized by this isoenzyme. In a small sample of quinidine treated patients, we found that one patient who was a PM for sparteine, had a lower 3-OH-quinidine/quinidine steadystate plasma concentration ratio than the remaining seven EM patients (Brinn et al., 1986). In contrast, Mikus et al. (1986), in a panel study, found no differences in quinidine kinetics be-
Correspondence: Dr Kim Br0sen, Department of Clinical Pharmacology, Odensc University, J.B. Winsl0ws Vej 19, DK-50(0 Odense C, Denmark
248
Short report tween three PM and three EM subjects, but the few subjects and the use of intravenous quinidine administration, with a lower fraction of quinidine being eliminated through metabolism (Rakhit et al., 1984), may have reduced the power of that study. Guengerich et al. (1986), using purified human P450 isoenzymes, found that 3-OHquinidine formation from quinidine was mediated by P450nf (mediating nifedipine oxidation), but not to any extent by P450dbl. To what extent these in vitro data can be related to the in vivo situation is not known, however. Finally, Otton et al. (1988) found in human liver microsome preparations that sparteine or debrisoquine added in concentrations up to 250 F.M, did not influence the rate of 3-OH-quinidine formation from quinidine or the overall disappearance rate of quinidine. However, using this technique, a minor involvement of P450dbl in the metabolism of quinidine cannot be excluded. In order to elucidate further the involvement of P450dbl in the oxidation of quinidine, we investigated the kinetics of quinidine after a single oral dose in panels of four EM and four PM of sparteine.
Methods Seven healthy men and one healthy woman, aged 21-38 years, participated in the study. All volunteers were drug free and none had a daily alcohol intake. Four subjects were phenotyped as sparteine EM (sparteine/dehydrosparteines in 12-h urine: 0.41-0.97) and four subjects as PM (sparteine/dehydrosparteines in 12-h urine: 58-161) (Br0sen et al., 1985). The volunteers consented to participate on the basis of verbal and written information, and the protocol was approved by the regional ethics committee. At 08.00 h after an overnight fast each volunteer took a single oral dose of 400 mg quinidine sulphate (conventional capsules). The capsules were swallowed with tap water and neither food nor drink was allowed for the following 3 h. Blood samples were drawn through a heparinized intravenous catheter (Venflon) at 30 min intervals for the first 3 h and then hourly for the following 13 h. Additional blood samples were drawn by venepuncture 24, 30 and 48 h after the test dose. Blood was collected in heparinized test-tubes and was centrifuged immediately. Plasma was kept frozen (-20° C) until analysis. Urine was collected at 4 h intervals for the first 16 h and then for 16-24 h, 24-30 h and 30-48 h. After recording urine volume, an aliquot was kept frozen (-20° C) until analysis. The sub-
249
jects were kept in the ward for the first 16 h, and apart from fasting they were permitted normal activity. The volunteers were under continual surveillance by one or two physicians and no side effects or adverse reactions were observed. Quinidine and 3-OH-quinidine in plasma and urine were assayed by quantitative thin layer chromatography. Briefly, 400 ,ul of the standard and subject samples, alkalinized with 150 ,ul 25% ammonia, were extracted with 500 RI dichlorethane (vortexed 30 min). The organic phase was split into two portions of 50 (or 100) and 350 ,ul respectively, and transferred to separate t.l.c. plates (Merck DC 5729) using a Desaga Autospotter. In this way linear standard curves up to concentrations of about 20 nmol ml-l and with a lower level of detection of about 0.05 nmol ml-' were constructed. Standard curves were constructed by adding known amounts of quinidine and 3-OH-quinidine to blank plasma and extracted as the other samples and run on each t.l.c. plate. Urines were diluted 1:10 with blank urine before assay. After development in a system of chloroform: acetone: diethylamine 25: 20: 7, the two compounds were separated with RFvalues of 0.67 for quinidine and 0.25 for 3-OH-quinidine. In situ scanning of the plates was carried out using a Zeiss KM3 densitometer with simultaneous transmission-reflectance measurement at 330 nm. All samples were assayed in duplicate and the day to day reproducibility (coefficient of variation) was 4-8% at concentrations above 0.1 nmol ml-'. The 4-8 h urines from all subjects were also assayed for quinidine + 3-OH-quinidine after treatment with 1-glucuronidase/arylsulphatase (Boehringer, Mannheim, FRG) at 370 C for 16 h. Identical concentrations were found with and without enzyme treatment, and control samples kept at 370 C for 16 h without enzyme showed no significant decay of either of the two compounds. The presence of glucuronide metabolites in the urine could therefore be excluded. Areas under the plasma concentration curves for quinidine, AUCq, and 3-OH-quinidine, AUC3OHq, were calculated using the linear trapezoidal rule with extrapolation to infinity using the elimination rate constant of the final slope (Xz) determined by log-linear regression analysis of measurements at 24, 30 and 48 h. On average the extrapolated area represented 4.4% of the total AUC in both EM and PM (range: 3.0-7.0%). The total clearance of quinidine was calculated from: CL Dose/AUCq (equation 1) =
K. Br0sen, F. Davidsen & L. F. Gram
250
The systemic availability of quiniidine was reported to be 70-90%, the loss beiing related to first-pass metabolism (Rakhit et al.., 1984; Ueda et al., 1976). This is in accordaince with the clearance values found in this and (earlier studies (Mikus et al., 1986; Ochs et al., 1 980; Ueda & Dzindzio, 1978). Renal clearance was calculated from: CLR =
quinidine excreted in uriine (0-48 h) AUCq (0-48 h
(equation 2) Metabolic clearance was calculaited from:
CLM = CL - CLR
(equation 3)
The partial clearance to 3-hydrroxyquinidine was calculated from: CLq 3OH = 3-OH-quinidine excreted in urine (0-48 h) AUCq (0-48 h) (equation 4) -
If 3-OH-quinidine is subject to,further metabolism in addition to renal eliminat -ion, the value obtained with equation 4 will unde restimate the true clearance by 3-hydroxylationl (see Discusi sion). The renal clearance of 3-OH-qiuinidine was calculated from:
t,,2
=
In2/X,
Discussion
(equation 5)
The clearance values found for quinidine in this study are in accordance with those reported by others (Ochs et al., 1980). This also applies to the high renal clearance values of 3-OH-quinidine we found which are close to those of Vozeh et al. (1985) who administered 3-OH-quinidine
half-Ife, l, t½, htfe,
to healthy subjects. directly The panel study ruled out
CLR(3OHq) = 3-OH-quinidine excreted in uri ne (0-48 h) AUC3 OH q (0-48 h Finally, the terminal elimination was calculated from:
total, 44-81% of the dose was recovered in the urine as quinidine + 3-OH-quinidine within 48 h. A biexponential decline in the log-plasma drug concentration curves was apparent in several subjects (Figure 1). Thus, the initial absorption- and distribution-phases continued up to 16-24 h, followed by an apparently monoexponential elimination phase. The mean terminal elimination half-life was 12 and 11 h in EM and PM, respectively (Table 1). The calculated pharmacokinetic parameters are listed in Table 1. The mean total clearance of quinidine (equation 1) was 14.8 1 h-' in EM and 10.71 h-' in PM (NS). The metabolic clearance was (equation 3): 9.6 1 h-' in EM vs 6.4 1 h- in PM (NS). In both phenotypes the partial clearance via 3-hydroxylation (equation 4) represented only about one third of the metabolic clearance, but was significantly lower in PM (mean: 2.4 1 h -) than in EM (mean: 3.1 I h-). In both phenotypes, the renal clearance of 3-OH-quinidine (equation 5) was about five times larger than its formation clearance. Accordingly, formation of 3-OH-quinidine was rate limiting such that AUC3 OH q was smaller than the AUCq and the terminal decline in the log concentration of the metabolite parallelled that of quinidine (Figure 1) (Rowland & Tozer, 1980).
f
(equation 6)
The Mann-Whitney test for uinpaired data used for the group compariisons, and a significance level of 5% was emplh oyed.
was
Results The mean 48 h recovery (% of dose ) of quinidine was 37% (27-53%) and of 3-OHI-quinidine it was 21% (16-26%). There was rio difference between phenotypes in these re-coveries. In
a major involvement of P450dbl in the metabolism of quinidine. However, the 20% lower formation clearance of 3-OH-quinidine found in PM compared with EM at a significance level of 5% suggests that a fraction of the quinidine dose might be metaother hand, it could bolised by P450dbl. On theerror because of the reflect a type I statistical small number of subjects studied. It is also possible that further metabolism of 3-OH-quinidine
might confound the calculation of its formation clearance. Although, like Vozeh et al. (1985),
we did not find any glucuronide of 3-OHquinidine in the urine, only 25% of a dose of 3-OH-quinidine given directly has been recovered in the urine.
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The finding of longer elimination half-lives than previously reported was an unexpected finding. Although the calculation of t./2 from only three measurements may be criticised, all three values, except the last one in one subject (EM 4) were well above the assay limit and were determined with good precision. Furthermore, the inclusion of measurements from 14 h generally yielded similar tl, values, and plasma 3-OH-quinidine declined at the same rate. Earlier studies in which the half-life of quinidine was estimated (Mahon et al., 1976; Mikus et al., 1986; Ochs et al., 1980; Rakhit et al., 1984; Ueda & Dzindzio, 1978) all used shorter sampling times. Furthermore, an undetected prolonged terminal t.12 may explain why observed steady-state drug concentrations during multiple dosing were found to be higher than those predicted from single dose kinetics of quinidine
(Mahon et al., 1976). Finally, our finding is in accordance with the long duration of debrisoquine or sparteine MR-elevation after even small doses of quinidine (Boobis et al., 1988; Nielsen et al., data to be published). A more definite evaluation of the elimination kinetics of quinidine should be carried out as a post multiple dose/steady-state study with more frequent sampling in the 15-50 h period and perhaps also with a longer sampling period. Such a study should be combined with further efforts to elucidate the possible involvement of P450dbl in the oxidation of quinidine. This study was supported by a grant from the Lundbeck Foundation and the P. Carl Petersen's Foundation. The authors wish to express their gratitude to Mrs G. Rodtborg Nielsen for performing the drug assays. The results described in this publication represent a contribution to the aims of COST B1.
References Boobis, A. R., Edwards, R. J., Singleton, A., Sesardic, D., Murray, B., Speirs, C. J., Murray, S., Kobayashi, S. & Davies, D. S. (1988). The contribution of polymorphic isozymes of cytochrome P450 to the pharmacokinetics and toxicity of foreign compounds in man. In Microsomes and drug oxidations. Proceedings of the 7th international symposium, Adelaide, Australia, 17-21 August 1987, eds. Miners, J. O., Birkett, D. J., Drew, R., May, B. K. & McManus, M. E., pp. 216-23. London: Taylor and Francis. Brinn, R., Br0sen, K., Gram. L. F., Haghfelt, T. & Otton, S. V. (1986). Sparteine oxidation is practi-
cally abolished in quinidine-treated patients. Br. J. clin. Pharmac., 22, 194-197. Br0sen, K., Otton, S. V. & Gram, L. F. (1985). Sparteine oxidation polymorphism in Denmark (1985). Acta Pharmac. Tox., 57, 357-360. Br0sen, K., Gram, L. F., Haghfelt, T. & Bertilsson, L. (1987). Extensive metabolizers of debrisoquine become poor metabolizers during quinidine treatment. Pharmac. Tox., 60, 313-314. Br0sen, K. & Gram, L. F. (1989). The clinical significance of the sparteine/debrisoquine oxidation polymorphism. Eur. J. clin. Pharmac., 36, 537547.
Short report Drayer. D. E., Lowenthal, D. T., Restivo, K. M., Schwartz, A., Cook. C. E. & Reidenberg, M. M. (1978). Steady-state serum levels of quinidine and active metabolites in cardiac patients with varying degrees of renal function. Clin. Pharmac. Ther.. 24, 31-39. Eichelbaum. M., Spannbrucker, N., Steincke, B. & Dengler, H. J. (1979). Defective N-oxidation of sparteine in man: a new pharmacogenetic defect. Eur. J. clin. Pharmac.. 16, 183-187. Evans, D. A.. Mahgoub, A., Sloan, T. P., Idle, J. R. & Smith, R. L. (1980). A family and population study of the genetic polymorphism of debrisoquine oxidation in a white British population. J. med. Genetics. 17, 102-105. Gonzalez, F. J.. Skoda, R. C., Kimura, S., Umeno, M., Zanger, U. M., Nebert, D. W., Gelboin, H. V., Hardwick, J. P. & Meyer, U. A. (1988). Characterization of the common genetic defect in humans deficient in debrisoquine metabolism. Nature, 331, 442-446. Guengerich, F. P., Miiller-Enoch, D. & Blair, I. A. (1986). Oxidation of quinidine by human liver cytochrome P450. Mol. Plharmac., 30, 287-295. Inaba, T., Nakano, M., Otton, S. V., Mahon, W. A. & Kalow, W. (1985). A human cytochrome P450 characterized by inhibition studies of the sparteinedebrisoquine monooxygenase. Can. J. Physiol. Plharmac.. 62, 860-862. Leemann, T., Dayer, P. & Meyer, U. A. (1986). Single dose quinidine treatment inhibits metoprolol oxidation in extensive metabolizers. Eur. J. clin. Pharmac., 29, 739-741. Mahon, W. A., Mayersohn, M. & Inaba, T. (1976). Disposition kinetics of two oral forms of quinidine. Clin. Pharmac. Ther., 19, 566-575. Mikus, G., Ha, H. R., Vozeh, S., Zekom, C., Follath, F. & Eichelbaum, M. (1986). Pharmacokinetics and metabolism of quinidine in extensive and poor metabolizers of sparteine. Eur. J. clin. Plharmac., 31, 69-72. Ochs, H. R., Greenblatt, D. J. & Woo, E. (1980). Clinical pharmacokinetics of quinidine. Clin. Pharmacokin., 5. 150-168.
253
Otton, S. V., Inaba, T. & Kalow, W. (1983). Inhibition of sparteine oxidation in human liver by tricyclic antidepressants and other drugs. Life Sci., 32, 795-800. Otton, S. V., Inaba, T. & Kalow, W. (1984). Competitive inhibition of sparteine oxidation in human liver by beta-adrenoceptor antagonists and other cardiovascular drugs. Life Sci., 34, 73-80. Otton, S. V., Brinn, R. & Gram, L. F. (1988). In vitro evidence against the oxidation of quinidine by the sparteine/debrisoquine monooxygenase of the human liver. Drug Metab. Disp., 16. 15-17. Rakhit, A., Holford, N. H. G., Guentert, T. G., Maloney, K. & Riegelman, S. (1984). Pharmacokinetics of quinidine and three of its metabolites in man. J. Pharmacokin. Biopharm., 12, 1-21. Rowland, M. & Tozer, T. N. (1980). Clinical pharmacokinetics: concepts and applications. Philadelphia: Lea and Febiger. Speirs, C. J., Murray, S., Boobis, A. R., Seddon, C. E. & Davies, D. S. (1986). Quinidine and the identification of drugs whose elimination is impaired in subjects classified as poor metabolizers of debrisoquine. Br. J. clin. Pharmac., 22, 739743. Ueda, C. T. & Dzindzio, B. S. (1978). Quinidine kinetics in congestive heart failure. Clin. Pharmac. Ther., 23, 158-164. Ueda, C. T., Williamson, B. J. & Dzindzio, B. S. (1976). Absolute quinidine bioavailability. Clin. Pharmac. Ther., 20, 260-265. Vozeh, S., Uematsu, T., Guentert, T. W., Riem, H. & Follath, F. (1985). Kinetics and electrocardiographic changes after oral 3-OH-quinidine in healthy subjects. Clin. Pharmac. Ther., 37, 575581. Zanger, U. M., Hauri, H. P., Loeper, J., Homberg, J. C. & Meyer, U. A. (1988). Absence of hepatic cytochrome P450bufI causes genetically deficient debrisoquine oxidation in man. Biochemistry, 27, 5447-5454.
(Received 11 May 1989, accepted 9 October 1989)
Quinidine kinetics after a single oral dose in relation to the sparteine oxidation polymorphism in man K. BR0SEN, F. DAVIDSEN & L. F. GRAM Department of Clinical Pharmacology, Odense University. J.B. Winsl0ws Vej 19, DK-5000 Odense C, Denmark
The kinetics at a single oral dose (400 mg) of quinidine were studied in four extensive metabolizers (EM) and four poor metabolizers (PM) of sparteine. The clearance of quinidine by 3-hydroxylation was significantly lower in PM than in EM, but the difference was small (25-30%). This finding suggests that 3-hydroxylation, in part, is catalyzed by the same isoenzyme of cytochrome P450, P450dbl which oxidizes sparteine. Otherwise, no significant phenotypic differences in total or metabolic clearance were found and it is concluded that the metabolism of quinidine is largely carried out by P450 isoenzymes different from P450dbl. A biexponential decline in the log plasma quinidine concentration vs time curves was observed in all subjects, and the mean elimination half-life was 11-12 h. This is about twice as long as generally reported in the literature.
Keywords quinidine sparteine genetic polymorphism metabolism pharmacokinetics Introduction The oxidation of the pharmacogenetic probe drugs sparteine and debrisoquine reflects the activity of a distinct isoenzyme of cytochrome P450, P450dbl (Gonzalez et al., 1988). The oxidations exhibit genetic polymorphism in vivo (Eichelbaum et al., 1979; Evans et al., 1980) and in Caucasians two phenotypes are clearly distinguished: about 7% are poor metabolizers (PM) and more than 90% are extensive metabolizers (EM). In the livers of PM, different forms of incorrectly spliced mRNA for P450dbl have been identified (Gonzalez et al., 1988), and this may explain why P450dbl is absent in the PM (Zanger et al., 1988). The elimination of more than 20 clinically used drugs is influenced by the sparteine/debrisoquine oxidation polymorphism (Br0sen & Gram, 1989). In preparations of human liver microsomes, quinidine has been shown to be a very potent competitive inhibitor of sparteine and debrisoquine oxidation (Guengerich et al., 1986; Inaba
et al., 1985; Otton et al., 1986). In accordance with this, clinical studies have shown that therapeutic doses of quinidine almost completely abolish the oxidation of sparteine and debrisoquine in EM subjects (Brinn et al., 1986; Br0sen et al., 1987; Leemann et al., 1986; Speirs et al., 1986). Many in vitro inhibitors of sparteine/debrisoquine oxidation, such as tricyclic antidepressants and beta-adrenoceptor antagonists have been shown to be themselves substrates for P450dbl (Inaba et al., 1985; Otton et al., 1983, 1984). Therefore, it could be expected that quinidine also is oxidized by this isoenzyme. In a small sample of quinidine treated patients, we found that one patient who was a PM for sparteine, had a lower 3-OH-quinidine/quinidine steadystate plasma concentration ratio than the remaining seven EM patients (Brinn et al., 1986). In contrast, Mikus et al. (1986), in a panel study, found no differences in quinidine kinetics be-
Correspondence: Dr Kim Br0sen, Department of Clinical Pharmacology, Odensc University, J.B. Winsl0ws Vej 19, DK-50(0 Odense C, Denmark
248
Short report tween three PM and three EM subjects, but the few subjects and the use of intravenous quinidine administration, with a lower fraction of quinidine being eliminated through metabolism (Rakhit et al., 1984), may have reduced the power of that study. Guengerich et al. (1986), using purified human P450 isoenzymes, found that 3-OHquinidine formation from quinidine was mediated by P450nf (mediating nifedipine oxidation), but not to any extent by P450dbl. To what extent these in vitro data can be related to the in vivo situation is not known, however. Finally, Otton et al. (1988) found in human liver microsome preparations that sparteine or debrisoquine added in concentrations up to 250 F.M, did not influence the rate of 3-OH-quinidine formation from quinidine or the overall disappearance rate of quinidine. However, using this technique, a minor involvement of P450dbl in the metabolism of quinidine cannot be excluded. In order to elucidate further the involvement of P450dbl in the oxidation of quinidine, we investigated the kinetics of quinidine after a single oral dose in panels of four EM and four PM of sparteine.
Methods Seven healthy men and one healthy woman, aged 21-38 years, participated in the study. All volunteers were drug free and none had a daily alcohol intake. Four subjects were phenotyped as sparteine EM (sparteine/dehydrosparteines in 12-h urine: 0.41-0.97) and four subjects as PM (sparteine/dehydrosparteines in 12-h urine: 58-161) (Br0sen et al., 1985). The volunteers consented to participate on the basis of verbal and written information, and the protocol was approved by the regional ethics committee. At 08.00 h after an overnight fast each volunteer took a single oral dose of 400 mg quinidine sulphate (conventional capsules). The capsules were swallowed with tap water and neither food nor drink was allowed for the following 3 h. Blood samples were drawn through a heparinized intravenous catheter (Venflon) at 30 min intervals for the first 3 h and then hourly for the following 13 h. Additional blood samples were drawn by venepuncture 24, 30 and 48 h after the test dose. Blood was collected in heparinized test-tubes and was centrifuged immediately. Plasma was kept frozen (-20° C) until analysis. Urine was collected at 4 h intervals for the first 16 h and then for 16-24 h, 24-30 h and 30-48 h. After recording urine volume, an aliquot was kept frozen (-20° C) until analysis. The sub-
249
jects were kept in the ward for the first 16 h, and apart from fasting they were permitted normal activity. The volunteers were under continual surveillance by one or two physicians and no side effects or adverse reactions were observed. Quinidine and 3-OH-quinidine in plasma and urine were assayed by quantitative thin layer chromatography. Briefly, 400 ,ul of the standard and subject samples, alkalinized with 150 ,ul 25% ammonia, were extracted with 500 RI dichlorethane (vortexed 30 min). The organic phase was split into two portions of 50 (or 100) and 350 ,ul respectively, and transferred to separate t.l.c. plates (Merck DC 5729) using a Desaga Autospotter. In this way linear standard curves up to concentrations of about 20 nmol ml-l and with a lower level of detection of about 0.05 nmol ml-' were constructed. Standard curves were constructed by adding known amounts of quinidine and 3-OH-quinidine to blank plasma and extracted as the other samples and run on each t.l.c. plate. Urines were diluted 1:10 with blank urine before assay. After development in a system of chloroform: acetone: diethylamine 25: 20: 7, the two compounds were separated with RFvalues of 0.67 for quinidine and 0.25 for 3-OH-quinidine. In situ scanning of the plates was carried out using a Zeiss KM3 densitometer with simultaneous transmission-reflectance measurement at 330 nm. All samples were assayed in duplicate and the day to day reproducibility (coefficient of variation) was 4-8% at concentrations above 0.1 nmol ml-'. The 4-8 h urines from all subjects were also assayed for quinidine + 3-OH-quinidine after treatment with 1-glucuronidase/arylsulphatase (Boehringer, Mannheim, FRG) at 370 C for 16 h. Identical concentrations were found with and without enzyme treatment, and control samples kept at 370 C for 16 h without enzyme showed no significant decay of either of the two compounds. The presence of glucuronide metabolites in the urine could therefore be excluded. Areas under the plasma concentration curves for quinidine, AUCq, and 3-OH-quinidine, AUC3OHq, were calculated using the linear trapezoidal rule with extrapolation to infinity using the elimination rate constant of the final slope (Xz) determined by log-linear regression analysis of measurements at 24, 30 and 48 h. On average the extrapolated area represented 4.4% of the total AUC in both EM and PM (range: 3.0-7.0%). The total clearance of quinidine was calculated from: CL Dose/AUCq (equation 1) =
K. Br0sen, F. Davidsen & L. F. Gram
250
The systemic availability of quiniidine was reported to be 70-90%, the loss beiing related to first-pass metabolism (Rakhit et al.., 1984; Ueda et al., 1976). This is in accordaince with the clearance values found in this and (earlier studies (Mikus et al., 1986; Ochs et al., 1 980; Ueda & Dzindzio, 1978). Renal clearance was calculated from: CLR =
quinidine excreted in uriine (0-48 h) AUCq (0-48 h
(equation 2) Metabolic clearance was calculaited from:
CLM = CL - CLR
(equation 3)
The partial clearance to 3-hydrroxyquinidine was calculated from: CLq 3OH = 3-OH-quinidine excreted in urine (0-48 h) AUCq (0-48 h) (equation 4) -
If 3-OH-quinidine is subject to,further metabolism in addition to renal eliminat -ion, the value obtained with equation 4 will unde restimate the true clearance by 3-hydroxylationl (see Discusi sion). The renal clearance of 3-OH-qiuinidine was calculated from:
t,,2
=
In2/X,
Discussion
(equation 5)
The clearance values found for quinidine in this study are in accordance with those reported by others (Ochs et al., 1980). This also applies to the high renal clearance values of 3-OH-quinidine we found which are close to those of Vozeh et al. (1985) who administered 3-OH-quinidine
half-Ife, l, t½, htfe,
to healthy subjects. directly The panel study ruled out
CLR(3OHq) = 3-OH-quinidine excreted in uri ne (0-48 h) AUC3 OH q (0-48 h Finally, the terminal elimination was calculated from:
total, 44-81% of the dose was recovered in the urine as quinidine + 3-OH-quinidine within 48 h. A biexponential decline in the log-plasma drug concentration curves was apparent in several subjects (Figure 1). Thus, the initial absorption- and distribution-phases continued up to 16-24 h, followed by an apparently monoexponential elimination phase. The mean terminal elimination half-life was 12 and 11 h in EM and PM, respectively (Table 1). The calculated pharmacokinetic parameters are listed in Table 1. The mean total clearance of quinidine (equation 1) was 14.8 1 h-' in EM and 10.71 h-' in PM (NS). The metabolic clearance was (equation 3): 9.6 1 h-' in EM vs 6.4 1 h- in PM (NS). In both phenotypes the partial clearance via 3-hydroxylation (equation 4) represented only about one third of the metabolic clearance, but was significantly lower in PM (mean: 2.4 1 h -) than in EM (mean: 3.1 I h-). In both phenotypes, the renal clearance of 3-OH-quinidine (equation 5) was about five times larger than its formation clearance. Accordingly, formation of 3-OH-quinidine was rate limiting such that AUC3 OH q was smaller than the AUCq and the terminal decline in the log concentration of the metabolite parallelled that of quinidine (Figure 1) (Rowland & Tozer, 1980).
f
(equation 6)
The Mann-Whitney test for uinpaired data used for the group compariisons, and a significance level of 5% was emplh oyed.
was
Results The mean 48 h recovery (% of dose ) of quinidine was 37% (27-53%) and of 3-OHI-quinidine it was 21% (16-26%). There was rio difference between phenotypes in these re-coveries. In
a major involvement of P450dbl in the metabolism of quinidine. However, the 20% lower formation clearance of 3-OH-quinidine found in PM compared with EM at a significance level of 5% suggests that a fraction of the quinidine dose might be metaother hand, it could bolised by P450dbl. On theerror because of the reflect a type I statistical small number of subjects studied. It is also possible that further metabolism of 3-OH-quinidine
might confound the calculation of its formation clearance. Although, like Vozeh et al. (1985),
we did not find any glucuronide of 3-OHquinidine in the urine, only 25% of a dose of 3-OH-quinidine given directly has been recovered in the urine.
Short report
251 4)
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The finding of longer elimination half-lives than previously reported was an unexpected finding. Although the calculation of t./2 from only three measurements may be criticised, all three values, except the last one in one subject (EM 4) were well above the assay limit and were determined with good precision. Furthermore, the inclusion of measurements from 14 h generally yielded similar tl, values, and plasma 3-OH-quinidine declined at the same rate. Earlier studies in which the half-life of quinidine was estimated (Mahon et al., 1976; Mikus et al., 1986; Ochs et al., 1980; Rakhit et al., 1984; Ueda & Dzindzio, 1978) all used shorter sampling times. Furthermore, an undetected prolonged terminal t.12 may explain why observed steady-state drug concentrations during multiple dosing were found to be higher than those predicted from single dose kinetics of quinidine
(Mahon et al., 1976). Finally, our finding is in accordance with the long duration of debrisoquine or sparteine MR-elevation after even small doses of quinidine (Boobis et al., 1988; Nielsen et al., data to be published). A more definite evaluation of the elimination kinetics of quinidine should be carried out as a post multiple dose/steady-state study with more frequent sampling in the 15-50 h period and perhaps also with a longer sampling period. Such a study should be combined with further efforts to elucidate the possible involvement of P450dbl in the oxidation of quinidine. This study was supported by a grant from the Lundbeck Foundation and the P. Carl Petersen's Foundation. The authors wish to express their gratitude to Mrs G. Rodtborg Nielsen for performing the drug assays. The results described in this publication represent a contribution to the aims of COST B1.
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(Received 11 May 1989, accepted 9 October 1989)
