Mechanisms of Ageing and Development, 65 (1992) 167-175
167
Elsevier Scientific Publishers Ireland Ltd.
INFLUENCE OF AGE ON STEREOSELECTIVE PHARMACOKINETICS AND METABOLISM OF HEXOBARBITAL IN THE RAT
AN M. VERMEULEN*, FRANS M. BELPAIRE, FRITS DE SMET, MARIE-THI~RESE ROSSEEL and MARC G. BOGAERT Heymans Institute of Pharmacology, University of Gent Medical School, Gent (Belgium) (Received February 26th, 1992)
SUMMARY
The influence of age on stereoselective pharmacokinetics and in vitro metabolism of R- and S-hexobarbital was studied in the rat. After intravenous administration of the racemate, the plasma concentrations of S-hexobarbital are markedly lower than those of R-hexobarbital. For S-hexobarbital the half-life is somewhat shorter and the volume of distribution and plasma clearance is higher than for its antipode. For both enantiomers an increase in AUC and half-life, and a decrease in clearance are observed with aging. These changes occur mainly between the 3rd and the 12th month and are slightly more pronounced for R- than for S-hexobarbital, as appears from the S/R ratios. The volume of distribution shows no changes with aging. In vitro disappearance rate in 3-month-old rats is significantly higher for S- than for R-hexobarbital. There is for both enantiomers an increase in disappearance rate in 12-month-old rats as compared to younger or older rats, but this is significant only for the R-enantiomer. There are pronounced differences in the kinetics and metabolism of both hexobarbital enantiomers; changes with aging occur, but are only slightly and not always significantly more important for R- than for Shexobarbital.
Key words: Aging; Hexobarbital; Enantiomers; Hepatocytes; Metabolism; Stereoselective pharmacokinetics
Correspondence to: An M. Vermeulen, Heymans Institute of Pharmacology, University of Gent Medical School, De Pintelaan 185, 9000 Gent, Belgium. *Research Assistant of the National Fund for Scientific Research. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
168
INTRODUCTION Aging can influence a drug's pharmacokinetics and pharmacodynamics. In the case of racemic mixtures, aging can affect the kinetics of the two enantiomers differently. Hexobarbital was the first drug for which such a differential effect as a consequence of aging was shown. Chandler et ai. [1] found in humans that after oral administration of racemic hexobarbital, the metabolic clearance of the S-enantiomer in the elderly was only half of that in the young group, while the metabolic clearance of the R-hexobarbital remained unaltered. Recently, a study by Smith et al. [2] showed that after rifampicin treatment, the increase in the oral clearance of R-hexobarbital was different between young and elderly subjects, while for S-hexobarbital the increase in oral clearance was the same in both age groups. In rats, enantioselectivity in the pharmacokinetics of hexobarbital was shown by Breimer and Van Rossum [31, by van der Graaff [4] and by Groen [5]. The purpose of our study was to investigate in the rat the influence of age on the enantioselective pharmacokinetics of hexobarbital after i.v. administration of the racemate. Furthermore, the in vitro disappearance rate of the hexobarbital enantiomers was studied in freshly isolated hepatocytes. METHODS
Materials Racemic hexobarbital (l,5-dimethyl-5-(1 '-cyclohexenyl)-barbituric acid) was obtained from Serva (Polylab, Antwerp). R-(-)- and S-(+)-hexobarbital were kindly donated by Prof. Dr. J. Knabe (Universit/it des Saarlandes, Germany). Heptabarbital and allobarbital were obtained from Brocacef (Maarssen, The Netherlands). All other reagents were from E. Merck (Darmstadt, Germany) and Carlo Erba (Milan, Italy). Collagenase (clostridiopeptidase A; EC 3.4.24.3) was obtained from Sigma (St. Louis, MO, USA). Animals and procedures Male Wistar rats (SPF) aged 3, 12 and 24 months were purchased from the breeding laboratories of the University of Leuven, Belgium. They were kept in a 12/12-h (light/dark) cycle and at a room temperature of + 25°C. The rats were fasted for about 16 h before the experiments, with free access to water. For the in vivo study, silicone catheters were implanted in both jugular veins under ether anaesthesia, without using anticoagulant. The ether anaesthesia was interrupted and 2 h later racemic hexobarbital (25 mg/kg in a volume of 2 ml/kg) was administered i.v. to the conscious animals via one of the catheters. Each rat was used only once. At different times after drug administration, blood samples (300 ~1) were withdrawn from the venous catheter not used for injection and collected in
169 heparinized tubes. After centrifugation, the plasma samples were stored at -20°C until analysis. For the in vitro metabolism study, hepatocytes were isolated from the rats by the perfusion technique described by Seglen [61, with minor modifications [7]. In contrast to Chindavijak et al. [7], we used 25 mg collagenase to isolate the cells and we increased the content of calcium chloride in the buffer to 3.81 mmol/l. Viability, as assessed with the Trypan blue dye exclusion method [6], was between 73 and 84%. The cell suspensions were diluted with Krebs-bicarbonate buffer to a final cell concentration of 2.5 x 106 viable cells per ml incubation mixture. The incubation conditions were chosen on the basis of linearity of the metabolizing activity with cell concentration and incubation time; the drug concentrations were lying around the Km of S-hexobarbital and around one-half of the Km of R-hexobarbital. The experiments were performed in a total volume of 3 ml at 37°C in a shaking water bath (120 oscillations/min), under an atmosphere of 95% 02/5% CO2, by gassing every 5-10 min. After preincubation for 5 min, the drug was added to give a final concentration of 127/zmol/1. After 15 min of incubation samples (in duplicate) were transferred in 100/~1 hydrochloric acid 0.1 N and kept at -20°C until analysis.
Assay of hexobarbital enantiomers The enantiomers of hexobarbital were quantified using HPLC with UV detection. For the assay of the enantiomers in plasma, a direct method with a chiral AAGcolumn was used [8]. For the assay of the enantiomers in the incubation mixture, a chiral mobile phase containing 15 mM/3-cyclodextrine was used. The extraction procedure followed was the same as for plasma, except that allobarbital (750 ng) was used as the internal standard. The residue was reconstituted in 100/~1 of mobile phase. The HPLC system consisted of a SP 8700 solvent delivery system, with a Rheodyne model 7010 injector (20/~1), a Spectra-Physics UV detector (210 nm) and a Spectra-Physics 4290 integrator. The column used was a Spherisorb 5 0 D S (250 x 4.6 mm) reversed phase column (Chrompack, Holland). Separation was accomplished at room temperature, using an isocratic mobile phase methanol/ethanol NaH2PO4" H20 50 mM (100/150/750 by vol.), at a flow rate of 1.0 ml/min. The inter-day coefficients of variation at concentrations of 300 ng/100/~1 (n = 15) and 1250 ng/100 /~1 (n = 15) were for R-hexobarbital 9.88% and 6.14% and for Shexobarbital 9.25% and 5.33%.
Measurement of cytochrome P-450 Total cytochrome P-450 concentrations were determined from the CO difference spectrum of the reduced protein [9].
Data analysis and statistics All results are expressed as pertaining to the sodium salt. Pharmacokinetic parameters were calculated model independently. AUC was calculated by the
170
trapezoidal method and extrapolated to infinity. Co was obtained by extrapolating the plasma concentration-time curve to time 0. Plasma clearance was calculated from C1--D/AUC. Half-life was calculated from the terminal part of the log concentration-time curve. Volume of distribution was calculated from Va = C1/ke. For the in vitro metabolism study, the disappearance rate was expressed as nanomoles of hexobarbital enantiomer disappearing per 106 viable cells per minute. Results are given as means ± S.E.M. Data for R- and S-hexobarbital of 3-monthold rats were subjected to the paired Wilcoxon rank test. For all parameters, ratios of S- over R-hexobarbital were calculated. To determine the effect of aging on the pharmacokinetic parameters and on these ratios, all data were subjected to one way analysis of variance (ANOVA): results in the 12- and 24-month-old rats were compared with those in 3-month-old rats. When ANOVA indicated significant differences (P < 0.05), the Student's t-test was applied to determine which age groups differed from one another. Significance was assumed when P < 0.05. RESULTS There were no significant differences in haematocrit values between the rats of the three age groups. A. Plasma concentration-time curves The mean plasma concentration-time curves for R- and S-hexobarbital in rats of 3, 12 and 24 months of age are shown in Fig. 1 and the corresponding pharmacokinetic parameters are given in Table I. In all age groups, the mean plasma concentrations of S-hexobarbital are significantly lower than those of R-hexobarbital and decline slightly faster. Volume of distribution and clearance are significantly higher for S- than for R-hexobarbital, whereas its half-life is somewhat lower. With aging, the most important changes occur between 3 and 12 months; further changes between 12 and 24 months are less pronounced. As compared with the 3month-old rats, there is a significant increase in both the AUC and the half-life of older rats. Clearances of both enantiomers are significantly decreased in rats of 12 and 24 months of age, whereas volume of distribution does not change. These changes upon aging are slightly more pronounced for R- than for Shexobarbitol, as shown by the S/R ratios (see Table II). B. In vitro drug metabolism study The disappearance rate of R- and S-hexobarbital in rats of different ages is shown in Table III. In all age groups, the disappearance rate of S-hexobarbital is significantly higher than that of R-hexobarbital. With increasing age, for both enantiomers an increased disappearance rate in 12month-old rats is observed and the values decline again in 24-month-old rats. The increase at 12 months was however significant only for the R-enantiomer. The
2'0
In =111
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3 m(n=111
12 m (n=111
x
~\E
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~
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TIME(min)
8b
~o
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2'0
Lo
PLASMACONCENTRATION 10 (pg/m[)
go
x 3m
~
TIME(rain)
(n=11)
Fig. 1. Plasma concentration-time curves of R- (left panel) and S-hexobarbital (right panel) in rats of different ages after i.v. administration o f racemic hexobarbital (25 mg/kg). Results are means -*- S.E.M.
o.~ ~)
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PLASMACONCENTRATION 10 ~ / m t )
172
TABLE I P H A R M A C O K I N E T I C PARAMETERS OF R- A N D S-HEXOBARBITAL IN RATS OF DIFFERENT AGES AFTER I.V. A D M I N I S T R A T I O N OF RAC-HEXOBARBITAL (25 mg/kg). RESULTS ARE MEANS ± S.E.M.
Weight (g)
3 months
12 months
24 months
( n = 11)
( n = 10)
( n = 11)
320 ± 3
580 + 17"**
611 ± 14"**
237 53.9 1.95 25.2
+ ± ± ±
419 32.7 2.03 47.4
± 52** ± 2.5*** 4- 0.14 ± 7.9*
490 27.8 1.95 51.8
+ ± ± ±
46*** 2.4*** 0.08 4.3***
139 92.6 2.98 22.6
4- 9 ± 4.5 4- 0.21 ± 1.5
220 59.5 2.89 35.0
± ± ± ±
253 52.5 2.62 35.9
± ± ± ±
22*** 3.7*** 0.16 2.8***
R-hexobarbital AUC (/zg.min/ml) Clp (ml/min/kg) Vd (l/kg) Tl/2 (min)
11++ 2.6++ 0.1++ 1.4+
S-hexobarbital AUC (tzg • min/ml) Clp (ml/min/kg) Vd (l/kg) Tl/2 (min)
16"** 4.0*** 0.19 3.4**
AUC, area under the plasma concentration-time curve; Clp, total plasma clearance; Vd, volume of distribution; TI/2, plasma half life. *P < 0.05; **P < 0.01, ***P < 0.001 different from 3-month-old rats (Student's t-test); +P < 0.01; ++P < 0.001 different from the values for S-hexobarbital in 3-month-old rats (paired Wilcoxon rank test).
TABLE II
S/R RATIOS FOR THE P H A R M A C O K I N E T I C PARAMETERS A N D FOR THE DISAPPEARANCE RATE OF THE E N A N T I O M E R S OF HEXOBARBITAL IN RATS OF D I F F E R E N T AGES. RESULTS ARE MEANS ± S.E.M.
3 months
12 months
24 months
0.546 1.861 1.426 0.780
0.532 1.952 1.340 0.714
1.1I. AUC CI e Vd
Tin
0.586 1.721 1.534 0.893
± ± ± 4-
0.018 0.052 0.070 0.033
± ± ± +
0.021 0.080 0.028 0.033*
± ± ± ±
0.034 0.118 0.042* 0.051"*
In vitro Disappearance rate
2.496 ± 0.102
2.253 ± 0.088
2.553 ± 0.143
AUC, area under the plasma concentration-time curve; Clp, total plasma clearance; Vd, volume of distribution; Tj/2, plasma half-life. *P < 0.05; **P < 0.01 different from 3-month-old rats (Student's t-test).
173 TABLE III CYTOCHROME P-450 CONTENT A N D DISAPPEARANCE RATE OF THE ENANT1OMERS OF HEXOBARBITAL IN ISOLATED HEPATOCYTES OF RATS OF D I F F E R E N T AGES. THE METABOLIZING ACTIVITY IS EXPRESSED AS N A N O M O L E OF D R U G DISAPPEARING PER l06 VIABLE CELLS PER MINUTE. DATA ARE MEANS 4- S.E.M.
3 months (n = 7)
12months
24 months
(n = 7 )
(n = 7)
Weight (g)
343 ± 10
531 ± 22***
539 ± 17"**
Disappearance rate (nmol/106 viable cells/min) R-hexobarbital S-hexobarbital
0.273 ± 0.030 ++ 0.675 ± 0.069
0.387 ± 0.037* 0.860 ± 0.053
0.266 + 0.038 0.656 4- 0.072
0.263 ± 0.037
0.337 ± 0.038
0.292 ± 0.019
Cytochrome P-450 (nmole/106 cells)
*P < 0.05; ***P < 0.001 different from 3-month-old rats (Student's t-test); ++P < 0.001 different from the values for S-hexobarbital in 3-month-old rats (paired Wilcoxon rank test).
changes with aging are not enantioselective, as is clear from the S/R ratios (Table II). The cytochrome P-450 content in 12-month-old rats is higher than in 3- and 24month-old rats, but the differences are not significant. DISCUSSION
.4. R- versus S-hexobarbital The clearance of S-hexobarbital was about twice and its volume of distribution about 1.5-times that of R-hexobarbital. The half-life in 3-month-old rats was slightly, but significantly higher for R-hexobarbital. In the rat, both hexobarbital enantiomers display a high extraction behaviour (Es = 0.95 and ER = 0.71) [4] and their clearance is therefore determined by liver blood flow, intrinsic clearance and free fraction. As the enantiomers were administered simultaneously, liver blood flow is the same for both enantiomers. Furthermore, the binding of racemic hexobarbital is not very pronounced (38-55%) [10,11] and no difference in binding to albumin between the two enantiomers was found [12]. Therefore, the difference in systemic clearance between the two enantiomers is probably due to a difference in intrinsic clearance, which is in accordance with some literature data [4,5,13-16]. Our in vitro results substantiate this explanation: a more than twofold difference in disappearance rate between the two enantiomers was found. The difference in Vd between the two enantiomers in our study could point to an enantioselective
174
difference in tissue binding, as in vitro no enantioselective differences in binding to albumin were found [12], but other data on binding are not available. Van der Graaff [4] and Groen [5] found a comparable difference in lid between the two enantiomers, while Breimer and Van Rossum [3] found no difference at all. The difference in half-life between the enantiomers is due to differences in both lid and CI. Small differences in half-life were also found by van der Graaff [4] and by Groen [5], while Breimer and Van Rossum [3] found a much more pronounced difference after administration of the separate enantiomers.
B. Influence of aging The plasma concentrations and the AUC of the hexobarbital enantiomers increase markedly from the 3rd to the 12th month, with little changes thereafter. The decrease in clearance of both enantiomers is also most pronounced between 3- and 12-monthold rats, which can probably be explained by a decrease in hepatic blood flow. It has been shown that liver blood flow decreases from 5 to 36 weeks and remains almost constant thereafter up to 104 weeks [16]. Vd in 24-month-old rats shows a slight decrease for S-hexobarbital, which is not significant, but enantioselective, as is clear from the S/R ratios (Table II). The half-life increases markedly for both enantiomers between 3 and 12 months and remains at the same level in 24-month-old rats. Our in vitro results show for both enantiomers an increase in disappearance rate at 12 months, which parallels the tendency towards higher cytochrome P-450 contents of these hepatocytes. At 24 months, the results are similar to those at 3 months. Thus, the capacity of hepatocytes to metabolize does not seem to decrease with aging. Boonstra-Nieveld and van Bezooijen [17] studied the pharmacokinetics of racemic hexobarbital in 3- and 30-month-old BN/BiRij rats. They found a significant decrease in intrinsic clearance of the racemate in 30-month-old rats compared to 3month-old rats, when expressed as a function of body weight or as a function of liver weight. In contrast, Groen [5] found no changes in metabolic clearance of both enantiomers between 6- and 24- and 6- and 30-month-old BN/BiRij rats. However, hepatic intrinsic clearance of hexobarbital in Wistar rats and BN/BiRij rats differs by a factor of about 10, so it is difficult to compare different rat strains. In our study, Vd shows no changes with aging, so probably, plasma and tissue binding also show no changes with aging. Boonstra-Nieveld and van Bezooijen [17] also found no changes in this parameter with aging for the racemate. The increase in half-life in our study, which is 50% for the racemate in the study of BoonstraNieveld and van Bezooijen [17], is a consequence of a combination of an unchanged Vd with a decreased C1. Groen [5] found in 24-month-old rats for both enantiomers a half-life twice as high as in 6-month-old rats. It can be concluded that in the rat, the enantioselectivity in hexobarbital kinetics is due to stereoselectivity in metabolism. Aging influences the kinetics of both R- and S-hexobarbital and this is slightly more pronounced for the R-enantiomer. Most
175
changes occur between 3 and 12 months of age, i.e., the period of maturation and up to 24 months no further changes are seen. ACKNOWLEDGEMENTS
We are grateful to Mrs. De Meulemeester for her help in the analytical work. This work was supported by a grant of the National Fund for Scientific Research (grant number 3.9006.87). A.M. Vermeulen was a grantee from the I.W.O.N.L. from 1-1089 to 30-09-90 and a Research Assistant of the National Fund for Scientific Research from 1-10-90 on. REFERENCES 1 M.H.H. Chandler, S.R. Scott and R.A. Blouin, Age-associated stereoselective alterations in hexobarbital metabolism. Clin. Pharmacol. Ther., 43 0988) 436-441. 2 D.A. Smith, M.H.H. Chandler, S.I. Shedlofsky, P.J. Wedlund and R.A. Blouin, Age-dependent stereoselective increase in the oral clearance of hexobarbitone isomers caused by rifampicin. Br. J. Clin. Pharmacol., 32 (1991) 735-739. 3 D.D. Breimer and J.R. Van Rossum, Pharmacokinetics of the enantiomers of hexobarbital studied in the same intact rat and in the same isolated perfused rat liver. Eur. J. Pharmacol., 26 (1974) 321-330. 4 M. Van der Graaff, Characterization andprediction of in vivo oxidative drug metabolizing enzyme activity. A re-evaluation of hexobarbital as a model substrate. Thesis, J.H. Pasmans B.V., 's-Gravenhage (1985). 5 K. Groen, The influence of ageing on P-450-mediated drug metabolism. (1991) Thesis. 6 P.O. Seglen, Preparation of rat liver cells. I. Effect of Ca 2÷ on enzymatic dispersion of isolated perfused liver. Exp. Cell Res., 74 (1972) 450-454. 7 B. Chindavijak, F.M. Belpaire and M.G. Bogaert, Effect of inflammation on the metabolism of antipyrine, lidocaine and propranolol in isolated rat hepatocytes. Pharmacology, 36 0988) 279-282. 8 A.M. Vermeulen, M.T. Rosseel and F.M. Belpaire, High-performance liquid chromatographic method for the simultaneous determination of R-(-)- and S-(+)-hexobarbital in rat plasma. J. Chromatogr., 567 (1991) 472-479. 9 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239 (1964) 2370-2378. l0 Y. Igari, Y. Sugiyama, S. Awazu and M. Hanano, Comparative physiologically based pharmacokinetics of hexobarbital, phenobarbital and thiopental in the rat. J. Pharmacokin. Biopharm., 10 0982) 53-75. 11 N.P.E. Vermeulen, M. Danhof, I. Setiawan and D.D. Breimer, Disposition of hexobarbital in the rat. Estimation of "first-pass" elimination and influence of ether anaesthesia. J. Pharmacol. Exp. Ther., 226 0983) 201-205. 12 D.D. Breimer and J.M. Van Rossum, Pharmacokinetics of (+)-, (-)- and ( -~)-hexobarbitone in man after oral administration. J. Pharm. Pharmacol., 25 0973) 762-763. 13 R.L. Furrier, J.S. McCarthy, R.E. Stitzel and M.W. Anders, Stereoselective metabolism of the enantiomers of hexobarbital. J. Pharmacol. Exp. Ther., 169 (1969) 153-158. 14 E. Degkwitz, V. Ullrich, H. Staudinger and W. Rummel, Metabolism and cytochrome P-450 binding spectra of(+)- and (-)-hexobarbital in rat liver microsomes. Hoppe-Seyler's Z. Physiol. Chem., 350 (1969) 547-553. 15 D.R. Feller and W.C. Lubawy, Interactions of the hexobarbital enantiomers with rat liver microsomes. Pharmacology, 9 (1973) 129-137. 16 K. Iwamoto, J. Watanabe, K. Araki, N. Deguchi and H. Sugiyama, Effect of age on the hepatic clearance of propranolol in rats. J. Pharm. Pharmacol., 37 0985) 466-470. 17 I.H.L Boonstra-Nieveld and C.F.A. van Bezooijen, Pharmacokinetics of hexobarbital in young and old rats. Mech. Ageing Devel., 50 0989) 289-298.
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Elsevier Scientific Publishers Ireland Ltd.
INFLUENCE OF AGE ON STEREOSELECTIVE PHARMACOKINETICS AND METABOLISM OF HEXOBARBITAL IN THE RAT
AN M. VERMEULEN*, FRANS M. BELPAIRE, FRITS DE SMET, MARIE-THI~RESE ROSSEEL and MARC G. BOGAERT Heymans Institute of Pharmacology, University of Gent Medical School, Gent (Belgium) (Received February 26th, 1992)
SUMMARY
The influence of age on stereoselective pharmacokinetics and in vitro metabolism of R- and S-hexobarbital was studied in the rat. After intravenous administration of the racemate, the plasma concentrations of S-hexobarbital are markedly lower than those of R-hexobarbital. For S-hexobarbital the half-life is somewhat shorter and the volume of distribution and plasma clearance is higher than for its antipode. For both enantiomers an increase in AUC and half-life, and a decrease in clearance are observed with aging. These changes occur mainly between the 3rd and the 12th month and are slightly more pronounced for R- than for S-hexobarbital, as appears from the S/R ratios. The volume of distribution shows no changes with aging. In vitro disappearance rate in 3-month-old rats is significantly higher for S- than for R-hexobarbital. There is for both enantiomers an increase in disappearance rate in 12-month-old rats as compared to younger or older rats, but this is significant only for the R-enantiomer. There are pronounced differences in the kinetics and metabolism of both hexobarbital enantiomers; changes with aging occur, but are only slightly and not always significantly more important for R- than for Shexobarbital.
Key words: Aging; Hexobarbital; Enantiomers; Hepatocytes; Metabolism; Stereoselective pharmacokinetics
Correspondence to: An M. Vermeulen, Heymans Institute of Pharmacology, University of Gent Medical School, De Pintelaan 185, 9000 Gent, Belgium. *Research Assistant of the National Fund for Scientific Research. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
168
INTRODUCTION Aging can influence a drug's pharmacokinetics and pharmacodynamics. In the case of racemic mixtures, aging can affect the kinetics of the two enantiomers differently. Hexobarbital was the first drug for which such a differential effect as a consequence of aging was shown. Chandler et ai. [1] found in humans that after oral administration of racemic hexobarbital, the metabolic clearance of the S-enantiomer in the elderly was only half of that in the young group, while the metabolic clearance of the R-hexobarbital remained unaltered. Recently, a study by Smith et al. [2] showed that after rifampicin treatment, the increase in the oral clearance of R-hexobarbital was different between young and elderly subjects, while for S-hexobarbital the increase in oral clearance was the same in both age groups. In rats, enantioselectivity in the pharmacokinetics of hexobarbital was shown by Breimer and Van Rossum [31, by van der Graaff [4] and by Groen [5]. The purpose of our study was to investigate in the rat the influence of age on the enantioselective pharmacokinetics of hexobarbital after i.v. administration of the racemate. Furthermore, the in vitro disappearance rate of the hexobarbital enantiomers was studied in freshly isolated hepatocytes. METHODS
Materials Racemic hexobarbital (l,5-dimethyl-5-(1 '-cyclohexenyl)-barbituric acid) was obtained from Serva (Polylab, Antwerp). R-(-)- and S-(+)-hexobarbital were kindly donated by Prof. Dr. J. Knabe (Universit/it des Saarlandes, Germany). Heptabarbital and allobarbital were obtained from Brocacef (Maarssen, The Netherlands). All other reagents were from E. Merck (Darmstadt, Germany) and Carlo Erba (Milan, Italy). Collagenase (clostridiopeptidase A; EC 3.4.24.3) was obtained from Sigma (St. Louis, MO, USA). Animals and procedures Male Wistar rats (SPF) aged 3, 12 and 24 months were purchased from the breeding laboratories of the University of Leuven, Belgium. They were kept in a 12/12-h (light/dark) cycle and at a room temperature of + 25°C. The rats were fasted for about 16 h before the experiments, with free access to water. For the in vivo study, silicone catheters were implanted in both jugular veins under ether anaesthesia, without using anticoagulant. The ether anaesthesia was interrupted and 2 h later racemic hexobarbital (25 mg/kg in a volume of 2 ml/kg) was administered i.v. to the conscious animals via one of the catheters. Each rat was used only once. At different times after drug administration, blood samples (300 ~1) were withdrawn from the venous catheter not used for injection and collected in
169 heparinized tubes. After centrifugation, the plasma samples were stored at -20°C until analysis. For the in vitro metabolism study, hepatocytes were isolated from the rats by the perfusion technique described by Seglen [61, with minor modifications [7]. In contrast to Chindavijak et al. [7], we used 25 mg collagenase to isolate the cells and we increased the content of calcium chloride in the buffer to 3.81 mmol/l. Viability, as assessed with the Trypan blue dye exclusion method [6], was between 73 and 84%. The cell suspensions were diluted with Krebs-bicarbonate buffer to a final cell concentration of 2.5 x 106 viable cells per ml incubation mixture. The incubation conditions were chosen on the basis of linearity of the metabolizing activity with cell concentration and incubation time; the drug concentrations were lying around the Km of S-hexobarbital and around one-half of the Km of R-hexobarbital. The experiments were performed in a total volume of 3 ml at 37°C in a shaking water bath (120 oscillations/min), under an atmosphere of 95% 02/5% CO2, by gassing every 5-10 min. After preincubation for 5 min, the drug was added to give a final concentration of 127/zmol/1. After 15 min of incubation samples (in duplicate) were transferred in 100/~1 hydrochloric acid 0.1 N and kept at -20°C until analysis.
Assay of hexobarbital enantiomers The enantiomers of hexobarbital were quantified using HPLC with UV detection. For the assay of the enantiomers in plasma, a direct method with a chiral AAGcolumn was used [8]. For the assay of the enantiomers in the incubation mixture, a chiral mobile phase containing 15 mM/3-cyclodextrine was used. The extraction procedure followed was the same as for plasma, except that allobarbital (750 ng) was used as the internal standard. The residue was reconstituted in 100/~1 of mobile phase. The HPLC system consisted of a SP 8700 solvent delivery system, with a Rheodyne model 7010 injector (20/~1), a Spectra-Physics UV detector (210 nm) and a Spectra-Physics 4290 integrator. The column used was a Spherisorb 5 0 D S (250 x 4.6 mm) reversed phase column (Chrompack, Holland). Separation was accomplished at room temperature, using an isocratic mobile phase methanol/ethanol NaH2PO4" H20 50 mM (100/150/750 by vol.), at a flow rate of 1.0 ml/min. The inter-day coefficients of variation at concentrations of 300 ng/100/~1 (n = 15) and 1250 ng/100 /~1 (n = 15) were for R-hexobarbital 9.88% and 6.14% and for Shexobarbital 9.25% and 5.33%.
Measurement of cytochrome P-450 Total cytochrome P-450 concentrations were determined from the CO difference spectrum of the reduced protein [9].
Data analysis and statistics All results are expressed as pertaining to the sodium salt. Pharmacokinetic parameters were calculated model independently. AUC was calculated by the
170
trapezoidal method and extrapolated to infinity. Co was obtained by extrapolating the plasma concentration-time curve to time 0. Plasma clearance was calculated from C1--D/AUC. Half-life was calculated from the terminal part of the log concentration-time curve. Volume of distribution was calculated from Va = C1/ke. For the in vitro metabolism study, the disappearance rate was expressed as nanomoles of hexobarbital enantiomer disappearing per 106 viable cells per minute. Results are given as means ± S.E.M. Data for R- and S-hexobarbital of 3-monthold rats were subjected to the paired Wilcoxon rank test. For all parameters, ratios of S- over R-hexobarbital were calculated. To determine the effect of aging on the pharmacokinetic parameters and on these ratios, all data were subjected to one way analysis of variance (ANOVA): results in the 12- and 24-month-old rats were compared with those in 3-month-old rats. When ANOVA indicated significant differences (P < 0.05), the Student's t-test was applied to determine which age groups differed from one another. Significance was assumed when P < 0.05. RESULTS There were no significant differences in haematocrit values between the rats of the three age groups. A. Plasma concentration-time curves The mean plasma concentration-time curves for R- and S-hexobarbital in rats of 3, 12 and 24 months of age are shown in Fig. 1 and the corresponding pharmacokinetic parameters are given in Table I. In all age groups, the mean plasma concentrations of S-hexobarbital are significantly lower than those of R-hexobarbital and decline slightly faster. Volume of distribution and clearance are significantly higher for S- than for R-hexobarbital, whereas its half-life is somewhat lower. With aging, the most important changes occur between 3 and 12 months; further changes between 12 and 24 months are less pronounced. As compared with the 3month-old rats, there is a significant increase in both the AUC and the half-life of older rats. Clearances of both enantiomers are significantly decreased in rats of 12 and 24 months of age, whereas volume of distribution does not change. These changes upon aging are slightly more pronounced for R- than for Shexobarbitol, as shown by the S/R ratios (see Table II). B. In vitro drug metabolism study The disappearance rate of R- and S-hexobarbital in rats of different ages is shown in Table III. In all age groups, the disappearance rate of S-hexobarbital is significantly higher than that of R-hexobarbital. With increasing age, for both enantiomers an increased disappearance rate in 12month-old rats is observed and the values decline again in 24-month-old rats. The increase at 12 months was however significant only for the R-enantiomer. The
2'0
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12 m (n=111
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TIME(min)
8b
~o
o.1 ~
2'0
Lo
PLASMACONCENTRATION 10 (pg/m[)
go
x 3m
~
TIME(rain)
(n=11)
Fig. 1. Plasma concentration-time curves of R- (left panel) and S-hexobarbital (right panel) in rats of different ages after i.v. administration o f racemic hexobarbital (25 mg/kg). Results are means -*- S.E.M.
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172
TABLE I P H A R M A C O K I N E T I C PARAMETERS OF R- A N D S-HEXOBARBITAL IN RATS OF DIFFERENT AGES AFTER I.V. A D M I N I S T R A T I O N OF RAC-HEXOBARBITAL (25 mg/kg). RESULTS ARE MEANS ± S.E.M.
Weight (g)
3 months
12 months
24 months
( n = 11)
( n = 10)
( n = 11)
320 ± 3
580 + 17"**
611 ± 14"**
237 53.9 1.95 25.2
+ ± ± ±
419 32.7 2.03 47.4
± 52** ± 2.5*** 4- 0.14 ± 7.9*
490 27.8 1.95 51.8
+ ± ± ±
46*** 2.4*** 0.08 4.3***
139 92.6 2.98 22.6
4- 9 ± 4.5 4- 0.21 ± 1.5
220 59.5 2.89 35.0
± ± ± ±
253 52.5 2.62 35.9
± ± ± ±
22*** 3.7*** 0.16 2.8***
R-hexobarbital AUC (/zg.min/ml) Clp (ml/min/kg) Vd (l/kg) Tl/2 (min)
11++ 2.6++ 0.1++ 1.4+
S-hexobarbital AUC (tzg • min/ml) Clp (ml/min/kg) Vd (l/kg) Tl/2 (min)
16"** 4.0*** 0.19 3.4**
AUC, area under the plasma concentration-time curve; Clp, total plasma clearance; Vd, volume of distribution; TI/2, plasma half life. *P < 0.05; **P < 0.01, ***P < 0.001 different from 3-month-old rats (Student's t-test); +P < 0.01; ++P < 0.001 different from the values for S-hexobarbital in 3-month-old rats (paired Wilcoxon rank test).
TABLE II
S/R RATIOS FOR THE P H A R M A C O K I N E T I C PARAMETERS A N D FOR THE DISAPPEARANCE RATE OF THE E N A N T I O M E R S OF HEXOBARBITAL IN RATS OF D I F F E R E N T AGES. RESULTS ARE MEANS ± S.E.M.
3 months
12 months
24 months
0.546 1.861 1.426 0.780
0.532 1.952 1.340 0.714
1.1I. AUC CI e Vd
Tin
0.586 1.721 1.534 0.893
± ± ± 4-
0.018 0.052 0.070 0.033
± ± ± +
0.021 0.080 0.028 0.033*
± ± ± ±
0.034 0.118 0.042* 0.051"*
In vitro Disappearance rate
2.496 ± 0.102
2.253 ± 0.088
2.553 ± 0.143
AUC, area under the plasma concentration-time curve; Clp, total plasma clearance; Vd, volume of distribution; Tj/2, plasma half-life. *P < 0.05; **P < 0.01 different from 3-month-old rats (Student's t-test).
173 TABLE III CYTOCHROME P-450 CONTENT A N D DISAPPEARANCE RATE OF THE ENANT1OMERS OF HEXOBARBITAL IN ISOLATED HEPATOCYTES OF RATS OF D I F F E R E N T AGES. THE METABOLIZING ACTIVITY IS EXPRESSED AS N A N O M O L E OF D R U G DISAPPEARING PER l06 VIABLE CELLS PER MINUTE. DATA ARE MEANS 4- S.E.M.
3 months (n = 7)
12months
24 months
(n = 7 )
(n = 7)
Weight (g)
343 ± 10
531 ± 22***
539 ± 17"**
Disappearance rate (nmol/106 viable cells/min) R-hexobarbital S-hexobarbital
0.273 ± 0.030 ++ 0.675 ± 0.069
0.387 ± 0.037* 0.860 ± 0.053
0.266 + 0.038 0.656 4- 0.072
0.263 ± 0.037
0.337 ± 0.038
0.292 ± 0.019
Cytochrome P-450 (nmole/106 cells)
*P < 0.05; ***P < 0.001 different from 3-month-old rats (Student's t-test); ++P < 0.001 different from the values for S-hexobarbital in 3-month-old rats (paired Wilcoxon rank test).
changes with aging are not enantioselective, as is clear from the S/R ratios (Table II). The cytochrome P-450 content in 12-month-old rats is higher than in 3- and 24month-old rats, but the differences are not significant. DISCUSSION
.4. R- versus S-hexobarbital The clearance of S-hexobarbital was about twice and its volume of distribution about 1.5-times that of R-hexobarbital. The half-life in 3-month-old rats was slightly, but significantly higher for R-hexobarbital. In the rat, both hexobarbital enantiomers display a high extraction behaviour (Es = 0.95 and ER = 0.71) [4] and their clearance is therefore determined by liver blood flow, intrinsic clearance and free fraction. As the enantiomers were administered simultaneously, liver blood flow is the same for both enantiomers. Furthermore, the binding of racemic hexobarbital is not very pronounced (38-55%) [10,11] and no difference in binding to albumin between the two enantiomers was found [12]. Therefore, the difference in systemic clearance between the two enantiomers is probably due to a difference in intrinsic clearance, which is in accordance with some literature data [4,5,13-16]. Our in vitro results substantiate this explanation: a more than twofold difference in disappearance rate between the two enantiomers was found. The difference in Vd between the two enantiomers in our study could point to an enantioselective
174
difference in tissue binding, as in vitro no enantioselective differences in binding to albumin were found [12], but other data on binding are not available. Van der Graaff [4] and Groen [5] found a comparable difference in lid between the two enantiomers, while Breimer and Van Rossum [3] found no difference at all. The difference in half-life between the enantiomers is due to differences in both lid and CI. Small differences in half-life were also found by van der Graaff [4] and by Groen [5], while Breimer and Van Rossum [3] found a much more pronounced difference after administration of the separate enantiomers.
B. Influence of aging The plasma concentrations and the AUC of the hexobarbital enantiomers increase markedly from the 3rd to the 12th month, with little changes thereafter. The decrease in clearance of both enantiomers is also most pronounced between 3- and 12-monthold rats, which can probably be explained by a decrease in hepatic blood flow. It has been shown that liver blood flow decreases from 5 to 36 weeks and remains almost constant thereafter up to 104 weeks [16]. Vd in 24-month-old rats shows a slight decrease for S-hexobarbital, which is not significant, but enantioselective, as is clear from the S/R ratios (Table II). The half-life increases markedly for both enantiomers between 3 and 12 months and remains at the same level in 24-month-old rats. Our in vitro results show for both enantiomers an increase in disappearance rate at 12 months, which parallels the tendency towards higher cytochrome P-450 contents of these hepatocytes. At 24 months, the results are similar to those at 3 months. Thus, the capacity of hepatocytes to metabolize does not seem to decrease with aging. Boonstra-Nieveld and van Bezooijen [17] studied the pharmacokinetics of racemic hexobarbital in 3- and 30-month-old BN/BiRij rats. They found a significant decrease in intrinsic clearance of the racemate in 30-month-old rats compared to 3month-old rats, when expressed as a function of body weight or as a function of liver weight. In contrast, Groen [5] found no changes in metabolic clearance of both enantiomers between 6- and 24- and 6- and 30-month-old BN/BiRij rats. However, hepatic intrinsic clearance of hexobarbital in Wistar rats and BN/BiRij rats differs by a factor of about 10, so it is difficult to compare different rat strains. In our study, Vd shows no changes with aging, so probably, plasma and tissue binding also show no changes with aging. Boonstra-Nieveld and van Bezooijen [17] also found no changes in this parameter with aging for the racemate. The increase in half-life in our study, which is 50% for the racemate in the study of BoonstraNieveld and van Bezooijen [17], is a consequence of a combination of an unchanged Vd with a decreased C1. Groen [5] found in 24-month-old rats for both enantiomers a half-life twice as high as in 6-month-old rats. It can be concluded that in the rat, the enantioselectivity in hexobarbital kinetics is due to stereoselectivity in metabolism. Aging influences the kinetics of both R- and S-hexobarbital and this is slightly more pronounced for the R-enantiomer. Most
175
changes occur between 3 and 12 months of age, i.e., the period of maturation and up to 24 months no further changes are seen. ACKNOWLEDGEMENTS
We are grateful to Mrs. De Meulemeester for her help in the analytical work. This work was supported by a grant of the National Fund for Scientific Research (grant number 3.9006.87). A.M. Vermeulen was a grantee from the I.W.O.N.L. from 1-1089 to 30-09-90 and a Research Assistant of the National Fund for Scientific Research from 1-10-90 on. REFERENCES 1 M.H.H. Chandler, S.R. Scott and R.A. Blouin, Age-associated stereoselective alterations in hexobarbital metabolism. Clin. Pharmacol. Ther., 43 0988) 436-441. 2 D.A. Smith, M.H.H. Chandler, S.I. Shedlofsky, P.J. Wedlund and R.A. Blouin, Age-dependent stereoselective increase in the oral clearance of hexobarbitone isomers caused by rifampicin. Br. J. Clin. Pharmacol., 32 (1991) 735-739. 3 D.D. Breimer and J.R. Van Rossum, Pharmacokinetics of the enantiomers of hexobarbital studied in the same intact rat and in the same isolated perfused rat liver. Eur. J. Pharmacol., 26 (1974) 321-330. 4 M. Van der Graaff, Characterization andprediction of in vivo oxidative drug metabolizing enzyme activity. A re-evaluation of hexobarbital as a model substrate. Thesis, J.H. Pasmans B.V., 's-Gravenhage (1985). 5 K. Groen, The influence of ageing on P-450-mediated drug metabolism. (1991) Thesis. 6 P.O. Seglen, Preparation of rat liver cells. I. Effect of Ca 2÷ on enzymatic dispersion of isolated perfused liver. Exp. Cell Res., 74 (1972) 450-454. 7 B. Chindavijak, F.M. Belpaire and M.G. Bogaert, Effect of inflammation on the metabolism of antipyrine, lidocaine and propranolol in isolated rat hepatocytes. Pharmacology, 36 0988) 279-282. 8 A.M. Vermeulen, M.T. Rosseel and F.M. Belpaire, High-performance liquid chromatographic method for the simultaneous determination of R-(-)- and S-(+)-hexobarbital in rat plasma. J. Chromatogr., 567 (1991) 472-479. 9 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem., 239 (1964) 2370-2378. l0 Y. Igari, Y. Sugiyama, S. Awazu and M. Hanano, Comparative physiologically based pharmacokinetics of hexobarbital, phenobarbital and thiopental in the rat. J. Pharmacokin. Biopharm., 10 0982) 53-75. 11 N.P.E. Vermeulen, M. Danhof, I. Setiawan and D.D. Breimer, Disposition of hexobarbital in the rat. Estimation of "first-pass" elimination and influence of ether anaesthesia. J. Pharmacol. Exp. Ther., 226 0983) 201-205. 12 D.D. Breimer and J.M. Van Rossum, Pharmacokinetics of (+)-, (-)- and ( -~)-hexobarbitone in man after oral administration. J. Pharm. Pharmacol., 25 0973) 762-763. 13 R.L. Furrier, J.S. McCarthy, R.E. Stitzel and M.W. Anders, Stereoselective metabolism of the enantiomers of hexobarbital. J. Pharmacol. Exp. Ther., 169 (1969) 153-158. 14 E. Degkwitz, V. Ullrich, H. Staudinger and W. Rummel, Metabolism and cytochrome P-450 binding spectra of(+)- and (-)-hexobarbital in rat liver microsomes. Hoppe-Seyler's Z. Physiol. Chem., 350 (1969) 547-553. 15 D.R. Feller and W.C. Lubawy, Interactions of the hexobarbital enantiomers with rat liver microsomes. Pharmacology, 9 (1973) 129-137. 16 K. Iwamoto, J. Watanabe, K. Araki, N. Deguchi and H. Sugiyama, Effect of age on the hepatic clearance of propranolol in rats. J. Pharm. Pharmacol., 37 0985) 466-470. 17 I.H.L Boonstra-Nieveld and C.F.A. van Bezooijen, Pharmacokinetics of hexobarbital in young and old rats. Mech. Ageing Devel., 50 0989) 289-298.
