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The urinary 6β-hydroxycortisol excretion in man on inducers and inhibitors of the hepatic mixed function oxidase
Pharmac. Ther. Vol. 33, pp. 197 to 199, 1987
0163-7258/87 $0.00 + 0.50
Printedin GreatBritain.All rightsreserved
Copyright© 1987PergamonJournalsLtd
Specialist Subject Editor: E. E. Om,~r~vs
THE URINARY 6/~'HYDROXYCORTISOL EXCRETION IN MAN ON INDUCERS AND INHIBITORS OF THE HEPATIC MIXED FUNCTION OXIDASE J. P. DESAGER, E. DUMONT and C. HAKVEIqGT Universitd Catholique de Louvain, Laboratoire de Pharmacothdrapie, 53, A venue E. Mounier, B-1200Brussels, Belgium
The relationship among four different in vivo measurements of microsomal enzyme activity has been well established for three drugs known to be enzyme inducers in man: antipyrine, phenobarbital and rifampicin (1). We were particularly interested in two of these four parameters: the total body clearance of antipyrine and the urinary excretion of @3-hydroxycortisol (6/3-OHF). We used these measurements in a group of healthy volunteers taking phenobarbital (100rag/day; see Table 1). The 24hr-urinary excretion of 6/3-OHF, relative to that of the 17-hydroxycorticosteroids, and the antipyrine clearance were significantly increased. A statistically significant difference was found between these 2 sets of data (p<0.05). By measuring the 6/3-OHF excretion on the last day of phenobarbital intake, just before the antipyrine clearance start and then throughout the 2 days of this last test, we observed an acute effect of antipyrine on the 6/3-OHF excretion, consisting of a twofold increase on the second day (24-48hr; Table 1). Thus, antipydne contributed indirectly to the increased 6/3-OHF excretion, measured after a treatment with an inducer. In other words, 6/3-OHF values obtained during an antipyrine clearance test are spurious. Nevertheless, both tests are complementary as published recently for sulfinpyrazone (2) and carbamazepine (3) which enhance significantly the 6/3-OHF excretion and the antipyrine clearance. The main advantage to the use of 6/3-OHF over some other tests, is that it represents the biotransformation of an endogenous substrate. This substrate is metabolized by the hepatic cytochrome P-450 system which is also intimately linked to the biotransformation of xenobiotics and drugs. The antipyrine test investigates some isoforms of cytochrome P-450 while 6/3-OHF is formed by other isoforms from cortisol. This specificity of the cytochrome P-450 is illustrated by primaquine which induces the isoforms active on antipyrine but not those on cortisol (Breckenridge, this symposium). The best way to express the results of 6/3-OHF excretion is not definitely established. Results are corrected either by dividing the amount of 6/3-OHF by the amount of cortisol or by the amount of 17-hydroxycorticosteroids measured in the same urine collection. When the 17-hydroxycorticosteroids have increased after a treatment, the origin of this increase is still being debated. Indeed, the investigated drug may increase cortisol production or displace cortisol from its binding sites, particularly from cortisol binding globulin. The measurement of 6/~-OHF was first performed by RIA (4) and thereafter by HPLC (5,6,7). Although value of 6B-OHF as the most sensitive index of induction of the hepatic mixed function oxidase (MFO) system is now well established, its use to detect an inhibition is controversial. The most typical inhibitors of the hepatic MFO are the H2-receptor blocking agents (8). Cimetidine reduces the antipyrine clearance and the urinary excretion of 6/~-OHF. The effect is more modest in terms of absolute values although highly statistically significant (Table 1). This seems to indicate that both tests are less sensitive in detecting an inhibition. For ranitidine, on the other hand, no significant effect was found on the 2 parameters (Table 1) and the level of significance was low (p>0.05). It would be reasonable to think that in an MFO system activated by a strong inducer, the effect 197
Phenobarbital Phenobarbital Antipyrine
Cimetidine Cimetidine Ranitidine Ranitidine
6/~ O H F / 1 7 O H C t Antipyrine Cl T 6/30HF/17OHC
68 O H F / 1 7 O H C Antipyrine Cl T 6/~ O H F / 1 7 O H C Antipyrine CI T
l g/day 1.2 g / d a y 300 m g / d a y 300 r a g / d a y
100 m g / d a y 100 r a g / d a y 18 m g / k g
Dose 2 weeks 2 weeks 0-24hr 24-48hr 1 week 2 weeks 2 weeks 2 weeks
Duration
*C1T = Total body clearance. t 6 / 3 0 H F = 6/3-hydroxycortisol; 17OHC = 17 hydroxy corticosteroids. ~:S.E.M. was used for all data.
Drug
Parameter 8 8 8 8 16 6 6 6
N 16.7 48.7 15.3 21.5 20.1 48.2 21.65 45.7
Before + 4444444-
2.5J/ 2.6 1.9 3 1.7 6.1 3.5 3.9
27.3 58.3 21.5 29.0 !4.6 29.9 19.7 44.8
After 44444444-
4.9 4.9 3 6 1.6 2.7 3 4.2
5 6 5 6 2 0 2 1
~
~ 1 1 2 0 0 0 2 4
~
Trends
TAn~ 1. Urinary 613-hydroxycortisol Excretion and Antipyrine Clearance* Measured in Healthy Volunteers
P 2 <0.05 1 <0.05 1 <0.05 2 <0.025 14 <0.025 6 <0.005 2 NS 1 NS
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ga
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Inhibition or induction evaluated by 6/3-hydroxycortisol excretion
199
of an inhibitor would be amplified. Such experiment has been described by Park (this symposium) in man receiving carbamazepine with a placebo or with cimetidine. No significant difference was found between the treatments, and it was concluded that 6/5-OHF was not sensitive enough to detect an inhibition. This conclusion is in contradiction with the results obtained with cimetidine alone. Very recently (9), the more potent inhibitory effect of cimetidine on MFO, five-fold higher than ranitidine, has been confirmed on human hepatic microsomes from uninduced patients. Results were similar in a liver with induced enzyme activity. It was concluded that the "inhibitory potency of the compounds seems to depend on the individual isozyme pattern of the hepatic microsomes". This suggests that induced liver may respond differently to ranitidine, leading to results different from those reported by Park with cimetidine. The in vitro concentrations used of both drugs are a hundred to a thousand fold higher than the circulating plasma levels in treated patients. The in vivo inhibitory effects are observed with far lower concentrations and clinically the same severity of unwanted effects have now been encountered with cimetidine and ranitidine. Pharmacogenetic investigations are thus needed in these patients suffering from acute effects to point out the specific target of these inhibitors. It can be concluded that too often only partial information is obtained and it does not allow clearcut conclusions from these drug interactions. If the urinary 6/3-OHF excretion and the antipyrine clearance test remain first line investigational tools, they need to be supported by pharmacogenetic tests. REFERENCES 1. OmCBAU$,E. E. and P ~ , B. K. (1979) Measurement of 6/3-hydroxycortisol excretion as an in vivo parameter in the clinical assessment of the microsomal enzyme-inducing capacity of antipyrine, phenobarbitone and rifampicin. Eur. J. Clin. Pharmacol. 15: 139-145. 2. STAIOER,C., ScmJcnT, F., WALTER,E., GUNDERT-RE~Y,U., H~D~RANDT, R., D~VI~s, J., WANG, N. S., Hxmaca~o, J. and W ~ x , E. (1983) Effect of single and multiple doses of sulphinpyrazone on antipyrine metabolism and urinary excretion of 6fl-hydroxycortisol. Eur. J. Clin. Pharma¢oi. 25: 797-801. 3. MOISt.AND, T. A., PARK, B. K. and RYI~NCE, G. W. (1982) Microsomal enzyme induction in children: the influence of carbamazepine treatment on antipyrine kinetics, 6fl-hydroxycortisol excretion and plasma 7-glutamyltranspeptidase activity. Br. J. clin. Pharmacol. 14: 861-865. 4. P ~ x , B. K. (1978) A direct radioimmuno-assay for 6~-hydroxycortisol in human urine. J. Steroid Biochem. 9: 963-966. 5. ROOTS, I., HOLBE, R., H O W ~ N , W., N I G h , S., H E n ~ ' t ~ R , G. and HIEDEBRANDT,A. G. (1979) Quantitative determination by HPLC of urinary 6/~-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs. Eur. J. Clin. Pharmacol. 16: 63-71. 6. Duuo)zr, E., SCLAVONS,M. and DESxtmR, J. P. (1984) Use of an internal standard to assay 6~-hydroxycottisul in urine. J. Liquid Chromatogr. 7: 2051-2057. 7. N ~ . J. and Y~,~TX, M. (1985) Determination of urinary cortisol and 6/~-hydroxycortisol by high performance liquid chromatography. Clin. Chim. Acta 149: 215-224. 8. IO.o~z, U. and REaOJ~rN, I. W. (1984) Drug interactions through binding to cytochrome P-450: the experience with H2-receptor blocking agents. Pharm. Res. 2: 59-62. 9. H o ~ s c n , H. P., Htrr~L, H., KmcH, W. and OmcnAus, E. E. (1985) Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine. Eur. J. Clin. Pharmacol. 29: 199-206.
0163-7258/87 $0.00 + 0.50
Printedin GreatBritain.All rightsreserved
Copyright© 1987PergamonJournalsLtd
Specialist Subject Editor: E. E. Om,~r~vs
THE URINARY 6/~'HYDROXYCORTISOL EXCRETION IN MAN ON INDUCERS AND INHIBITORS OF THE HEPATIC MIXED FUNCTION OXIDASE J. P. DESAGER, E. DUMONT and C. HAKVEIqGT Universitd Catholique de Louvain, Laboratoire de Pharmacothdrapie, 53, A venue E. Mounier, B-1200Brussels, Belgium
The relationship among four different in vivo measurements of microsomal enzyme activity has been well established for three drugs known to be enzyme inducers in man: antipyrine, phenobarbital and rifampicin (1). We were particularly interested in two of these four parameters: the total body clearance of antipyrine and the urinary excretion of @3-hydroxycortisol (6/3-OHF). We used these measurements in a group of healthy volunteers taking phenobarbital (100rag/day; see Table 1). The 24hr-urinary excretion of 6/3-OHF, relative to that of the 17-hydroxycorticosteroids, and the antipyrine clearance were significantly increased. A statistically significant difference was found between these 2 sets of data (p<0.05). By measuring the 6/3-OHF excretion on the last day of phenobarbital intake, just before the antipyrine clearance start and then throughout the 2 days of this last test, we observed an acute effect of antipyrine on the 6/3-OHF excretion, consisting of a twofold increase on the second day (24-48hr; Table 1). Thus, antipydne contributed indirectly to the increased 6/3-OHF excretion, measured after a treatment with an inducer. In other words, 6/3-OHF values obtained during an antipyrine clearance test are spurious. Nevertheless, both tests are complementary as published recently for sulfinpyrazone (2) and carbamazepine (3) which enhance significantly the 6/3-OHF excretion and the antipyrine clearance. The main advantage to the use of 6/3-OHF over some other tests, is that it represents the biotransformation of an endogenous substrate. This substrate is metabolized by the hepatic cytochrome P-450 system which is also intimately linked to the biotransformation of xenobiotics and drugs. The antipyrine test investigates some isoforms of cytochrome P-450 while 6/3-OHF is formed by other isoforms from cortisol. This specificity of the cytochrome P-450 is illustrated by primaquine which induces the isoforms active on antipyrine but not those on cortisol (Breckenridge, this symposium). The best way to express the results of 6/3-OHF excretion is not definitely established. Results are corrected either by dividing the amount of 6/3-OHF by the amount of cortisol or by the amount of 17-hydroxycorticosteroids measured in the same urine collection. When the 17-hydroxycorticosteroids have increased after a treatment, the origin of this increase is still being debated. Indeed, the investigated drug may increase cortisol production or displace cortisol from its binding sites, particularly from cortisol binding globulin. The measurement of 6/~-OHF was first performed by RIA (4) and thereafter by HPLC (5,6,7). Although value of 6B-OHF as the most sensitive index of induction of the hepatic mixed function oxidase (MFO) system is now well established, its use to detect an inhibition is controversial. The most typical inhibitors of the hepatic MFO are the H2-receptor blocking agents (8). Cimetidine reduces the antipyrine clearance and the urinary excretion of 6/~-OHF. The effect is more modest in terms of absolute values although highly statistically significant (Table 1). This seems to indicate that both tests are less sensitive in detecting an inhibition. For ranitidine, on the other hand, no significant effect was found on the 2 parameters (Table 1) and the level of significance was low (p>0.05). It would be reasonable to think that in an MFO system activated by a strong inducer, the effect 197
Phenobarbital Phenobarbital Antipyrine
Cimetidine Cimetidine Ranitidine Ranitidine
6/~ O H F / 1 7 O H C t Antipyrine Cl T 6/30HF/17OHC
68 O H F / 1 7 O H C Antipyrine Cl T 6/~ O H F / 1 7 O H C Antipyrine CI T
l g/day 1.2 g / d a y 300 m g / d a y 300 r a g / d a y
100 m g / d a y 100 r a g / d a y 18 m g / k g
Dose 2 weeks 2 weeks 0-24hr 24-48hr 1 week 2 weeks 2 weeks 2 weeks
Duration
*C1T = Total body clearance. t 6 / 3 0 H F = 6/3-hydroxycortisol; 17OHC = 17 hydroxy corticosteroids. ~:S.E.M. was used for all data.
Drug
Parameter 8 8 8 8 16 6 6 6
N 16.7 48.7 15.3 21.5 20.1 48.2 21.65 45.7
Before + 4444444-
2.5J/ 2.6 1.9 3 1.7 6.1 3.5 3.9
27.3 58.3 21.5 29.0 !4.6 29.9 19.7 44.8
After 44444444-
4.9 4.9 3 6 1.6 2.7 3 4.2
5 6 5 6 2 0 2 1
~
~ 1 1 2 0 0 0 2 4
~
Trends
TAn~ 1. Urinary 613-hydroxycortisol Excretion and Antipyrine Clearance* Measured in Healthy Volunteers
P 2 <0.05 1 <0.05 1 <0.05 2 <0.025 14 <0.025 6 <0.005 2 NS 1 NS
$
ga
oo
Inhibition or induction evaluated by 6/3-hydroxycortisol excretion
199
of an inhibitor would be amplified. Such experiment has been described by Park (this symposium) in man receiving carbamazepine with a placebo or with cimetidine. No significant difference was found between the treatments, and it was concluded that 6/5-OHF was not sensitive enough to detect an inhibition. This conclusion is in contradiction with the results obtained with cimetidine alone. Very recently (9), the more potent inhibitory effect of cimetidine on MFO, five-fold higher than ranitidine, has been confirmed on human hepatic microsomes from uninduced patients. Results were similar in a liver with induced enzyme activity. It was concluded that the "inhibitory potency of the compounds seems to depend on the individual isozyme pattern of the hepatic microsomes". This suggests that induced liver may respond differently to ranitidine, leading to results different from those reported by Park with cimetidine. The in vitro concentrations used of both drugs are a hundred to a thousand fold higher than the circulating plasma levels in treated patients. The in vivo inhibitory effects are observed with far lower concentrations and clinically the same severity of unwanted effects have now been encountered with cimetidine and ranitidine. Pharmacogenetic investigations are thus needed in these patients suffering from acute effects to point out the specific target of these inhibitors. It can be concluded that too often only partial information is obtained and it does not allow clearcut conclusions from these drug interactions. If the urinary 6/3-OHF excretion and the antipyrine clearance test remain first line investigational tools, they need to be supported by pharmacogenetic tests. REFERENCES 1. OmCBAU$,E. E. and P ~ , B. K. (1979) Measurement of 6/3-hydroxycortisol excretion as an in vivo parameter in the clinical assessment of the microsomal enzyme-inducing capacity of antipyrine, phenobarbitone and rifampicin. Eur. J. Clin. Pharmacol. 15: 139-145. 2. STAIOER,C., ScmJcnT, F., WALTER,E., GUNDERT-RE~Y,U., H~D~RANDT, R., D~VI~s, J., WANG, N. S., Hxmaca~o, J. and W ~ x , E. (1983) Effect of single and multiple doses of sulphinpyrazone on antipyrine metabolism and urinary excretion of 6fl-hydroxycortisol. Eur. J. Clin. Pharma¢oi. 25: 797-801. 3. MOISt.AND, T. A., PARK, B. K. and RYI~NCE, G. W. (1982) Microsomal enzyme induction in children: the influence of carbamazepine treatment on antipyrine kinetics, 6fl-hydroxycortisol excretion and plasma 7-glutamyltranspeptidase activity. Br. J. clin. Pharmacol. 14: 861-865. 4. P ~ x , B. K. (1978) A direct radioimmuno-assay for 6~-hydroxycortisol in human urine. J. Steroid Biochem. 9: 963-966. 5. ROOTS, I., HOLBE, R., H O W ~ N , W., N I G h , S., H E n ~ ' t ~ R , G. and HIEDEBRANDT,A. G. (1979) Quantitative determination by HPLC of urinary 6/~-hydroxycortisol, an indicator of enzyme induction by rifampicin and antiepileptic drugs. Eur. J. Clin. Pharmacol. 16: 63-71. 6. Duuo)zr, E., SCLAVONS,M. and DESxtmR, J. P. (1984) Use of an internal standard to assay 6~-hydroxycottisul in urine. J. Liquid Chromatogr. 7: 2051-2057. 7. N ~ . J. and Y~,~TX, M. (1985) Determination of urinary cortisol and 6/~-hydroxycortisol by high performance liquid chromatography. Clin. Chim. Acta 149: 215-224. 8. IO.o~z, U. and REaOJ~rN, I. W. (1984) Drug interactions through binding to cytochrome P-450: the experience with H2-receptor blocking agents. Pharm. Res. 2: 59-62. 9. H o ~ s c n , H. P., Htrr~L, H., KmcH, W. and OmcnAus, E. E. (1985) Isolation of human hepatic microsomes and their inhibition by cimetidine and ranitidine. Eur. J. Clin. Pharmacol. 29: 199-206.