Europ.J.clin.Pharmacol. 9, 219-227 (1975) © by Springer-Verlag 1975

Induction of Drug Metabolism in Man after Rifampicin Treatment Measured by Increased Hexobarbital and Tolbutamide Clearance W. Zilly a, D. D. Breimer 2 and E. Richter 1 IDepartment of Internal Medicine, University of WHrzburg, Federal Republic of Germany, and 2Department of Pharmacology, Subfaculty of Pharmacy, University of Leiden, The Netherlands Received: February 3, 1975, accepted: May 15, 1975

Summary. Five healthy volunteers took 1.2 g rifampicin daily for 8 days, and before and afterwards each received hexobarbital (7.32 mg/kg) and tolbutamide (20 mg/kg) by i.v. infusion on two consecutive days. The plasma concentrations of the two drugs were determined during and after infusion. The average elimination half-life of hexobarbital had decreased from 325 to 122 min and of tolbutamide from 418 to 183 min following rifampicin treatment. It was calculated that the metabolic clearance of hexobarbital had increased about three-fold and that of tolbutamide more than two-fold. Significant changes in the distribution kinetics of the two drugs were not observed. The results suggest that rifampicin is capable of inducing drug metabolism in man, which leads to an increased rate of elimination of drugs that undergo biotransformation in the liver. Key words: Rifampicin, induction of drug metabolism, hexobarbital kinetics, tolbutamide kinetics, plasma concentrations, man.

At present rifampicin is frequently used for antituberculous therapy. The kinetics of the drug alone and in combination with para-aminosalicyiic acid or isoniazid have recently been studied (Acocella et al., 1972a and b; Curci et al., 1972; Boman, 1974). It became apparent from these studies that the half-life of rifampicin was reduced after a few days of treatment. Needle liver biopsies from patients who had received rifampicin showed a distinct increase in cytochrome P-450 and in the activities of several microsomal enzymes (Schoene et al., 1972; Remmer et al., 1973). In addition, Jezequel et al. (1971) found that rifampicin caused proliferation of the s m o o t h endoplasmic reticulum in liver cells of guinea pig and man. In combination these findings suggest that rifampicin is able to induce drug metabolizing enzyme systems in the liver. The present investigation was undertaken to obtain further evidence of this hypothesis. The influence of rifampicin treatment upon the pharmacokinetics of two test substances was studied, both of which are completely

metabolized in the liver by microsomal enzymes (hexobarbital: Bush and Weller, 1972; tolbutamide: Thomas and Ikeda, 1966). MATERIAL AND METHODS Drugs

Hexobarbital-Na (Evipan®) was purchased from Bayer, Leverkusen (West Germany); tolbutamide (Rastinon@) from Hoechst, Frankfurt/Main (West Germany); 35S-tolbutamide-Na from Buchier & Co., Amersham (England); rifampicin (Rifa 300 @ ) from GrHnenthal (West Germany); and indocyanin green (Cardio Green ®) from Hynson, Westcott, Dunning (USA). Vol un reefs

The investigations were performed in 5 fully consenting healthy male volunteers, aged 22-24 years. Approximately 8 weeks before the experiments they were recruited from the students of the medical faculty. The

220

Table I. Liver function tests and plasma creatinine prior to (a) and after (b) rifampicin treatment (1,2 g/d for 8 days) in five healthy male volunteers Name

W.G.

Age (years)

Body weight (kg)

23

a) 80

Height (cm)

SGPT (mU/ml)

Bilirubin (~g/IOO ml)

iCG-half Creatinine life (min)a (mg/IOO ~i)

183

a) 40

a) 0,4

a) 4,30

a) O,6

b) 30

b) 0,8

b) 2,80

b) 1,0

b) 80 B.B.

22

a) 73

180

b) 75 D.A.

24

a) 69

22

a) 50

a) 0,2

a) 3,17

a) 0,8

b) 0,3

b) 2,45

b) 0,8

172

b) 68 S.G.

a) 10 b)

a) IO

a) 0,4

a) 4,40

a) 0,8

b) 18

b) 0,6

b) 1,14

b) O,8

167

b) 50 B.E.

23

a) 69 b) 68

a

8

181

a) 20

a) 0,5

a) 5,49

a) I,o

b) 12

b) 0,7

b) 2,91

b) 0,9

a) 12

a) 1,5

a) 4,62

a) 1,0

b) 12

b) 1,4

b) 3,48

b) 1,0

Indocyanin green; dose 0,5 mg/kg; determined by ear-densitometry

m e d i c a l i n v e s t i g a t o r s p e r s o n a l l y inf o r m e d each v o l u n t e e r a b o u t the purpose, methods, d o s a g e regimen, r a d i o a c t i v i t y , p o s s i b l e risks and side e f f e c t s of the study, to e n a b l e each v o l u n t e e r to m a k e his own j u d g e m e n t about his w i l l i n g n e s s and a b i l i t y to p a r t i c i p a t e . Each s u b j e c t was also i n f o r m e d that he was free to r e t i r e at any time d u r i n g the c o u r s e o f the e x p e r i m e n t . A f t e r r e v i e w i n g the l i t e r a t u r e and from their own e x p e r i e n c e s it was the authors' o p i n i o n that rif a m p i c i n g i v e n alone w o u l d p r o b a b l y not be h e p a t o t o x i c ; for example, H a k i m et al. (1971) did not o b s e r v e c h a n g e s in liver f u n c t i o n tests w h e n t r e a t i n g p a t i e n t s w i t h 20 m g / k g r i f a m p i c i n daily, even in c o m b i n a t i o n w i t h i s o n i a z i d and s t r e p t o mycin. All v o l u n t e e r s had n o r m a l liver and renal function, as j u d g e d by n o r m a l s e r u m levels of t r a n s a m i n a s e (SGPT), b i l i r u b i n and c r e a t i n i n e and a n o r m a l i n d o c y a n i n g r e e n h a l f - l i f e (Table I). The b i o c h e m i c a l tests w e r e r e p e a t e d a f t e r t r e a t m e n t w i t h r i f a m p i c i n . The v o l u n t e e r s had not r e c e i v e d any r e g u l a r m e d i c a t i o n d u r i n g the two m o n t h s p r e c e d i n g the e x p e r i m e n t s . Treatment

On Day I of the i n v e s t i g a t i o n , a f t e r an o v e r n i g h t fast, the v o l u n t e e r s r e c e i v e d h e x o b a r b i t a l - Na (7.32 m g / k g free acid) by l i n e a r i.v. i n f u s i o n for 60 min. A r e l a t i v e l y slow i n f u s i o n p r o c e d u r e for i.v. d r u g a d m i n i s t r a t i o n was p r e f e r r e d

in o r d e r not to d i s c o m f o r t the volunteers and to avoid the severe CNS d e p r e s s i o n that f o l l o w s r a p i d i n j e c t i o n of anaest h e t i c b a r b i t u r a t e s . V e n o u s b l o o d samples 5 ml w e r e c o l l e c t e d in h e p a r i n i z e d tubes at 10, 20, 30, 40, 50 and 60 min d u r i n g the infusion, and at 10, 20 and 30 min, and I, 2, 4, 6, 8, 10, 12, 18 and 24 h after it. In addition, a 24 h p o o l e d urine sample was collected. Plasma and urine samples were k e p t frozen until a n a l y s e d w i t h i n 6 weeks. On Day 2 3 5 S - t o l b u t a m i d e - N a (20 mg/kg; sp act O.12 ~Ci/mg) was a d m i n i s t e r e d to the same v o l u n t e e r s by a linear i.v. i n f u s i o n lasting 20 min. They r e c e i v e d a c o n t i n e n t a l b r e a k f a s t shortly b e f o r e the e x p e r i m e n t in o r d e r to avoid severe h y p o g l y c a e m i a . Venous b l o o d samples were taken at 5, 10, 15 and 20 m i n d u r i n g the i n f u s i o n and at 0.5, I, 2, 3, 4, 6, 8, 12, 20, 24, 30, 36 and 47 h a f t e r it. Urine was c o l l e c t e d at f r e q u e n t intervals up to 47 hours in o r d e r to d e t e r m i n e the u r i n a r y e x c r e t i o n h a l f - l i f e of t o l b u t a m i d e m e t a b o l i t e s . P l a s m a and urine samples w e r e k e p t frozen u n t i l a n a l y s e d w i t h i n 4 weeks. D u r i n g Day 3-10 each v o l u n t e e r r e c e i v e d 1.2 g r i f a m p i c i n o r a l l y each day. A r e l a t i v e l y high dose of r i f a m p i c i n was used, since it has b e c o m e a p p a r e n t f r o m o t h e r s t u d i e s that the s t i m u l a t i n g e f f e c t of a d r u g on h e p a t i c drug m e t a b o l i s m seems to be dose dependent. A p o s s i b l e e f f e c t m i g h t be p r o d u c e d by a h i g h e r d o s e d u r i n g a rel a t i v e l y short p e r i o d of treatment. On Day 11 the e x p e r i m e n t w i t h h e x o b a r b i t a l ,

221

and on Day 12 that w i t h t o l b u t a m i d e was repeated. The doses a d m i n i s t e r e d and the s a m p l i n g s c h e d u l e s were the same as in the first trial. Assay of Drugs

P l a s m a and urine c o n c e n t r a tions of h e x o b a r b i t a l were a n a l y s e d a c c o r d i n g to the m e t h o d of B r e i m e r and van R o s s u m (1974), u s i n g gas c h r o m a t o graphy w i t h a n i t r o g e n s e l e c t i v e detector. 2.0 ml p l a s m a or 10.O ml u r i n e was e x t r a c t e d twice w i t h 10 ml light petro!eum (40 - 600) - a m y l a l c o h o l (1OO : 2); m e t h o h e x i t a l (3.0 ~g) was used as the internal standard. The e x t r a c t was conc e n t r a t e d to d r y n e s s and the r e s i d u e d i s s o l v e d in O.1 ml abs. ethanol. A b o u t 2 - 5 ul of the s o l u t i o n was i n j e c t e d into a H e w l e t t - P a c k a r d gas c h r o m a t o g r a p h (Model 5750), e q u i p p e d w i t h a n i t r o g e n d e t e c t o r (Hewlett-Packard, Model 15161A). C o l u m n conditions: glass (1.8 m x 3 m m I.D.), p a c k e d w i t h 3% OV 17 on G a s - C h r o m Q, 60 - 80 mesh, temp. 230o C. Hexobarbital.

3'-Keto-hexobarbital. The 3 ' - k e t o - h e x o b a r b i t a l c o n c e n t r a t i o n in urine was also d e t e r m i n e d by gas c h r o m a t o g r a p h y (Breimet, 1974). 2.0 ml urine was e x t r a c t e d twice with 10 ml of d i s t i l l e d ethyl acetate. The e x t r a c t was c o n c e n t r a t e d to dryness and to the r e s i d u e was added O.1 ml abs. ethanol, c o n t a i n i n g 60 ~g codeine base as an e x t e r n a l standard. A f t e r e v a p o r a t i o n of the e t h a n o l the residue was d i s s o l v e d in 0.5 ml acetone, and 5 ~i of this s o l u t i o n was i n j e c t e d into the gas c h r o m a t o g r a p h , e q u i p p e d w i t h the n i t r o g e n detector. C o l u m n c o n d i t i o n s were the same as for hexobarbital. The identity of the m e t a b o l i t e was c h e c k e d by gas c h r o m a t o g r a p h y - mass s p e c t r o m e t r y (LKB 9000) by c o m p a r i s o n with the s y n t h e t i c r e f e r e n c e compound. Tolbutamide. P l a s m a concentrations of t o l b u t a m i d e were m e a s u r e d by the c o l o r i m e t r i c m e t h o d of S p i n g l e r (1957), as well as by c o u n t i n g 35S-radioactivity. P a r a l l e l m e a s u r e m e n t s of t o l b u t a m i d e levels by the two m e t h o d s were performed, since the limit of s e n s i t i v i t y of S p i n g l e r ' s m e t h o d is about 30 mg/l. The results of the two m e t h o d s a g r e e d s a t i s f a c t o r i l y at c o n c e n t r a t i o n s h i g h e r than 30 mg/l, w h i c h i n d i c a t e d that the r a d i o a c t i v i t y of 35S r e p r e s e n t e d the time c o n c e n t r a t i o n of tolbutamide. M e t a b o i i t e s of t o l b u t a m i d e do not interfere w i t h the c o l o r i m e t r i c d e t e r m i n a t i o n of u n c h a n g e d drug (Spingler, 1957). In urine, total t o l b u t a m i d e m e t a b o l i t e c o n c e n t r a t i o n s were a n a l y z e d by the

m e t h o d of N e l s o n et al. (1960), as a c o n t r o l for m e a s u r e m e n t s of r a d i o a c t i v i ty. B i n d i n g of t o l b u t a m i d e to p l a s m a p r o t e i n s was d e t e r m i n e d by u l t r a c e n t r i f u g a t i o n (Scholtan, 1965). P l a s m a samples 6 ml w h i c h had b e e n c o l l e c t e d 15 min after s t a r t i n g the t o l b u t a m i d e infusion were t r a n s f e r r e d to high speed c e n t r i f u g a t i o n tubes, and were run for about 17 h at 40,000 r p m in a p r e p a r a tive u l t r a c e n t r i f u g e (Beckman Spinco L 50, Rotor 50 Ti). The c o n t e n t s of the tubes were s e p a r a t e d into 12 x 0.5 ml fractions, and the t o l b u t a m i d e c o n c e n tration was e s t i m a t e d in r e l a t i o n s h i p to p r o t e i n c o n c e n t r a t i o n . U n b o u n d tolbutamide was c a l c u l a t e d by r e g r e s s i o n analysis. Calculation of Pharmacokinetic Parameters

The p o s t - i n f u s i o n p l a s m a c o n c e n t r a t i o n s of h e x o b a r b i t a l and t o l b u t a m i d e were c o m p u t e r fitted by n o n - l i n e a r least squares r e g r e s s i o n a n a l y s i s a c c o r d i n g to the f o l l o w i n g equation, w h i c h is v a l i d for t w o - c o m p a r t m e n t k i n e t i c s after linear i.v. i n f u s i o n (Loo and Riegelman, 1970): AI -sT e (T-t) Cp = --~ • (1-e )- e + A2 -BT 8 (T-t) B-T " (1-e )'e w h e r e Cp is the p o s t - i n f u s i o n p l a s m a c o n c e n t r a t i o n , A I and A 2 are the hypothetical i n t e r c e p t s with the o r d i n a t e for an i.v. bolus injection, e and are the rate c o n s t a n t s for the rapid and slow decay of the p l a s m a c o n c e n t r a tion, r e s p e c t i v e l y , T is the linear infusion time, and t is time. D e t a i l s of the fitting p r o c e d u r e have b e e n described e l s e w h e r e in a p a p e r on the k i n e t i c s of h e x o b a r b i t a l in h e a l t h y s u b j e c t s (Breimer et al., 1975). A f t e r h a v i n g o b t a i n e d the best fits for the c o e f f i c i e n t s A I and A2, and the rate c o n s t a n t s e and 8, the p a r a m e t e r s i n t r i n s i c to the t w o - c o m p a r t m e n t open m o d e l were c a l c u l a t e d a c c o r d i n g to van R o s s u m (1971). In the case of t o l b u t a m i de after r i f a m p i c i n treatment, the plasma c o n c e n t r a t i o n d e c a y could be d e s c r i b e d by o n e - c o m p a r t m e n t kinetics. RESULTS H e x o b a r b i t a l and t o l b u t a m i d e i n f u s i o n s were well t o l e r a t e d by the v o l u n t e e r s . H y p o g l y c e m i a o c c u r r e d to a m o d e r a t e degree (blood g l u c o s e 30-40 m g / I O O ml)

222

^d•

m.~i icl

7~

~n

u

5.1 ~' \

o~

3.~

~ E

2.1

m

585-6mg hexobarbitat finear intravenous infusion 60min two compartment modet

~\

"÷~.

~

201~% 126-~ \.?'+.+

7s{

1600mg tolbutamide linear intravenous infusion 20rain

\

"-.+

~+-

U

cD~

ter rifampici

E

+

o (.)

o 0

0.t

I

0

0

60 t20 t80 240 300 360 /+20 480 5&O 600 660 720 780 Time {mini

g

8

12

16 20 2& Time [hours)

28

32

36

Fig. i. Plasma concentrations of hexobarbital plotted semi-logarithmically during and after a 60 min zero-order i.v. infusion. The upper curve was obtained prior to and the lower curve after rifampicin treatment for 8 days (1.2 g/d). The post-infusion concentrations were fitted according to two-compartment kinetics. The concentrations during infusion (triangles and circles) ha~e not been used in the fitting procedure; the curves representing this phase were calculated on basis of the parameters deduced from the post-infusion data. The figure was taken directly from a computer plot

Fig. 2. Tolbutamide plasma concentrations plotted semi-logarithmically during and after a 20 min zero-order i.v. infusion. The upper curve was obtained prior to and the lower curve after rifampicin treatment for 8 days (1.2 g/d). For the former the post-infusion concentrations were fitted according to two-compartment kinetics and in the latter according to single compartment kinetics. The concentrations during infusion were not used in the fitting procedure. The figure was taken directly from a computer plot

Table 2. Pharmacokinetic parameters of hexobarbital treatment with rifmnpicin (1,2 g/d) a

in five volunteers before (a) and after (b) 8 days

Hexobarbital

e

8

(min-])

t~

(min-l)

Vp

(min)

VI

(ml x min -I x kg -I

VDs s

(i x kg -I)

(l x kg -I)

HB-output in 24-hurine (% of dose)

Keto-HBoutput in 24-h-urine (% of dose)

a)

b)

a)

b)

a)

b)

a)

b)

a)

b)

a)

b)

a)

b)

a)

b)

W+G.

O,O235

0,0543

O,OO16

O,OO51

441

138

2,7

10,3

0,44

0,52

1,40

1,49

O,31

0,12

46,2

45,7

B.H.

0,0299

0,0653

0,0027

0,0070

256

99

3,9

9,8

0,52

0,52

[,20

1,13

0,26

0,02

41,4

19,3

D.A.

0,0355

0,595

0,0033

0,0070

208

99

4,5

11,O

0,35

0,40

0,98

I,O3

0,48

0,13

66,3

91,6

S.G.

O,0123

O,O192

0,0020

O,O05~

351

136

2,5

8,3

0,63

0,88

1,05

1,25

0,41

0,20

40,5

47,2

B.E.

0,0083

0,0224

O,OO19

O,OO51

367

138

2,5

8,8

0,73

0,81

1,O7

1,29

0,26

O,11

30,9

57,4

mean

0,O219

0,0437

0,0023

0,0059

324,6

122

3,22

9,64

0,53

0,63

1,14

1,24

0,34

O,12

45,1

52,3

SD

0,O114

O,0212

O,OOO7

O,OO10

92,7

21

0,92

1,O9

0,15

O,21

O,17

0,]'7

0, IO

0,06

13,1

26,1

and 6 are the rate constants for the two compartment open model; t~ is the .half-l~fe of the ~ phase, where t~ = 0.69/6; Vp is the plasma clearance calculated according to van Rossum (1971), where Vp=D/(AI/e+A2/6); V I is the volume of the initial dilution space, where V 1 = D/(A I + A2); VDs s is the volume of distribution at steady state, where VDs s = D(AI/e2 + A2/~2) / (AI/~ + A2/6)2; A I and A 2 are the intercepts with the ordinate for an i.v. bolus injection of the same amount of drug, which w~re Calculated from the experimental intercepts after infusion according to the method of Loo and Riegelman (19707. aThe individual plasma concentrations

are available on request from the authors.

4O

223 about 30 min after the end of the tolbutamide infusion, but additional glucose administration was not required. The volunteers often displayed typical signs of CNS depression during hexobarbital infusion (drowsiness, incoherent speech, and sometimes they slept.) Recovery occurred within two or three hours of the infusion, as judged by subjective feelings. After 8 days of rifampicin treatment conventional liver function tests showed no particular alterations (Table I). A reduction in the half-life of indocyanin green was observed in every volunteer, which was comparable to the previous findings of Melikian et al. (1972), who studied the influence of various drugs on indocyanin green elimination. Due to the experimental procedure, i.e. single injection technique and ear densitometry, accurate clearance values could not be obtained, and it is difficult, therefore , to speculate on the meaning of influence of rifampicin on indocyanin green elimination. It is suggested that the present results can only be interpreted in terms of no disturbance of hepatic dye excretion after rifampicin treatment. Rifampicin was well tolerated by the volunteers and adverse reactions due to continuous treatment, as described for instance by Scheuer et al. (1974), were not observed. The hypoglycaemic reaction in terms of blood glucose level 30 min after tolbutamide administration had not changed significantly after rifampicin treatment.

Hexobarbi tal

The post-infusion concentration-time curves exhibited two distinct phases in semi-logarithmic plots, which indicates that two-compartment kinetics were valid (Fig. I). Hexobarbital plasma concentrations were distinctly lower after rifampicin treatment and, in addition, the plasma concentration decay was much faster than in the control study (Fig.

trial (average increase from 3.22 ml. min-1"kg -1 to 9.64 ml. min-1"kg-1). The 24-hour urinary excretion of unchanged hexobarbital was less than 0.5% in the control experiment, and it fell to less than 0.2% after rifampicin treatment (Table 2). On the other hand, excretion of one of the major metabolites of hexobarbital, 3'-keto-hexobarbitai, mostly increased after rifampicin treatment (Table 2). Tolbutamide

Two-compartment kinetics were valid for tolbutamide, although a better fit was obtained for the data after rifampicin treatment by use of single compartment kinetics (Fig. 2). As in the case of hexobarbital results, the plasma concentrations of toibutamide were distinctly lower in the second trial (Fig. 2). The average half-life of to!butamide had decreased from 418 to 183 min after rifampicin treatment. This could mainly be attributed to increased clearance of tolbutamide, since the volume of distribution remained unchanged (Table 3). In each volunteer the clearance value was more than twice as high as in the first trial; average increase from 0.21 ml. min-l.kg -I to 0.47 ml-min-l.kg-1. The cumulative urinary excretion of tolbutamide metabolites during the first 6 h after drug administration increased considerably after rifampicin treatment (Table 3). The half-life of tolbutamide metabolites calculated from the excretion data was 352 ± 89 min on the first and 233 ± 20 min on the second trial in the five volunteers. It appeared that there was a significant correlation between the plasma half-life of unchanged tolbutamide and the urinary excretion half-life of its metabolites (correlation coefficient 0,92). Binding of tolbutamide to plasma proteins did not change significantly after rifampicin treatment; approximately 95% was bound as determined by the ultracentrifugation method on the two occasions.

I). The average hexobarbital half-life in the slowest phase (represented by the rate constant ~, where 8:O.59 min -I) had decreased from 325 to 122 min following rifampicin treatment. This was apparently due to an increase in the metabolic clearance of hexobarbital, since significant changes in the other pharmacokinetic parameters did not occur (Table 2). In each volunteer the clearance value was from two to more than three-fold higher than in the first

DISCUSSION Several investigations have indicated that rifampicin stimulates its own metabolic degradation during continuous treatment, as judged by shortening of its half-life (Acocella et al., 1972a; Boman, 1974; Virtanen and Tala, 1974). The inductive effect of the compound on metabolism of other drugs administered concomitantly has not been well documented. Acocella et al. (1972a) were

224 Table 3. Pharmacokinetic parameters of tolbutamide in 5 volunteers before (a) and after (b) 8 days treatment with rifampicin (I~2 g/d) a Tolbutamide

~ (min-I)

a)

~ (min-I)

t~ (min)

b)

a)

b)

a)

b)

Vp (ml x min -I x kg -I )

vI (i x kg-i)

VDs s i x kg-I)

To .-metabolites in urine (% of dose) after after 2 h 6 h

a)

a)

a)

a)

b)

b)

b)

b)

a)

b)

W.G.

0,0230

-

0,0019

O,OO41

361

172

O , 2 1 0,46

0,069

0, I0 0, II

2 3 , 3 43,8

5 4 , 1 82,5

B.B.

0,0595

-

O,0016

0,oo41

442

171

O,19

O,122

0,12

15,5 28,1

4 2 , 6 65,9

0,51

0,13

D.A.

0,0303

-

O,0019

O,OO42

364

166

0,26

0,56

O,081 -

0,13

O,13

25,4 39,4

5 4 , 9 81,7

S.G.

O,O216

-

O,OO19

0,0040

372

175

0,23

0,50

0,096

0,12

0,13

16,2 34,6

42,0

76,7

B.E.

0,0476

-

0,OO13

0,0030

550

229

O,16

0,34

0,083

0,12

0,11

11,7 20,I

3 1 , O 46,9

Mean

0,0364

-

0.0.917 0,0039

418

183

O , 2 1 O,47

0,090

O,118 0,122

18,4 3 3 , 2

4 4 , 9 70,7

± SD

O,O165

-

0,0003

81

26

O,O3

0,020

O,011 0,011

0,0005

0,08

5,7

9,4

9,9

14,9

The parameters of experiment (a) were fitted according to two compartment kinetics and of experiment (b) by single compartment kinetics. In the latter case, VDss was calculated by dividing the given dose by the corrected intercept with the ordinate, and Vp was calculated by multiplying VDs s by 8. aThe individual plasma concentrations are available on request from the authors.

u n a b l e to s h o w s u c h a n i n t e r a c t i o n between rifampicin and isoniazid after daily treatment for one week. However, M i c h o t et al. (1970) o b s e r v e d that higher doses of anticoagulants were necessary during rifampicin t h e r a p y . It h a s a l s o been reported that women taking oral contraceptives during such treatment developed abnormalities of the menstrual cycle and occasionally pregnancy ocet al., 1 9 7 3 ) . curred (Nocke -Finck Since hexobarbital is a l m o s t c o m p l e t e ly e l i m i n a t e d by hepatic metabolism (Bush and Weller, 1972), and has been w i d e l y u s e d as a m o d e l s u b s t r a t e in studies of changes of drug metabolism in animal experiments, this compound ought to be suitable for similar experim e n t s in m a n . F o r c o m p a r a t i v e reasons, tolbutamide w h i c h is a l s o c o m p l e t e l y metabolized in the liver (Thomas and I k e d a , 1 9 6 6 ) , a n d is w i d e l y e m p l o y e d in practical pharmacotherapy, was also included in the present investigation. In o r d e r t o a v o i d t h e b i o a v a i l a b i l i t y problems which may be encountered after oral administration, e.g. due to the pharmaceutical formulation or to a "first-pass e f f e c t " w h i c h is n o t i m probable under conditions of important stimulation of drug metabolism, the compounds were given intravenously. This permitted reliable estimation of pharmacokinetic parameters, and the crossover design of the study created the possibility for comparison of results from each individual. Although the dramatic decrease in the plasma half-lives of both drugs observed after rifampicin treatment indicated

greatly increased rates of metabolism, t h i s c a n o n l y b e r e g a r d e d as c e r t a i n if t h e r e is n o s i m u l t a n e o u s change in the apparent volume of distribution. The elimination half-life of almost every d r u g is m a i n l y d e p e n d e n t on the apparent volume of distribution and on its renal or metabolic clearance (van R o s s u m , 1 9 7 1 ) . C l e a r a n c e m a y b e r e g a r d e d as a measure of the functional ability of a substance to be removed by the eliminating organ ( R o w l a n d et el., 1 9 7 3 ; R i e g e l man and Rowland, 1973). The distribution behaviour of hexobarbital and tolbutamide was not significantly influenced by rifampicin, as j u d g e d b y t h e v o l u m e s of d i s t r i b u t i o n f o u n d in the two trials in each individual ( T a b l e s 2 a n d 3). T h e s h o r t e n e d halflife must be attributed entirely to increased clearance of the drugs which provides evidence for extensive stimulation of microsomal drug metabolism by rifampicin. A significant correlation was found between the clearances of both substances if t h e d a t a b e f o r e a n d a f t e r r i f a m p i c i n treatment were taken into consideration (Fig. 3). From the individual clearance data and the mean values given in Tables 2 and 3 it can be deduced that rifampicin stimulated the elimination of hexobarbital to a slightly greater extent than tolbutamide. This observation may suggest that the rifampicin effect was more pronounced on the activity of the hexobarbital metabolizing enzyme system in the liver. However, it s h o u l d b e c o n sidered that the second hexobarbital

225

i n v e s t i g a t i o n was p e r f o r m e d 24 h after the last dose of r i f a m p i c i n , w h e r e a s the t o l b u t a m i d e test was done after a further 24 h. P e r h a p s the s t i m u l a t o r y e f f e c t of r i f a m p i c i n on drug m e t a b o l i s m had d e c r e a s e d to some e x t e n t by that time. F u r t h e r p r e l i m i n a r y i n v e s t i g a t i o n s have r e v e a l e d that the i n d u c t i v e effect had d i s a p p e a r e d in m o s t v o l u n t e e r s within 14 days after the last r i f a m p i c i n dose (Zilly et al., 1975). The a b s o l u t e c l e a r a n c e v a l u e s for h e x o b a r b i t a l w e r e found to be 10 - 20 fold h i g h e r than for tolbutamide. F o l l o w i n g r i f a m p i c i n treatment hexobarbital clearance increased to about 10 ml. m i n . - 1 . k g -I. Since liver p l a s m a flow is only s l i g h t l y h i g h e r (12 - 15 ml. m i n - l . k g - 1 ; B r a d l e y et al. 1952), it may be a n t i c i p a t e d that to some e x t e n t h e x o b a r b i t a l c l e a r a n c e becomes d e p e n d e n t on liver b l o o d flow B r a n c h et al. (1974) (Gillette, 1 9 7 1 ) . r e c e n t l y d e m o n s t r a t e d that p h e n o b a r b i t a l t r e a t m e n t w h i c h is also k n o w n to be a p o t e n t e n z y m e - i n d u c e r , c a u s e d an increase in liver b l o o d flow. They showed that the f l o w - d e p e n d e n t p r o p r a n o l o l p l a s m a c l e a r a n c e i n c r e a s e d to a h i g h e r d e g r e e than did the n o n - f l o w - d e p e n d e n t c l e a r a n c e of antipyrine. A l s o lidocaine, a s u b s t a n c e w i t h a r e l a t i v e l y high m e t a b o l i c clearance, has been shown to have a d i m i n i s h e d p l a s m a c l e a r a n c e in patients with reduced cardiac output (Thomson et al°, 1973). By analogy, it cannot be e x c l u d e d that a r i f a m p i c i n induced i n c r e a s e in liver b l o o d flow is an a d d i t i o n a l factor in the o b s e r v e d high c l e a r a n c e of h e x o b a r b i t a l . In c o n s e q u e n c e of the a c c e l e r a t e d rate of m e t a b o l i s m of tolbutamide, an increase in c u m u l a t i v e u r i n a r y excretion of m e t a b o l i t e s was found d u r i n g the first 2 or 6 h after drug administration (Table 3). A good c o r r e l a t i o n was o b s e r v e d b e t w e e n the c u m u l a t i v e 2 hour e x c r e t i o n of m e t a b o l i t e s and the p l a s m a c l e a r a n c e of u n c h a n g e d drug (Fig. 4). As m e n t i o n e d earlier, the u r i n a r y e x c r e t i o n h a l f - l i f e of total m e t a b o l i t e s had s i g n i f i c a n t l y d e c r e a s e d after r i f a m p i c i n t r e a t m e n t w h i c h was in a c c o r d a n c e w i t h the i n d u c t i o n phenomenon. A s i m i l a r finding was r e p o r t e d by R e m m e r et al. (1973), who found an i n c r e a s e d rate of u r i n a r y a m i n o p h e n a z o n e e x c r e t i o n in p a t i e n t s i n j e c t e d with n o v a m i n s u l f o n u m and t r e a t e d with rifampicin. The finding of a d i m i n i s h e d 24 hour c u m u l a t i v e e x c r e t i o n of unchanged h e x o b a r b i t a l after r i f a m p i c i n was in a g r e e m e n t w i t h its s h o r t e n e d plasma h a l f - l i f e caused by a c c e l e r a t e d m e t a b o l i s m . M e a s u r e m e n t of the 24 hour e x c r e t i o n of 3 ' - k e t o - h e x o b a r b i t a l was i n c l u d e d in the i n v e s t i g a t i o n in o r d e r

12 I

.z

10

E

8

6 .,;>

4 "5 ~5 ,Z3 0

2

3i

0

_ . . _ / i 1............... I I 0,1 0,2 0.3 0.4 0.5 Vp ( m l . m i ~ l . k g -t ) Tolbutamide

I 0.6

Fig. 3. Plot of calculated plasma clearances of hexobarbital versus those of tolbutamide for five volunteers before (x) and after (.) rifampicin treatment (1,2 g/d for 8 days)

0.6 05 .X

"T

g

O.&

03 0.2 0.1

"(3

~

/

~

r = 0.85 ..........

aO

#_

~ before rifampicin • after rifampicin

i 10

t 20

J 30

I 40

1 50

Urinary rnetobotites excreted within the f i r s t 2 h o u r s (°/oof dose)

Fig. 4. Plot of the calculated tolbutamide plasma clearance versus cumulative 2 hour urinary excretion of tolbutamide metabolites in five volunteers before (x) and after (-) rifampicin treatment (1.2 g/d for 8 days)

226 to e s t a b l i s h p o s s i b l e q u a l i t a t i v e c h a n g e s in h e x o b a r b i t a l m e t a b o l i s m . T h e 3'-keto-derivative a p p e a r s to be an imp o r t a n t m e t a b o l i t e of h e x o b a r b i t a l in man, since the normal average cumulative e x c r e t i o n is 45% (Table 2). B u s h et el. (1953) w e r e t h e f i r s t t o i s o l a t e t h i s compound from the urine of dogs, and it w a s i d e n t i f i e d s u b s e q u e n t l y in h u m a n u r i n e ( T s u k a m o t o et ai. 1957;, F r e y , 1959). T h e p r e s e n t g a s c h r o m a t o g r a p h i c method provides a relatively simple procedure for quantitative determination of it in h u m a n u r i n e ( B r e i m e r , 1974). The results after rifampicin treatment were variable, suggesting that unpredicta b l e q u a l i t a t i v e c h a n g e s in h e x o b a r b i t a l metabolism may also have occurred(Table 2). Tolbutamide is l a r g e l y b o u n d to 1973), so plasma proteins (Held et al., c h a n g e s in p r o t e i n b i n d i n g c o u l d e a s i l y l e a d to an a l t e r e d p h a r m a c o k i n e t i c behaviour. For example, a faster decline in t o l b u t a m i d e p l a s m a c o n c e n t r a t i o n s h a s b e e n f o u n d in p a t i e n t s w i t h a c u t e or chronic liver disease ( S ~ d h o f et al., 1958; H e l d et al., 1973) w h i c h w a s a t t r i b u t e d to r e d u c e d p r o t e i n b i n d i n g caused by elevated bilirubin and/or hypalbuminaemia. However, rifampicin d o e s n o t s e e m to i n f l u e n c e the e x t e n t of p r o t e i n b i n d i n g o f t o l b u t a m i d e . The results of this investigation h a v e s h o w n t h a t r i f a m p i c i n is c a p a b l e of inducing drug metabolism in man w h i c h l e a d s to a n i n c r e a s e d r a t e of elimination of drugs that undergo biotransformation in t h e l i v e r b y m i c r o somal enzymes. T h e m e c h a n i s m by w h i c h t h e i n d u c t i o n t a k e s p l a c e is u n c e r t a i n a n d is e s pecially interesting since rifampicin i t s e l f is m a i n l y m e t a b o l i z e d by n o n microsomal desacetylation (Maggi et al., 1969). T h e t y p e of d r u g i n t e r a c t i o n e s t a b l i s h e d in t h e p r e s e n t i n v e s t i g a t i o n s h o u l d be g i v e n c o n s i d e r a t i o n w h e n r i f a m p i c i n is p r e s c r i b e d c o n c o m i t a n t l y with other drugs. Interference with anticoagulant d r u g t h e r a p y ( M i c h o t et al., 1970) a n d w i t h c o n t r a c e p t i v e pills (Nocke - F i n c k et al., 1973) h a v e a l ready indicated that the interaction may be of clinical significance. In a d d i t i o n , E d w a r d s et al. (1974) h a v e r e ported that patients with Addison's disease require larger daily doses of hydrocortisone when treated simultaneo u s l y w i t h r i f a m p i c i n . It is i m p o r t a n t to n o t e t h a t A c o c e l l a et al. (1972a) a n d B o m a n (1974) d i d n o t o b s e r v e c h a n g e s in the k i n e t i c s of i s o n i a z i d w h e n it w a s c o m b i n e d w i t h r i f a m p i c i n in t h e t r e a t ment of tuberculous patients. On the other hand, the absorption of rifampicin itself was significantly reduced when it w a s a d m i n i s t e r e d w i t h p a r a - a m i n o s a l i c y l i c a c i d (Boman, 1974).

The authors wish to thank Mrs. C.P.W.G.M. Verweij-van Wissen for help with determination of hexobarbital in plasma, and Mrs. R. Gerhardt, Miss S. KSppl and Miss D. Oschmann for their aid with determination of tolbutamide concentrations. The investigation was partly supported by a grant from M e Netherlands' Foundation for Medical Research, FUNGO.

Acknowledgements.

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