EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1994, Vol. 19, No.4, pp. 311-317
Influence of rifampicin on the toxicity and the analgesic effect of acetaminophen S. DIMOVA and Ts. STOYTCHEV Department of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Received for publication: June 22, 1993
Keywords: Rifampicin, acetaminophen, toxicity, analgesic effect, HPLC
SUMMARY The influence of rifampicin on the toxicity, analgesic effect and pharmacokinetics of acetaminophen was studied in male albino mice. Repeated administration of rifampicin (50 mglkg i.p. daily for 6 days) shortened hexobarbital sleeping time and increased liver weight, microsomal cytochrome P-450 and heme contents, NADPH-cytochrome c reductase and ethylmorphine-N-demethylase activities. Aniline hydroxylase activity was decreased and glucuronidation of p-nitrophenol was unaffected. Rifampicin pretreatment changed neither the LDsoof acetaminophen nor the hepatic glutathione level nor the glutathione depletion provoked by the toxic dose of acetaminophen (737 mg/kg p.o.). This suggests that rifampicin has no influence on the amount of acetaminophen toxic metabolites formed in the liver. Rifampicin decreased the acetaminophen analgesic effect in mice. Rifampicin decreased the Cmax, the half-time, the MRT and the AUC of acetaminophen and accelerated its clearance. The plasma concentration of acetaminophen glucuronide and acetaminophen sulfate was increased. It is assumed that the most probable mechanism by which rifampicin decreases acetaminophen analgesia is the accelerated acetaminophen elimination.
INTRODUCTION Acetaminophen (paracetamol) is a commonly used analgesic and antipyretic which causes hepatic necrosis in both laboratory animals and humans after massive doses (1). Hepatotoxicity is thought to result from the cytochrome P-450 mediated oxidation of acetaminophen to N-acetyl-p-benzoquinone imine (NAPQI), which binds covalently to tissue macromolecules thus causing cell necrosis (2,3). NAPQI is scavenged by reduced glutathione (GSH) leading to a decrease of hePlease send reprint requests to : Dr Svetlana Dimova, Department of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. BI. 23, 1113 Sofia, Bulgaria.
patic GSH concentration after acetaminophen (4). Hepatic necrosis develops when 70-80% of the GSH in the liver is depleted (4). The modulation of the microsomal monooxygenase activity induces changes in acetaminophen toxicity (1). A large body of research has indicated that rifampicin, an antibiotic mainly used in the treatment of tuberculosis, stimulates drug metabolism in humans and laboratory animals (5-7). Rifampicin enhances elimination and reduces the pharmacological effects of many drugs primarily cleared by metabolic oxidation, such as digitoxin, anticoagulants, oral contraceptives, cortisol, etc. (10). The aim of the present study was to investigate the influence of rifampicin on the toxicity and the analgesic effect of acetaminophen.
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Table I : Effect of rifampicin pretreatment on hexobarbital sleeping time, liver weight, some microsomal components and on the in vitro drug metabolism. Parameter Hexobarbital sleeping time (min) Liver weight (gllOg b.w.) Cyt P-450 (nmol/mg microsomal protein) Cyt bs (nmol/mg microsomal protein) Total heme (nmol/mg microsomal protein) NADPH-cytochrome c reductase activity (umol/min/rng microsomal protein) EMD (nmol/min/mg microsomal protein) AH (nmol/min/mg microsomal protein) UDPGT (nmol/min/mg microsomal protein)
n 8 18 5 5 5 5 5 5 5
Control 53.50 ± 2.90 0.613 ± 0.011 0.619 ± 0.047 0.284 ± 0.051 1.330 ± 0.072 0.221 2.185 0.678 5.410
± 0.006 ±0.159 ± 0.030 ± 0.299
Rifampicin
Change (%)
31.33 ±4.14** 0.678 ± 0.013*** 0.988 ± 0.018*** 0.315 ±0.085 1.680 ± 0.030**
-41 +11 +60 +11 +26
0.279 ± 0.020* 3.050 ± 0.084** 0.452 ± 0.034** 5.250 ± 0.142
+26 +39
-33 -3
Animals were sacrificed 24 h after the last rifampicin injection (50 mglkg i.p. daily for 6 days). AH - aniline hydroxylase activity; EMD - ethylmorphine-N-demethylase activity; UDPGT - UDP-glucuronyltransferase activity. Data are the mean ± SEM. • p < 0.05; up < 0.002; u.p < 0.001 compared to the corresponding control by using Students' t-test,
MATERIALS AND METHODS Animals The experiments were carried out on male albino mice weighing 20-26 g. Animals were allowed free access to standard laboratory diet and tap water. The animals were divided in groups, depending on the method of their treatment.
Rifampicin pretreatment Solutions of rifampicin (Pharmachim, Bulgaria) were prepared immediately before use. The drug was dissolved in redistilled water and HCI (pH 3.0). The mice were injected i.p. with 50 mg/kg rifampicin for 6 successive days. Controls received HCI (pH 3.0) only. In all experiments acetaminophen was administered 24 h after the last rifampicin injection.
tivity (13); ethylmorphine-N-demethylase activity (14); and UDP-glucuronyltransferase activity, using pnitrophenol as substrate (15). Hexobarbital sleeping time was measured 24 h after the last rifampicin administration, as the time between the loss and the recovery of the righting reflex after i.p. injection of 100 mglkg hexobarbital.
Acetaminophen acute toxicity Acetaminophen (Chemapol, Prague) was administered orally at 7 different doses (200-1000 mglkg) at 2 p.m. The drug was dissolved in Tween-80 and 0.9% NaCl. The animals were deprived of food 4 h before the oral administration of acetaminophen. Lethality was followed until the 7th day. The Litchfield-Wilcoxon method was used to calculate the average lethal dose (LD50) of acetaminophen.
Hepatic glutathione level Drug-metabolizing enzymes Microsomes were prepared 24 h after the last rifampicin administration, using the method of differential centrifugation (9). The livers of 3 animals were combined in one sample. Protein concentration was determined by the Biuret method, using bovine serum albumen standards. The following parameters were studied: cytochrome P-450 and cytochrome bs contents (10); total microsomal heme (II); NADPH-cytochrome c reductase activity (12); aniline hydroxylase ac-
The level of liver reduced glutathione (GSH) was determined at the lst, 2nd, 3rd and 4th h after oral treatment with 737 mglkg acetaminophen, using the fluorimetric method of Hissin and Hilf (16). A fluorescence spectrophotometer MPF-44B Perkin-Elmer was used.
Analgesic effect of acetaminophen To evaluate the analgesic effect, we used the method
S. Dimova & Ts. Stoychev, Rifampicin on acetaminophen
modified by Singh et al. (17) for inducing pain stimulation by i.p. injection of 1% solution of acetic acid at a dose of 0.1 ml/lOg b.w. Acetic acid was injected 30 min after oral administration of 5 different doses of acetaminophen (50-250 mg/kg), The number of writhings was recorded for 20 min after acetic acid injection.
313
Effect of rifampicin on acetaminophen acute toxicity Acetaminophen acute toxicity, evaluated by the LD50, was not significantly changed by rifampicin pretreatment (Table II).
Acetaminophen pharmacokinetics
Effect of rifampicin on the hepatic glutathione depletion caused by a toxic dose of acetaminophen
The blood samples were collected at 0, 10, 20, 30, 40, 50, 60, 120, 180 and 240 min post oral administration of 737 mg/kg acetaminophen. The plasma was separated by centrifugation and stored at -20°C before HPLC analysis. The plasma levels of the unchanged acetaminophen and its metabolites - acetaminophen glucuronide and acetaminophen sulfate - were measured using the HPLC method of Bhargava et al.(18). High pressure liquid chromatography was performed with a Beckman 114M Solvent Delivery Module, 163 Variable Wavelength Detector (Beckman) and CI8 analytical column (Ultraspher" XL-ODS, Beckman). The plasma concentration/time data were fitted by the nonlinear least squares program for PC HP-85 (19).
Acetaminophen applied alone at a dose of 737 mg/kg (p.o.), caused a significant depletion of liver reduced glutathione (GSH) in all intervals studied, reaching the maximum at the 2nd h. The GSH level decreased to 14% of the level in the controls. Rifampicin pretreatment induced no changes in the depletion of liver GSH provoked by the toxic dose of acetaminophen. The content of GSH at the 3rd and 4th h was significantly higher in rifampicin injected animals treated with acetaminophen as compared to that in animals receiving only acetaminophen. Rifampicin per se provoked no changes in the hepatic glutathione level (Fig. 1).
Statistical analysis Student's t-test was used to determine the significant difference between the groups. The results are presented as mean values ± SEM.
RESULTS Effect of rifampicin on drug-metabolizing enzymes The rifampicin pretreatment (50 mg/kg, i.p. for 6 days) affected the components of the mixed function oxidase system as well as the enzymatic activity. As seen from Table I, liver weight, cytochrome P-450, total heme and NADPH-cytochrome c reductase were significantly increased. The in vitro microsomal drug metabolism was differently affected by rifampicin: ethylmorphine metabolism significantly increased (by 39%), while aniline hydroxylation decreased (by 33%). The glucuroconjugation of p-nitrophenol was unaffected.
Effect of rifampicin on acetaminophen analgesia Rifampicin administered for 6 days decreased the analgesic effect of all acetaminophen doses (Fig. 2). The differences were significant at 50, 100 and 250 mg/kg acetaminophen. In the rifampicin-pretreated groups the acetaminophen exerted an analgesic effect, only in the highest doses of acetaminophen (200 and 250 mg/kg). Rifampicin per se had no effect on the writhing counts, caused by acetic acid. Table Il : Effect of rifampicin pretreatment on the LD50 of acetaminophen. W50 ofacetaminophen (mg/kg) Hour
Acetaminophen
Rifampicin Acetaminophen
24 48 72 168
766 716 654 654
898 875 875 875
(534 + (569 + (478 + (478 +
1101) 901) 895) 895)
(616 + (580 + (580 + (580 +
1309) 1320) 1320) 1320)
Rifampicin - 50 mglkg i.p. daily for 6 days. Acetaminophen was administered orally at 7 different doses (2()()...-1000 mglkg) 24 h after the last rifampicin injection.
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induced liver GSH depletion. Acetaminophen (737 mglkg p.o.) was administered 24 h after the last rifampicin injection (50 mg/kg i.p. daily for 6 days). Controls (filled circles), rifampicin treated animals (open circles), rifampicin injected animals treated with acetaminophen (filled squares), animals receiving only acetaminophen (open squares). Data are the means ± SEM. Each point is the mean from 6 animals. *P < 0.05 significant difference between rifampicin injected animals treated with acetaminophen and animals receiving only acetaminophenby using Students' t-test.
Effect of rifampicin on acetaminophen pharmacokinetics
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: Effect of rifampicin pretreatment on the acetaminophen analgesic effect. Acetaminophen was administered 24 h after the last rifampicin injection (50 mglkg i.p. daily for 6 days). The results are presented as mean ± SEM values of the writhing counts (for 20 min) induced by i.p. administration of 1% acetic acid. Each point is the mean from 10-12 mice. *p < 0.02; **p < 0.01 significant difference between rifampicin injected animals treated with acetaminophen (filled squares) and animals receiving only acetaminophen (open squares) by using Students' t-test.
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The plasma concentration/time curves for acetaminophen after single oral administration of acetaminophen (737 mg/kg) in control and rifampicin-pretreated animals are presented in Figure 3. The pharmacokinetic parameters are given in Table III. The maximum plasma concentration (C max) of unchanged acetaminophen in controls was higher than that in rifampicin-pretreated mice. Rifampicin pretreatment increased the elimination rate constant (Ket) of plasma acetaminophen which led to a lower half-time (TIIz) and a higher total body clearance (CL) as compared to controls. These changes in acetaminophen elimination decreased the area under the curve (AUC) of acetaminophen plasma concentration and shortened the mean residence time (MRT) of acetaminophen. The acetaminophen absorption rate constant (Ka), the Tmax and the volume of distribution (Vd) were not changed by rifampicin. In the rifampicin-pretreated animals, the plasma concentration of acetaminophen glucuronide was significantly higher at the 20th, 30th and 50th min (Fig. 4A), while the level of acetaminophen sulfate was
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Acetaminophen (mg/kg)
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and theoretical concentration/time curves for acetaminophen after single oral administration of acetaminophen in control (open squares) and rifampicin-pretreated mice (filled squares). Acetaminophen (737 mglkg) was administered 24 h after the last rifampicin injection (50 mglkg i.p. daily for 6 days). Each point represents the mean from 6 animals.
higher at 30th, 40th and 120th min (Fig. 4B) as compared to the controls.
315
S. Dimova & Ts. Stoychev, Rifampicin on acetaminophen
Table III : Pharmacokinetics of acetaminophen in mice after single oral administration of acetaminophen (737 mg/kg) alone and 24 h after the last rifampicin injection (50 mg/kg i.p. daily for 6 days). Acetaminophen
Parameter
335.25 24.87 0.079 0.016
C ma • (ug/rnl) T ma• (min) Ka (min-I) Kel (min-I) TI/2 (Ke1) (min) CL (ml/min) Vd (rnl) AUCtotal (ug.min/rnl) MRT(min)
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TIME (min) Fig. 4 : Plasma concentration of acetaminophen glucuronide (A) and acetaminophen sulfate (8) after a single oral administration of acetaminophen in control (open squares) and rifampicin-pretreated mice (filled squares). Acetaminophen (737 mg/kg) was administered 24 h after the last rifampicin injection (50 mg/kg i.p. daily for 6 days). The results are presented as acetaminophen equivalents. Each point is the mean from 5-6 animals. *p < 0.05; **P < 0.001 compared to the corresponding control by using Students' t-test.
Repeated i.p. injection of rifampicin (50 mg/kg daily for 6 days) in mice caused enzyme induction, demonstrated by shortened hexobarbital sleeping time and increased liver weight, cytochrome P-450 and total heme contents and NADPH-cytochrome c reductase activity. These results are in agreement with the data of other authors showing that rifampicin treatment induces hepatic mixed function oxidase activity (5,6). In our experiments, rifampicin pretreatment induced ethylmorphine-N-demethylase activity and inhibited aniline hydroxylase activity. This finding confirmed the results of Van de Brook et al. (6) and suggests that rifampicin is an atypical inducer with a differential inductive/repressive effect on particular P-450 isoenzymes. Lang et al. (7) have reported that rifampicin administration to rabbits induces an LM3 (IlIA) type and represses an LM4 (IA2) type cytochrome P450 isoenzyme . Acetaminophen toxicity is provoked by a highly reactive metabolite (NABQI) formed by cytochrome P-450-dependent enzyme systems in the liver (2,3). The present results showed that the induction of microsomal monooxygenases by rifampicin did not change the acetaminophen toxicity, determined by the LDso of acetaminophen and by the hepatic glutathione depletion. Rifampicin per se did not significantly affect the level of liver glutathione, suggesting no influence of rifampicin on liver glutathione synthesis. Thus it is assumed that rifampicin has no influence upon the amount of the acetaminophen toxic metabolite formed in the liver. Our results are in agreement with those of Prescott et al. (20), who have found that the urinary excretion of cysteine and mercapturic acid conjugates of acetaminophen (degradation products of acetaminophen glutathione conjugate) is not increased in patients on chronic rifampicin therapy. There are
316
Eur. J. Drug Metab. Pharmacokinet., 1994, No.4
two possible explanations of the unchanged acetaminophen toxicity in rifampicin pretreated mice. First, our results and those of Lange et al (7) suggest that rifampicin increases some and represses other cytochrome P-450 isoenzymes involved in acetaminophen bioactivation. Secondly, it is possible that rifampicin pretreatment influences the formation of acetaminophen glucuronide and acetaminophen sulfate. In contrast to the cytochrome P-450 mediated activation of acetaminophen, conjugation with glucuronic acid and sulfate represents detoxication pathways of the acetaminophen metabolism. It is known that the sensitivity to acetaminophen-induced toxicity is determined by the balance between toxication and detoxication metabolic pathways (21). In the present study rifampicin pretreatment significantly increased the maximum plasma concentration (C max) of acetaminophen glucuronide and acetaminophen sulfate and accelerated the clearance of nonmetabolized acetaminophen. This is in line with the results of Bock et al. (22) who observed a significantly increased metabolite/acetaminophen ratio in patients on rifampicin therapy. The induction of nontoxic glucuronidation and sulfation pathways results in a decreased proportion of the dose available for P-450 oxidation, which leads to an unchanged acetaminophen toxicity in rifampicinpretreated mice. UDP-glucuronyltransferase represents a family of enzymes, which exhibits distinct but overlapping substrate specificities (23). We found that rifampicin pretreatment did not change glucuronidation of p-nitrophenol (substrate of I, II, III and IV UDPglucuronyltransferase isoforms). Adachi et al. (24) have shown that rifampicin induces glucuronidation of bilirubin (specific substrate of isoform V of UDP-glucuronyltransferase) in the rat. This could explain why rifampicin does not influence the p-nitrophenol glucuronidation in vitro but increases the amount of acetaminophen glucuronide in vivo. The biochemical mechanism by which rifampicin increases the glucuronidation and sulfation capacity is still unclear: it could be either an increased transferase activity or an increased availability of the cosubstrates - UDP-glucuronic acid and 3'-phospho-adenosine-5'-phosphosulfate. Acetaminophen, at therapeutic doses, is mainly metabolised to glucuronide and sulfate conjugates and the pharmacological effects of acetaminophen are due to the nonmetabolized acetaminophen (25). There is a direct relationship between acetaminophen plasma levels and its analgesic effect (26). In our experiments, rifampicin pretreatment decreased the maximum plasma concentration, the half-time, MRT and AUC of acetaminophen and accelerated its clearance. Based on
these results we suggest that the most probable mechanism by which rifampicin decreases acetaminophen analgesia is the accelerated elimination of nonmetabolized acetaminophen due to the increased rate of acetaminophen glucuronide and acetaminophen sulfate metabolites formation. In conclusion, rifampicin pretreatment does not change acetaminophen toxicity and it reduces the acetaminophen analgesic effect in mice. This may have important implications for patients on chronic rifampicin therapy ingesting acetaminophen. It might be suggested that acetaminophen is not the most suitable analgesic-antipyretic for these patients.
ACKNOWLEDGEMENTS This work was supported by Grant (L-29) from the Bulgarian National Science Fund. The authors thank Prof. D. Mihailova and Z. Zivkova, Ph.D., for help with the calculation of the pharmacokinetics parameters.
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