Disposition Of Paracetamol Research Articles

Multiscale modelling approach combining a kinetic model of glutathione metabolism with PBPK models of paracetamol and the potential glutathione-depletion biomarkers ophthalmic acid and 5-oxoproline in humans and rats

A key role of the antioxidant glutathione is detoxification of chemically reactive electrophilic drug metabolites within the liver. Therefore glutathione depletion can have severe toxic consequences. Ophthalmic acid and 5-oxoproline are metabolites involved in glutathione metabolism, which can be measured readily in the blood and urine and have been proposed as candidate biomarkers of hepatic glutathione content. However, currently it is unclear whether their concentrations in plasma exhibit a robust correlation with hepatic glutathione content. To explore this important question, we have developed a novel approach which combines a physiologically based pharmacokinetic (PBPK) model of metabolism and disposition of paracetamol (acetaminophen) with a previously developed mathematical systems model of hepatic glutathione homeostasis. Paracetamol is metabolised to reactive intermediates which deplete glutathione and cause toxicity when given at high doses. Our model correctly predicted that hepatic glutathione depletion following paracetamol administration resulted in elevated concentrations of 5-oxoproline and ophthalmic acid in blood and of 5-oxoproline in urine. However, we also found from the model that concentrations of both of the compounds were likely to be influenced by prolonged administration of paracetamol and by the concentrations of intracellular metabolites such as methionine. We conclude that care must be taken when extrapolating from concentrations of these biomarkers to hepatic glutathione status.

Pharmacokinetics and ocular disposition of paracetamol and paracetamol glucuronide in rabbits with diabetes mellitus induced by alloxan

Background:This study evaluates the pharmacokinetics (PK) and ocular disposition of paracetamol and paracetamol glucuronide in diabetic rabbits.Methods:Thirty two New Zealand rabbits were divided into four groups: control group (I, n=8), control group with diabetes (II, n=8), rabbits with diabetes receiving paracetamol (III, n=8), rabbits without diabetes receiving paracetamol (IV, n=8). To induce diabetes mellitus, alloxan was administrated intravenously (iv) in the dose of 90mg/kg body weight (b.w.) to 16 rabbits (groups II and III). Eight weeks post induction of the diabetic state, paracetamol was administrated via the ear vein at a dose of 35mg/kg b.w. to groups III and IV. Blood and aqueous (ocular fluid) samples were collected after drug administration. PK calculations were made based on non-compartmental analysis.Results:Significant differences were observed in PK of paracetamol between the studied groups. Lower value of the area under the concentration – time curve and enhanced clearance of paracetamol were noted in the diabetic group. In the case of paracetamol glucuronide, the area under the concentration – time curve was also little lower; however, no changes in the elimination rate were observed. Simultaneously, diminished ocular disposition of paracetamol was obtained in the diabetic group, whereas no changes were noted according to the penetration of paracetamol glucuronide.Conclusions:The PK as well as ocular disposition of paracetamol may be altered in non-treated diabetes mellitus The glucuronidation does not seem to be the process responsible for these changes.

Influence of water deprivation on the disposition of paracetamol.

The effect of acute (96 h) water deprivation on the disposition of paracetamol (acetaminophen) has been examined in male Sprague-Dawley rats. Plasma and urinary concentrations of the drug and its two major metabolites, the glucuronide and sulphate, were determined by a sensitive and specific high performance liquid chromatographic assay. Following an intravenous dose of 100 mg kg-1 of paracetamol, no significant changes were found in the elimination rate constant (k), the mean residence time (MRT), total plasma clearance (Cl) and the apparent volume of distribution at steady-state (Vss). However, rats deprived of water for 96 h excreted a larger percentage of the administered dose as the glucuronide conjugate (15.3 vs 7.9%) and a smaller percentage as unchanged paracetamol (7.3 vs 20.7%) in the urine. In addition, there was a significant two-fold increase in the partial metabolic clearance to paracetamol glucuronide. Water deprivation also led to a significant reduction in the renal clearance of paracetamol accompanied by an increase in the renal clearance of the glucuronide.

Poloxamer 407 Increases the Recovery of Paracetamol in the Isolated Perfused Rat Liver

The role of the fenestrated liver sinusoidal endothelial cells (LSECs) in the hepatic disposition of paracetamol was investigated in isolated perfused livers from rats treated with poloxamer 407 (P407), a surfactant that causes extensive defenestration of the LSECs. Bolus doses containing tracer amounts of (14)C-paracetamol and reference markers (Evans Blue, (3)H-sucrose) were injected into control rats and rats that had been administered P407 via intra-peritoneal injection 24 h prior to experimentation. Scanning electron microscopy confirmed a reduced number of fenestrations in rats treated with P407. The recovery of paracetamol was significantly increased in P407 rats compared to controls (0.72 ± 0.07 P407 vs. 0.67 ± 0.04 control, p < 0.05) and the volume of distribution of paracetamol as a fraction of the sucrose volume was significantly reduced. The permeability-surface area (PS) product for the transfer of paracetamol across the LSECs was also reduced in the P407 rats (0.032 ± 0.009 P407 vs. 0.043 ± 0.007 mL/s/g controls, p < 0.05). P407 treatment resulted in increased recovery and decreased PS product of paracetamol following a single pass through the isolated perfused liver. This is consistent with exclusion of paracetamol from the space of Disse due to defenestration of the LSECs.

Paracetamol disposition in renal allograft recipients.

To evaluate the disposition of paracetamol in renal allograft recipients. Eight fasting renal allograft recipients were given 1 g soluble paracetamol orally. Paracetamol was absorbed rapidly, and the mean plasma half-life from 2 h to 8 h was 2.6 +/- 0.5 h. After that, disappearance of paracetamol from the plasma was slower. Both the glucuronide and sulphate conjugates of paracetamol had slow elimination half-lives of 15.1 +/- 8.8 h and 26.2 +/- 14.6 h, and there were residual amounts of both conjugates in the plasma at 24 h. The renal clearances of both conjugates were low (21 +/- 14.2 ml/min and 32.4 +/- 31.4 ml/min) and there was a significant negative correlation between total amount of paracetamol recovered in the urine in 24 h and serum creatinine (r = -0.89, P<0.01). Paracetamol disposition is abnormal in renal allograft recipients and seems to relate to abnormal renal function in these patients.

In vitro cleavage of paracetamol glucuronide by human liver and kidney β-glucuronidase: determination of paracetamol by capillary electrophoresis

A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human β-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.25 μ M (38 ng/ml) and a limit of quantitation of 1 μ M (151 ng/ml). The suitability of the method has been shown for enzyme kinetic studies using different liver and kidney homogenates, respectively. Our data clearly demonstrate that paracetamol glucuronide is cleaved by human β-glucuronidase thereby releasing paracetamol. The CE method presented is not only a valuable tool for measuring human β-glucuronidase activity, but also allows investigation of the contribution of deglucuronidation of paracetamol glucuronide to the disposition of paracetamol.

Effect of polysaccharide peptide (PSP) on in vivo sulphation and glucuronidation of paracetamol in the rat.

The effect of polysaccharide peptide (PSP), an immunomodulator isolated from Coriolus versicolor COV-1, on the disposition of paracetamol was investigated in the rat. PSP (100 and 200 mg/kg, i.v.) was administered 30 min before a moderate dose (100 mg/kg, i.v.) of paracetamol was given. Plasma and bile concentrations of paracetamol, paracetamol glucuronide and paracetamol sulphate were measured by high performance liquid chromatography. The pharmacokinetics of paracetamol (100 mg/kg) alone was consistent with those reported previously, using a one-compartment model. PSP (200 mg/kg) significantly (P < 0.05) increased the clearance (controls, 19.06 +/- 2.74 ml/min/kg: PSP treated, 26.22 +/- 0.84 ml/min/kg) and volume of distribution (controls, 1.35 +/- 0.11 l/kg: PSP treated, 1.61 +/- 0.04 l/kg) of paracetamol by 37% and 21%, respectively. These changes were associated with concomitant increases in the glucuronide and sulphate metabolites in plasma, with significant increases in the Cmax and Tmax for both metabolites. The biliary excretion rate of paracetamol glucuronide and paracetamol sulphate were also measured. The Cmax values of paracetamol sulphate were significantly (P < 0.01) increased by 2.4-fold from 907.8 +/- 157.7 micrograms/ml (controls) to 3061 +/- 331 micrograms/ml after PSP treatment. The lower dose of PSP (100 mg/kg) had no significant effect on the disposition of paracetamol in this study, which agreed with previous reports that a low dose of PSP (100-200 mg/kg, i.p.) was less effective in the protection against paracetamol-induced hepatotoxicity. The time course of the increase in paracetamol sulphate in plasma and bile in this study coincided with the transient perturbation of glutathione (GSH) turnover by a similar dose range of PSP previously described, such that more cysteine was available for oxidation to inorganic sulphate. This increase in sulphate conjugation by PSP would, in part, contribute to the increase in disposition of paracetamol and may be related to the ability of PSP to decrease the covalent binding of paracetamol to microsomal proteins previously reported. Further studies are necessary to understand the mechanism(s) involved in the PSP-induced increases in paracetamol glucuronide and paracetamol sulphate formation and biliary excretion.

High resolution NMR spectroscopic studies on the metabolism and futile deacetylation of 4-hydroxyacetanilide (paracetamol) in the rat

Paracetamol (4-hydroxyacetanilide, acetaminophen) was synthesized with the acetyl group labelled with C 2H 3 (paracetamol-C 2H 3, and dosed to rats i.p. at 25 mg/kg (N = 5) and 40 mg/kg (N = 3) body weight. Paracetamol, with a 13CH 3 in the acetyl group (paracetamol- 13CH 3) was also synthesized and dosed to rats i.p. at 40 mg/kg (N = 3). The metabolism and excretion of the 2H-labelled compound was followed in the rat using 600 MHz 1H and 92.1 MHz 2H NMR spectroscopy of urine collected 0–8, 8–24, 24–32 and 32–48 hr post-dosing. The metabolism of paracetamol- 13CH 3 was also monitored using 600 MHz 1H NMR spectroscopy of urine collected 0–8, 8–24 and 24–48 hr post-dosing. For paracetamol-C 2H 3 the total recovery of the sulphate, glucuronide and N-acetyl cysteinyl metabolites via the urine accounted for 61.2 ± 14.1% of the 25 mg/kg dose and 61.4 ± 8.8% of the 40 mg/kg dose. For paracetamol- 13CH 3 the recovery was 102.7 ± 3.7% indicating that the low % urinary recovery with the C 2H 3-labelled drug is the result of isotope effects on the disposition of paracetamol. In the case of the paracetamol-C 2H 3, quantitative 1H NMR analysis of urine showed that 13.3 ± 0.5 and 10.0 ± 1.2 mole% (25 and 40 mg/kg, respectively) of the urinary paracetamol sulphate recovered following dosing of the deuterium labelled drug had the C 2H 3 acetyl groups replaced by C 1H 3 acetyl groups from endogenous sources. In the case of the paracetamol- 13CH 3 8.9 ± 0.7 mole% of the sulphate conjugate had also been transacetylated to paracetamol- 12CH 3. There was no significant difference between the level of futile deacetylation observed for the deuterated and 13C-labelled drug. Overall these data indicate a high level of deacetylation followed by reacetylation (i.e. futile deacetylation) prior to excretion of paracetamol via the nephrotoxic intermediate 4-aminophenol. The level of deacetylation is much higher than has previously been thought which may cast new light on the role of 4-aminophenol in the development of paracetamol induced nephrotoxicity.

The disposition of paracetamol and its conjugates during multiple dosing in patients with end-stage renal failure maintained on haemodialysis.

The disposition of oral paracetamol (1.0 g 3 times daily for 10 days) was studied in 6 patients with end-stage renal failure (creatinine clearance < 5 ml x min-1) maintained on haemodialysis 2 or 3 times per week. Blood was sampled daily for 10 days. The time of sampling depended on whether the patients were dialysed in the morning or afternoon but was always within 5 h of the last dose of paracetamol. On dialysis days samples were taken at the start of the session. The mean plasma concentration of paracetamol was 6.8 mg x l-1 after the first 24 h and subsequently varied little throughout the 10 days. Apparent steady-state plasma concentrations of 60.0 mg x l-1 and 54.5 mg x l-1 were reached for the glucuronide and sulphate conjugate of paracetamol respectively by the 2nd day of treatment with little variation throughout the remainder of the study. These steady-state concentrations of paracetamol glucuronide and sulphate were much lower than predicted. The steady-state plasma concentrations of the retained cysteine and mercapturate conjugates of paracetamol were low (5.7 and 3.7 mg x l-1, respectively) and there was no evidence of accumulation of these potentially toxic metabolites. It is not clear why regular dosing with paracetamol in haemodialysis patients did not cause the accumulation of paracetamol glucuronide or sulphate as predicted. There may be enterohepatic elimination of retained paracetamol conjugates or depletion of substrates such as inorganic sulphate during chronic dosing.

Impaired absorption of paracetamol in vegetarians.

1. The absorption and disposition of paracetamol was investigated in 10 healthy male Thai vegetarians and 10 similar non-vegetarians following an oral dose of 20 mg kg-1. 2. The absorption rate of paracetamol was significantly impaired in the vegetarians compared with the non-vegetarians as shown by a lower mean Cmax (11.7 +/- 1.4 vs 15.6 +/- 1.6 mg l-1; 95% confidence interval of the difference 2.49 to 5.36), increased tmax (median 1.75, range 0.75 to 3 h compared with 0.75 and 0.25 to 2 h) and an increase in the time for input of 50% of the total amount absorbed (0.54 +/- 0.38 compared with 0.20 +/- 0.10 h; 95% confidence interval of the difference 0.063 to 0.61). 3. A significantly lower total 24 h urinary recovery of paracetamol and metabolites (72.1 +/- 5.4 vs 86.4 +/- 5.4% of the dose; 95% confidence interval of the difference 8.0 to 20.6) indicated a decrease in the extent of absorption in the vegetarians also, although the total AUC values did not differ significantly between the two groups. 4. The plasma paracetamol half-life, partial metabolic clearances and fractional urinary excretion of the glucuronide, sulphate, cysteine and mercapturic acid conjugates of paracetamol were similar in the vegetarians and non-vegetarians.

The disposition of paracetamol and the accumulation of its glucuronide and sulphate conjugates during multiple dosing in patients with chronic renal failure

We have compared the disposition of oral paracetamol (1.0 g t.d.s. for 10 days) in 6 healthy volunteers and 6 conservatively-managed patients with chronic renal failure (mean plasma creatinine 451 mumol.l-1). Blood was sampled daily for 10 days before the morning dose of paracetamol. Each day the pretreatment plasma concentrations of paracetamol were higher in the renal failure patients than in the volunteers, with mean values over the 10 days of 3.1 and 1.1 mg.l-1 respectively. The mean daily plasma concentrations of the sulphate and glucuronide conjugates of paracetamol were markedly higher in the renal failure group and apparent steady-state concentrations of about 25 and 85 mg.l-1 were reached on the 2nd and 6th days respectively. The mean steady-state plasma concentrations of the glucuronide conjugate on the 7th to 10th days of treatment were positively correlated with the plasma creatinine concentration (r = 0.97), but this relationship did not hold for the sulphate conjugate. Cysteine and mercapturate conjugates could only be detected in one patient. Predictions of steady-state concentrations based on previous studies with single doses of paracetamol in renal failure patients were remarkably accurate for the glucuronide but not for the sulphate conjugate. These results are consistent with some extra-renal elimination of retained paracetamol conjugates in patients with chronic renal failure, with limited regeneration of the parent compound. The sulphate metabolite did not accumulate as predicted, possibly because of depletion of inorganic sulphate.

Effect of diethylether on the formation of paracetamol sulphate and glucuronide in isolated rat hepatocytes.

Diethylether has previously been shown to inhibit several pathways of drug metabolism, including conjugation of paracetamol in isolated rat hepatocytes. Since overall paracetamol conjugation consists of pathways of different subcellular localization (cytosolar sulphation and microsomal glucuronidation) the response of both pathways to diethylether was tested. The elimination of paracetamol (160 mumol/l, initial concentration) and the formation of paracetamol sulphate and glucuronide were measured (high-performance liquid chromatography) in suspensions of isolated rat hepatocytes from fasted and fed animals over 1 h in the absence and presence of diethylether (30 mmol/l). Approximately 90% of the paracetamol elimination was by sulphation and nearly 10% by glucuronidation both in the controls and in the presence of ether. The overall disposition of paracetamol and the formation of sulphate were both reduced by about 50% in the presence of ether compared to the controls while the formation of glucuronide was reduced by 70%. The results were not influenced by the nutritional state of the animals before sacrifice. It is concluded that the inhibitory effect of ether on total paracetamol metabolism was mainly caused by reduced sulphation. Since microsomal glucuronidation was also inhibited by ether, both cytosolar and microsomal enzyme systems were sensitive to diethylether.