Gen Pharmac Vol 23, No 2, pp 155-158, 1992 Pnnted m Great Britain All rights reserved

0306-3623/92 $500+000 Copyright © 1992 Pergamon Press plc

PLASMA CONCENTRATION OF PARACETAMOL AND ITS MAJOR METABOLITES AFTER p.o. DOSING WITH PARACETAMOL OR CONCURRENT ADMINISTRATION OF PARACETAMOL AND ITS N-ACETYL-DL-METHIONINE ESTER IN MICE L A SKOGLUND,I K INGEBRIGTSEN,IP LAUSUND2 and I. NAFSTAD1 ~Department of Pharmacology and Toxicology, Norwegian College of Vetennary Medlcme, P O Box 8146 Dep, N-0033 Oslo 1 and 2Department of Laboratory Ammal Sciences, National Institute of Pubhc Health, Geitmyrsvn 75, N-0462 Oslo 4, Norway (Recewed 20 August 1991) Abstract--1 Single doses of paracetamol 400 (PAR 400) and 800mg/kg (PAR 800), SUR 2647 combination (free paracetamol + paracetamol-N-acetyl-DL-methtonate, paracetamol/methlonme ratio 2 1) equivalent to PAR 400 (SURe 400) and PAR 800 (SURe 800) were given p o to male Born NMRI mice 2 The objectwe was to compare the plasma concentrations of free paracetamol and the major metabohtes paracetamol-sulphate and paracetamol-glucuromde for a 6 hr penod after each test drug 3 There was no slgmficant difference between PAR 400 and SURe 400 with respect to plasma paracetamol, paracetamol-glucuromde and paracetamol-sulphate concentratmn with the exception of lower plasma paracetamol concentration (P < 0 03) at 3 hr following PAR 400 4 There was no slgmficant difference between PAR 800 and SURe 800 with respect to plasma paracetamol, paracetamol-glucuromde and paracetamol-sulphate concentrations with the excepuon of lower plasma paracetamol-glucuromde concentration (P < 0 03) at 4 hr after dosing following SURe 800 5 Combining free paracetamol and its methlomne ester does not seem to alter the pattern of plasma paracetamol, paracetamol-glucuronlde and paracetamol-sulphate compared to equal doses of free paracetamol alone after p o administration of toxic doses to male Born NMRI mice

INTRODUCTION

McLean (1974) proposed the a d d m o n of methlonme or cysteme to the paracetamol (acetammophen, U.S A ) formulation as a means of enhancing the hepatic glutathlone (GSH) conjugation capacity m case of paracetamol mtoxlcauons. S U R 2647 is the N-acetyl-DL-methlonme ester of paracetamol Prehmmary studies in rats have shown the analgesic properties of S U R 2647 to be of a longer duraUon but w~th a slower onset of action than eqmmolar doses of paracetamol. Hence, S U R 2647 was mixed with free paracetamol tn a N-acetyl-DLmethionlne/paracetamol ratio of 1 2 to gwe a drug formulation named S U R 2647 combination Prewous studies have demonstrated the hepatoprophylactlc efficiency of S U R 2647 combination by elevating hepatic reduced glutathlone pools m mice after admlmstratlon of toxic doses (Skoglund et al, 1986, 1988) The purpose of the present study was to compare toxic doses of S U R 2647 combmaUon versus free paracetamol w~th respect to plasma paracetamol, paracetamol-sulphate and paracetamol-glucuromde m m~ce after p o. administration. MATERIALS AND METHODS

Ammals Ammals were male BOM NMRI mice 30°50 g (age > 20 days) generally kept 6 in each macrolon cage (type 3) and

acchmatlzed to laboratory surroundings for at least 2 weeks Lighting in animal quarters was controlled (12 12 dark hght cycles), temperature 20-21°C and hurmdtty 50-55% The ammals were fed commercial rat pelleted diet (Ewos, Sodertalje, Sweden) and water ad hbztum untd the commencement of the experiments Thereafter the ammals had access to water only throughout the trial A total of 80 mice were used m this study Drugs Drugs used were SUR 2647 combination which Is a mixture of paracetamol (N-acetyl-p-ammophenol) and SUR 2647 (p-acetamldophenyl, 2-carbamoyl, 4-methylthlobutanoate) m a molecular ratio of 1 1, paracetamol or SUR 2647 alone The test drugs were gwen m an 1% w/v aqueous suspension of methyl cellulose, grade 15, (NMD, Oslo, Norway) The test drug suspensions were made within 1 hr prior to each expertment and kept continuously sUrred until admm~strauon All expenments started between 07 00 and 08 00 hr The test drug suspensions were admtmstered by gastric tube according to the following dosage scheme, paracetamol 800 mg/kg body wt (PAR 800), SUR 2647 combination eqmvalent to paracetamol 800 mg/kg body wt (SURe 800), paracetamol 400 mg/kg body wt (PAR 400) and SUR 2647 combination eqmvalent to paracetamol 400 mg/kg body wt

(SURc 40o) Blood samphng and paracetamol/metabohte assay Blood samples were obtained by heart puncture dunng methoxyflurane (Penthrane ~ Abbott Lab, Kent, U.K ) inhalation anaesthesia and transferred to glass tubes contaming 65 U S P umts of sodmm hepanne (Venoject~, 155

L A SKOGLUNDet al

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Terumo C o , Tokyo, Japan) Blood samples were obtained 0 5, 1 0, 3 0, 4 0, 6 0 and 8 0 after medlcatmn with test drugs The ammals were sacrificed by cerwcal dlslocatmn after blood samphng After centrffugauon with 3000 rpm at 4°C for 8 mm m a Beckman TJ-6R cooled centrifuge (Beckman Instruments Inc, Fullerton, Cahf, U S A ), plasma was frozen at - 2 0 ° C for I week awaltmg paracetamol, paracetamol-glucuromde and paracetamol-sulphate analysis Plasma concentratmns of paracetamol, ~ts sulfate and glucuromde metabohtes were analyzed using a HPLC assay To 1 ml of plasma (or ddute of avadable plasma to 1 ml) was added 20gl mternal standard metacetamol (N-acetyl-mammophenol 1 mg/ml) and dlstdled water to compensate for the sptked volume in the standard hne ( l l 0 / d ) After vortexmg, 50 #1 72% perclonc amd (BDH Ltd, Poole, U K ) was added to preclp~tate plasma protein and the sample was centrifuged (Beckman J 6B, Beckman Instruments) for I0 mm at 1500 rpm A 5 #1 ahquot of the clear supernatant was rejected into the HPLC system (Model 6000 pump, U6K rejector, fixed wavelength UV absorbance detector at 254 nm, Waters Assoc Inc, Milford, Mass, U S A ) which was attached to a 5cm stainless steel (6 25mm O D ) pre-column packed w~th CO-Pell ODS (Whatman Inc, Chfton, N J , U S A ) and connected to a 15 cm stainless steel analytical column (4 5 mm I D ) packed with 5 m/~ Hypersd ODS (Whatman) The system was operated at 20°C and the elutlng solvent being a m~xture of 5 ml lsopropyl alcohol (BDH) and 295 ml 0 1% formic acid (BDH) m 0 1 M potassmm d~hydrogen phosphate (BDH) A flow rate of I 5 ml/mln was employed Under operatmg condmons single peaks are obtained for paracetamol and ~ts metabohtes The following retenuon ttmes were obtained for the internal standard metacetamol (11 1 mm), paracetamol (6 4 mm), paracetamol-glucuromde (2 4 mln), and paracetamol-sulphate (4 8 mm) HPLC chromatograms from various ttme points are shown in F~g 1 The concentrations of paracetamol and its metabohtes were estimated from a regression curve of peak height ratm (component/internal standatd) with respect to concentratmn from approprmtely constructed multi-level cahbratmn curves The paracetamol-glucuromde and paracetamol-sulphate results were expressed as their paracetamol equwalents

Stattstwal analysts The data were analyzed by z2-test (Bhattacharyya and Johnson, 1977) The stgmficance level was set at 5% The results are generally expressed as mean (SEM) values of 3-4 ammals w~th the exceptmn of 1 case (SURc 400,

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Fig 2 Plasma paracetamol concentraUons vs Ume after p o dosing with paracetamol 400 mg/kg (PAR 400) or eqmvalent amount of SUR 2647 combmatmn (SURe 400) are shown as means of 3-4 mice (SEM) paracetamol-sulphate 6 hr) In this case the data from one mouse is used due to the lack of sufficient plasma from the remaining animals w~thm the group permRtmg analysis

RESULTS

Plasma paracetamol, paracetamol-sulphate and paracetamol-glucuromde levels after single dosage of SUR 2647 eombmat~on or PAR 400mg/kg There was n o staUstlcally slgmficant difference between P A R 400 a n d S U R e 400 0 5, 1.0, 4 0 a n d 6 0 hr after dosing w~th respect to p l a s m a paracetamol c o n c e n t r a t i o n (Fig. 2) There was a difference m plasma p a r a c e t a m o l c o n c e n t r a t i o n between the two groups at 3 hr after dosing ( P < 0 03) There was no statistically significant difference between the two groups with respect to p l a s m a p a r a c e t a m o l - g l u c u r o m d e (Fig 3) o r p a r a c e t a m o l sulphate (Fig 4) c o n c e n t r a U o n t h r o u g h o u t the 6 h r observaUon period

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Fig 1 Drawing of representattve HPLC chromatograms of paracetamol (APAP), paracetamol-glucuronlde (A-gluc) and paracetamol-sulphate (A-SO4) extracted from plasma with SUR 2647 combination equwalent to paracetamol 800 mg/kg obtained 0 5 hr (B) and 8 0 hr (C) dosing Chromatograms of control plasma extract spiked w~th mternal standard is shown (A)

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Fig 3 Plasma paracetamol-sulphate concentrations vs Ume after p o dosing with paracetamol 400 mg/kg (PAR 400) or eqmvalent amount of SUR 2647 combmaUon (SURe 400) are shown as means of 3-4 mice (SEM) except for SURe 400 at 6 hr ( # ) where the data from one mouse ~s presented

Plasma paracetamol and metabohtes m mice E

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Fig 4 Plasma paracetamol-glucuromde concentratmns after p o dosing with paraeetamo1400 mg/kg (PAR 400) or equwalent amount of SUR 2647 combmatmn (SURc 400) are shown as means of 3-4 mice (SEM)

Plasma paracetamol, paracetamol-sulphate and paracetamol-glucuromde levels after smgle dosage of SUR 2647 combmatwn or PAR 800mg/kg There was no statlslacally slgmficant &fference at any time throughout the 6 hr observauon penod vath respect to plasma paracetamol (Fig 5) or paracetamol-sulphate (F~g 6) concentratmn for the two test groups The SUR 2647c group had less ( P < 0 0 3 ) plasma paracetamol-glucuromde (Fig 7) than the paracetamol group at 4 hr after dosing There was no &fference between the groups prior to or later than 4 hr dunng the 6 hr observalaon period DISCUSSION In rmce paracetamol is easdy absorbed after p o. admmlstralaon and the major plasma metabohtes, paracetamol-sulphate and paracetamol-glucuromde, are mainly eliminated directly by the kidney into the unne (Fischer et al, 1981; Wong et al, 1981, Domdar et al., 1985)

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F*g 6 Plasma paracetamol--sulphateconcentratmns vs time after p o dosing with paracetamol 800 mg/kg (PAR 800) or equwalent amount of SUR 2647 comhnaUon (SURc 800) are shown as means of 3-4 mice (SEM) The doses of paracetamol and SUR 2647 combination used m this study are sufficiently h,gh to saturate the blotransformalaon and ehmmalaon processes (Slegers et al., 1978). The data do not indicate that plasma paracetamol after dosing with a mixture of paracetamol-N-acetyl-DL-metluonate and free paracetamol differs stgmficantly from that obtained using eqmmolar amounts of free paracetamol alone. Possible factors which may have caused different absorption charactenslacs are &fferences m molecular weight (paracetamol 151 16, paracetamol-N-acetyloL-methlonate 324.4) and possible interactions with the test drug vehicle However, methylcellulose has a very low complexing tendency (Jfirgensen-Elde and Spelser, 1967) The plasma paracetamol profile of SUR combination eqmvalent to 800 mg paracetamol/kg compared to that of the same amount free paracetamol may suggest a shght lame delay before peak plasma concentralaon ~s obtamed dunng the first hour after

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Fig 5 Plasma paracetamol concentratmns vs Ume after p o dosing wRh paraeetamol 800 mg/kg (PAR 800) or eqmvalent amount of SUR 2647 combination (SURc 800) are shown as means of 3-4 mice (SEM)

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Fig 7 Plasma paracetamol-glueuromde concentraUons vs time after p.o dosing wRh paracetamol 800 mg/kg (PAR 800) or equwalent amount of SUR 2647 combmataon (SURc 800) are shown as means of 3-4 mlc¢ (SEM)

158

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dosing Varlat~ons in plasma concentrations among ammals recewmg the same drug dose with ~dent~cal time pomts of sacrifice due to mterlndlwdual absorption differences may have contributed to this observation The time points for blood samphng m the early phase of the present study are too few to allow for a precise determmatxon of pharmacoklnetlc data including peak plasma concentrations for the test drugs It has been suggested that the lnclus~on of lnorgamc sulphate (Gahnsky et a l , 1979) or N-acetylcysteme (Gahnsky and Levy, 1979), which is metabohzed to inorganic sulphate (Sheffner et a l , 1966), may stimulate paracetamol sulphat~on and thus reduce the amount of toxic paracetamol metabohtes The addmon of reduced glutathlone precursors such as meth~onlne, detoxlfy active paracetamolmetabohtes by conjugation with the glutathione end product Paracetamol-glutath~one conjugate is excreted into the bile from the liver (Grafstrom et a l , 1979, Wong et a l , 1979) to the intestine where paracetamol-glutathlone may be converted to paracetamol-cysteme and reabsorbed to through enterohepaUc circulation (Grafstrom et a l , 1979) Methlonlne, which is rapidly metabohzed to cysteme, should indirectly" also be able to increase the sulphat~on pathway of paracetamol There was no s~gmficant difference between the two test drugs with respect to plasma glucuromde and sulphate pattern compared to free paracetamol alone The present study does not support the suggestion of facdltated paracetamol sulphatlon after concommltant administration of methlonlne Acknowledgements--The authors are grateful to Ms P Cassldy for excellent technical assistance and to SterhngWinthrop R&D, Alnwlck, U K for the statistical analyses and HPLC assays of paracetamol and its metabohtes REFERENCES

Bhattacharyya G K and Johnson R A (1977) Stattstwal

Concepts and Methods Wdey, New York Domdar S M, Boor P J and Ahmed A E (1985) Potentiation of the hepatotoxlc effect of acetammophen by prior administration of sahcylate J Pharmac Exp Ther 233, 242-248 Fischer L J, Green M D and Harman A W (1981) Levels of acetammophen and Its metabohtes m mouse tissues after a toxic dose J Pharmac Exp Ther 219, 281-286 Gahnsky R E and Levy G (1979) Effect ofn-acetylcysteme on the pharmacokmetlcs of acetamlnophen in rats L~fe Sct 25, 693-700 Gallnsky R E, Slattery J T and Levy G (1979) Effect of sodium sulfate on acetammophen ehmmatlon by rats J Pharmaceut Sct 68, 803-805 Grafstrom R, Ormstad K, Moldeus P and Orrenms S (1979) Paracetamol metabolism in the isolated perfused rat liver with further metabolism of a bthary paracetamol conjugate by the small intestine Bwchem Pharmac 28, 3573-3579 Jurgensen-Eide G and Spelser P (1967) Interaction between drugs and non-ionic macromolecules I Acta Pharm Suec 4, 185-200 McLean A E M (1974) Prevention ofparacetamol poisonmg Lancet i, 729 SheffnerA L , M e d l e r E M , B a d e y K R , G a l I o D G , Mueller A J and Sarett H P (1966) Metabolic studies with acetylcysteme Btochem Pharmac 15, 1523-1535 Slegers C - P , Strubelt O and Schutt A (1978) Relations between hepatotoxlclty and pharmacokmetlcs of paracetamol in rats and mice Pharmacology 16, 273-278 Skoglund L A, Ingebngtsen K , Nafstad I and Aalen O (1986) Efficacy of paracetamol-estenfied methlonme versus cysteme or methlomne on paracetamol-mduced hepatic gsh depletion and plasma alat level in mice Bwchem Pharmac 35, 3071-3075 Skoglund L A, Ingebrlgtsen K, Nafstad I and Aalen O (1988) In wvo studies of concurrent administration of paracetamol and Its N-acetyl-DL-methlonme ester (SUR 2647 combination) Gen Pharmac 19, 213-217 Wong L T, Whltehouse L W, Solomonraj G and Paul C J (1981) Pathways of acetammophen conjugates m the mouse Toxic Lett 9, 145-151 Wong L T, Whltehouse L W, Solomonraj G , Paul C J and Thomas B H (1979) Separation and quantltatton of acetammophen and Its metabohtes m the bde of mice J Analyt Toxic 3, 260-262