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Br. J. Phannac. Chemother. (1967), 29, 150-157.
A KINETIC STUDY OF DRUG ELIMINATION: THE
EXCRETION OF PARACETAMOL AND ITS
METABOLITES IN MAN
BY
A. J. CUMMINGS,^ M.^ L.^ KING^ AND^ B.^ K.^ MARTIN
From the Nicholas Research Institute, Slough, Bucks.
(Received May 25, 1966)
In the preceding paper (Cummings, Martin & Park, 1967) theoretical considerations
have been presented relating to a drug which is eliminated by apparent first^ order
processes of urinary excretion and metabolism. These considerations have indicated
that when the excretion rate constants of drug metabolites are^ greater than^ the^ elimination
rate constant of the drug, plots of log rate of excretion of^ drug^ and of drug metabolite
eventually become linear and parallel to each other. Experimental verification of this
is now attempted with a study of the excretion of paracetamol and its metabolites in man.
When paracetamol is administered or is formed in the body from other drugs, such^ as
acetanilide and phenacetin, it is eliminated by metabolism and to a^ small extent^ by
excretion (Greenberg & Lester, 1946; Brodie & Axelrod, 1948, 1949).^ Although^ its
metabolism in man does not appear to have been studied in^ detail, paracetamol is^ usually
considered to be mainly metabolized to the^ sulphuric acid^ and^ glucuronic^ acid conjugates
(Smith, 1958). It is also metabolized to a^ small^ extent^ to^ S-(l-acetamido-4-hydroxy-
phenyl) cysteine (Jagenburg & Toczko, 1964). Lester & Greenberg (1947) report that
determinations of^ the increased excretion^ of^ ethereal^ sulphates^ and^ glucuronic^ acid in
urine after the administration of acetanilide showed that approximately two-thirds of^ the
p-aminophenol was excreted as sulphuric acid ester and one-third as^ glucuronide. The
isolation of paracetamol sulphate from urine of patients who^ had received^ acetanilide
was reported by Morner (1889). There^ appears to^ be^ no^ report of^ the^ isolation^ of
paracetamol glucuronide from human^ urine, although^ it^ has^ been^ isolated^ from the^ urine
of rabbits dosed with paracetamol or with a drug which gives rise to paracetamol (Smith
& Williams, 1949).
Studies of paracetamol elimination in^ man^ have been^ based^ on^ the^ determination of
the free paracetamol and total paracetamol conjugates in urine.^ The half-life of
paracetamol, calculated from the results of such studies, has^ been^ reported to^ be^ within the range 1.6 to 2.8 hr (Nelson & Morioka, 1963).
The present report gives details of the separate estimation^ of^ the^ rate^ of excretion of
paracetamol, and of its glucuronide and sulphate in^ urine.
KINETIC STUDY OF DRUG ELIMINATION
METHODS Chemical methods The following compounds were used as standards in the analytical procedures: (i) Paracetamol B.P. (ii) Paracetamol sulphate, potassium (^) salt monohydrate. Potassium p-nitrophenylsulphate was prepared by (^) the method of Burkhardt & Wood (1929) and reduced to the p-aminophenylsulphate by hydrogenation in aqueous solution using 5% palladium on charcoal at room temperature and atmospheric pressure. This was acetylated with acetic anhydride and recrystallized from aqueous ethanol to give p-acetamidophenylsulphate, which was isolated as the potassium salt. Analysis of the product gave: C, 33.0; H, 3.51; N, 4.91; H20, 6.27%. CH3CO, 15.0%. (Calc. for CsHsNOS, K, H20: C, 33.4; H, 3.51; N, 4.88; H20, 6.35%. CH3CO, 15.3%). (iii) Paracetamol (^) glucuronide, potassium salt, was isolated by the method of Kamil, Smith & Williams (1952) from the urine of rabbits which had been dosed with paracetamol. The product was dissolved in ethanol, 2 N-potassium hydroxide in methanol was added until the solution was slightly alkaline (pH 8). The precipitate was dissolved in a small volume of methanol, 0.5 vol. of ethanol was added and the precipitate collected. The product which was a white powder was dried and stored (^) in a desiccator. Analysis gave: C, 45.6; H, 5.3; N, 3.8%. (Calc. for C14H17NOs, K: C, (^) 45.9; H, 4.7; N, 3.8%).
Separation of the standard compounds The separation of paracetamol, paracetamol sulphate and paracetamol glucuronide standards was effected by thin-layer chromatography on silica gel using the solvent system, ethyl acetate, methanol, water, acetic acid (60:30:9: 1, v/v). With an ascending development, the RF values of these compounds approximated to 0.82, 0.63 and 0.25 respectively.
The determination of paracetamol and of two of its metabolites in urine after dosage with paracetamol Total urine collections were made at 1.5 hr intervals for 15 hr after the administration of para- cetamol (12 mg/kg body-weight) to four men. Urine was also collected over four 1.5 hr periods on the day before the study, for the determination of an average "^ blank "^ value for the drug and the metabolites for (^) each man.
Thin-layer chromatography of the urine collected after dosage with paracetamol revealed three substances with RF values respectively corresponding to those of paracetamol, paracetamol sulphate and paracetamol glucuronide. Further evidence for the identity of the substances isolated from the chromatograms, was obtained by comparison of their respective infra-red absorption spectra with those of the standard compounds. Paracetamol, paracetamol sulphate and paracetamol glucuronide (^) were determined in the 1.5 hr urine samples as follows. The urine (0.75% (^) of volume) was applied to form a narrow 18 cm band near the bottom of a 20 x 20 cm thin-layer (^) chromatography plate spread with a 0.3 mm layer of silica-gel (Merck, GF 254). The chromatograms were developed with the (^) solvent described above until the front had travelled 12 cm, then paracetamol and the two metabolites (^) were revealed as dark bands against a fluorescent background by examination under ultraviolet light. (^) Horizontal lines were drawn across the plates to isolate three strips of silica gel each (^) containing one (^) of these compounds and for any one compound the width of the strip was kept constant; this procedure ensured (^) a constant blank value. The whole of the silica gel within this strip was scraped off and quantitatively transferred into S ml. of water. Then, after shaking for (^30) min the silica gel was removed by centrifuging and the extinction of the supernatant solution was measured at 240 mP against a reagent blank. All three compounds exhibited maximum absorption at this (^) wavelength. Standard solutions of (^) paracetamol, paracetamol sulphate and paracetamol glucuronide were treated in the same way as the urines. (^) Plots of extinction against concentration for the three compounds were linear over the required range. The (^) reagent blank depended upon the amount of silica gel removed from the chromatogram, the (^) average values were equivalent to 22, 46 and 121
KINETIC STUDY OF DRUG (^) ELIMINATION
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A. J. CUMMINGS, M. L. KING and B. K. MARTIN
" Sigma-minus " plots (Martin, 1967). The total amount of paracetamol and of each of the metabolites which were excreted in the urine was calculated from the extrapolated
rate plots.^ This^ procedure^ is^ not entirely free^ from^ criticism,^ for it^ involves^ the^ assump-
tion that the excretion of these substances continues to be first order. The results of the present investigation are considered to be explicable in^ terms^ of the
theoretical considerations of the preceding paper (Cummings et al., 1967). Typically,
the maximum rate of excretion of paracetamol in urine was observed within a period of
3 hr after drug administration (Table 1). After a further short period of time its decline
could be interpreted as log-linear, indicating that the process of drug absorption had then
become negligible and that the process of elimination was first order. The rate of
excretion of paracetamol glucuronide continued to increase for periods of 3 to 6 hr after
drug administration^ and^ reflects^ the^ accrual^ of this^ metabolite^ in the^ body.^ In^ all^ four
subjects the observed maximum rate of excretion of paracetamol glucuronide occurred
later than that of paracetamol sulphate and in two subjects the maximum rate^ of
excretion of paracetamol sulphate was delayed relative to the maximum rate of excretion
of paracetamol (Table 1).
After attaining their respective maximum values the rate of excretion^ of^ both
metabolites decreases and plots of the log rate of excretion eventually appear to^ become
linear (Fig. 1 (a)). A close correlation then exists between the slope of^ the^ plots^ of^ the
log rate of excretion of the metabolites and of the^ drug,^ and^ statistical analysis^ of^ the
results indicates that the terminal sections of these plots can^ be^ interpreted^ as^ linear and
parallel. It may therefore be deduced that both^ paracetamol sulphate and^ paracetamol
glucuronide have excretion rate constants which^ are^ appreciably^ larger^ than the^ elimina-
tion rate constant for the drug.
The evaluation of rate^ constants
The elimination rate^ constant^ (K)^ for^ paracetamol^ was^ obtained^ by^ calculation of^ the
slope of^ the^ linear^ parts of^ the^ plots^ of^ log^ rate^ of excretion of^ paracetamol^ or^ its
metabolites (Fig. 1 (a)), or^ from^ the^ slope^ of^ the^ corresponding^ "^ Sigma-minus^ "^ plots
(Fig. 1 (b)).
The elimination constant consists of the sum of the rate constants which respectively
govern the^ excretion^ of^ unchanged^ drug^ and the formation of each^ metabolite,^ thus:
K=kD+k'p+k"I.F................. (1)
where kD and kF are the^ rate^ constants^ for^ drug^ excretion and metabolite formation
respectively.
The individual rate constants may be^ calculated^ from^ the^ relationships: kF (^) MU (^) .(2) kD (^) Duso
kD= ................K.^ (3) DAL where (^) DIOO and (^) Muw respectively represent the total^ amount^ of^ drug^ and^ of^ metabolite ultimately excreted and D represents the total^ amount^ of^ drug which^ participates^ in the
A. J. CUMMINGS, M. L. KING and B. K. MARTIN
the rate of excretion of drug in the urine. The proportion of the dose of paracetamol
which is excreted unchanged is small and this tends to diminish the accuracy of the
results in this instance. DISCUSSION
The results of the study of the excretion of paracetamol, paracetamol sulphate and
paracetamol glucuronide conform closely with the theoretical considerations previously
advanced (Cummings et al., 1967). The plots of log rate of excretion of the metabolites
are at first curves but their terminal sections can be interpreted as linear and
parallel to the plot for paracetamol and the elimination (^) rate constant for paracetamol
can in theory be calculated from the slope of any of these plots. However, relatively few
experimental values are available for the urinary excretion of paracetamol and the slope
of the plot of its log rate of excretion cannot be determined with great precision, the
equations for the set of three parallel straight lines (Fig. 1 (a)) are therefore based largely
upon the metabolite results. The question then arises whether the rate of excretion
of the metabolites achieves a true log-linear decline within the period of the experiment.
If this is not achieved, both the elimination rate constant of paracetamol (K) and the
excretion rate constants of the metabolites (ku) will be underestimated. The time required
for the log " Rate " plots of the metabolites to become linear is related to the values of
K and ku, and in the present studies the smallest difference is observed between the value
of K and the (^) value of ku of paracetamol glucuronide. When theoretical (^) log "Rate"
plots are constructed from the urinary excretion results of a model drug and one of its
metabolites, using the rate constants appropriate to paracetamol and paracetamol
glucuronide, it is found that the slope of the metabolite plot is within 10% of its terminal
value from about 9 hr after drug administration. This is in fair agreement with the
experimental results of the present studies and indicates that the error in the calculation
of K from the slope of the metabolite plots is likely to be less than 10%.
The corresponding "^ Sigma-minus "^ plots of the results become apparently linear from
about 6 hr after dosage, and the elimination rate constant of paracetamol may also be
calculated from their slope. This might seem to be the best method of calculating these
rate constants, but it must be noted that in this instance, the calculated total amount
of drug and total amount of metabolite ultimately excreted, on which the " Sigma-minus"
plots are based, were dependent upon the extrapolation of the terminal linear parts of
the log "^ Rate" plots.
The values of the elimination rate constant of paracetamol calculated from the results
of the present studies are in good agreement with those calculated by Nelson & Morioka
(1963) from the results of their study of the excretion of paracetamol and " total para-
cetamol metabolites" in (^) urine.
Greenberg & Lester (1946) reported that paracetamol was the major metabolite of
acetanilide in man and that this was excreted in the urine mainly as sulphate. In the
present study, however, paracetamol was eliminated predominantly (49%) as its glucuronic
acid conjugate.
SUMMARY
1. Paracetamol, paracetamol sulphate and paracetamol glucuronide excreted in the
urine after the administration of paracetamol, have been separated by thin-layer
chromatography and estimated spectrophotometrically.
KINETIC STUDY^ OF^ DRUG^ ELIMINATION
2. Plots of the log of the rate of excretion in urine of paracetamol, of paracetamol
sulphate and of paracetamol glucuronide against time eventually become linear and
parallel.
3. Two methods have been used to calculate the urinary excretion constants of
paracetamol sulphate and paracetamol glucuronide.
4. The results are considered to be in accord with theory in respect of a drug having
a first order elimination rate constant appreciably smaller than the first order excretion
rate constants of its metabolites.
The authors wish to thank Miss B.^ Croxford^ for her technical^ assistance,^ Mr. M. Longsdale and Mr. M.^ Rogers for the^ elemental^ and^ infra-red^ analyses, Mr. J.^ Verge for^ the^ synthesis of paracetamol sulphate and Miss A. Wylie for the statistical analyses.
REFERENCES BRODiE, B. B. & AXELROD, J. (1948). The fate of acetanilide in man. J. Pharmac. exp. Ther., 94,29-38. BRODiE, B. B. & AxELROD, J. (1949). The fate of^ acetophenetidin (phenacetin) in man, and methods for the estimation of^ acetophenetidin and its metabolites in^ biological^ material.^ J.^ Pharmac.^ exp. Ther., 97, 58-67. BURKHARDT, G. N. & WooD, H. (1929). Nitro-arylsulphuric acids and their reduction products. J. Chem. Soc., 141-152. CuMMINGS, A. J., MARTIN, B.^ K.^ & PARK, G. S.^ (1967). Kinetic^ considerations^ relating to^ the^ accrual^ and elimination of^ drug metabolites.^ Br. J.^ Pharmac.^ Chemother. 29, 136-149. GREENBERG, L. A. & LESTER, D. (1946). The metabolic fate of acetanilid and other aniline derivatives. I. (^) Major metabolites of acetanilid appearing in the urine. J. Pharmac. exp. Ther., 88, 87-98. JAGENBURG, 0. R. & TOCZKO, K.^ (1964). The^ metabolism of^ acetophenetidine. Isolation^ and^ characteriza- tion of (^) S-(l-acetamido-4-hydroxyphenyl)-cysteine, a metabolite of acetophenetidine. Biochem. J., 92, 639-643. KAMIL, I. A., SM1TH, J. N. & WILLIAMS, R. T. (1952). Studies in detoxication. 41. A study of the optical rotations of the amides and triacetyl methyl esters of some biosynthetic substituted phenylglucuronides. Biochem. J., 50, 235-240. LESTER, D. & GREENBERG, L.^ A.^ (1947).^ The metabolic^ fate^ of^ acetanilid^ and^ other^ aniline^ derivatives. II. Major metabolites^ of acetanilid^ appearing in the^ blood.^ J.^ Pharmac.^ exp. Ther., 90, 68-75. MARIN, B. K. (1967). Drug urinary excretion studies-a new method of^ treating the data. Nature (Lond.) In press. M6RNER, K. A. H. (1889). Stoffwechselproducte des^ Acetanilids^ im^ menschlichen^ Korper. Hoppe-Seyler's Z. physiol. Chem, 13, 12-25. NELsoN, E. & MORIoKA, T. (1963). Kinetics of the metabolism of acetaminophen by humans. J. pharm. Sci., 52, 864-868. SM1TH, J. N. & WILLIAMS, R. T.^ (1949). Studies^ in^ detoxication.^ 22.^ Metabolism^ of^ phenacetin (p-ethoxy- acetanilide) in the^ rabbit^ and^ a^ further observation^ on^ acetanilide^ metabolism.^ Biochem.^ J., 44,239-242. SMITH, P. K. (1958). Metabolism and^ intermediary products. In^ Acetophenetidin, 15-19. Interscience, London.
