Figure 1 The structure of antipyrine (1 -phenyl-2,3- groups, have looked at the factors which influence. antipyrine metabolism in man.

Transcription

1 Br. J. clin. Pharmac. (1977),, THE FIRST LILLY PRIZE LECTURE UNIVERSITY OF DUNDEE, JULY 1976 FACTORS INFLUENCING ANTIPYRINE ELIMINATION I.H. STEVENSON Department of Pharmacology and Therapeutics, University of Dundee, Ninewells Hospital, Dundee Antipyrine (phenazone) was one of the first important synthetic drugs. The uses to which it has been put, since its synthesis in Germany in 188 by a pupil of Emil Fischer, are summarized in Table 1. Since there was no requirement at that time for phase I trials, submissions to the Medicines Commision or to the F.D.A., antipyrine went into widespread clinical use as an antipyretic the same year it was synthesized. Two years after its introduction, reports began to appear of its analgesic effects and in the succeeding years, as the use of antipyrine as an antipyretic declined, it gained considerable popularity as an analgesic. It remained as such until the 1930s when it went out of favour due to a combination of toxicity, particularly of some related compounds, and perhaps in addition to the vagaries of prescribing as newer analgesics became available. As an indication of its extensive use during this period an early monograph (Greenberg, 1950) lists over 1700 references to this drug. Subsequently, antipyrine has proved to be of considerable value as a research tool in two related ways (Table 1). Firstly, its properties and distribution in the body are such that it can be used to measure total body water, a method first introduced by Soberman, Brodie, Levy, Axelrod, Hollander & Steele (199). Shortly afterwards, Brodie & Axelrod (1950) described its metabolism and provided much of the evidence on which the antipyrine plasma half-life test as we now know it is based. There was then something of a hiatus with regard to the use of antipyrine in human studies and only in the last 8 years or so has there been a resurgence of interest in this drug, led initially by Vesell and his colleagues (Vesell & Page, 1968). They, along subsequently with several other Table Uses of antipyrine 188~ Antipyretic/analgesic Measurement of total body water (Soberman et al., 199) Plasma antipyrine half-life procedure (Brodie & Axelrod, 1950) Factors influencing antipyrine metabolism (Vesell & Page, 1968) HC = C-CH3 O=C5 j 31 2N-CH3 N 06 H5 Figure 1 The structure of antipyrine (1 -phenyl-2,3- dimethylpyrazol-5-one) groups, have looked at the factors which influence antipyrine metabolism in man. The chemical structure of antipyrine is relatively simple (Figure 1) and as shown by Brodie & Axelrod (1960), it is metabolized extensively prior to excretion, the main product being -hydroxyantipyrine or its glucuronide or sulphate conjugate. Later (Yoshimura, Shimeno & Tsukamoto, 1968), its metabolism was shown to be rather more complicated, there being two further primary metabolites-3-hydroxymethyl and 3- carboxyantipyrine. A further possibility also exists that some of the drug is converted to norphenazone, with loss of the the methyl group in the 2 position (Baty & Price-Evans, 1973). The properties of antipyrine are listed in Table 2. It is a weak base of pka 1. which after oral administration is rapidly absorbed and distributed throughout total body water, with the absorption and distribution Table 2 Properties of antipyrine 1 Weak base, pka 1. 2 Rapidly absorbed from gastrointestinal tract 3 Distributed in total body water Less than 100% bound to plasma proteins 5 Extensively metabolized by liver microsomal enzymes 6 Kinetics-one compartment model system 7 Elimination not greatly influenced by increased hepatic blood flow

2 262 I.H. STEVENSON normally complete within 2 hours. It is less than 10% bound to plasma proteins and is extensively metabolized by cytochrome P50 dependent liver microsomal enzymes and only 5% of the unchanged drug appears in urine. The plasma disappearance of antipyrine follows first-order kinetics according to a simple one-compartment model system. There is no appreciable first-pass effect and it has a sufficiently low hepatic clearance that its elimination is not greatly influenced by increased hepatic bloodflow (Branch, Shand, Wilkinson & Nies, 197). These authors demonstrated that the more rapid elimination of antipyrine following phenobarbitone treatment was brought about 85% by increased microsomal enzyme activity and only 15% was attributable to increased hepatic blood flow. In examining the plasma disappearance of antipyrine, the method as originally outlined (Brodie, Axelrod, Soberman & Levy, 199) required multiple blood sampling with spectrophotometric assay of unchanged antipyrine in plasma. While this is still a widely used procedure, there have been several refinements of it in recent years (Table 3). The introduction of a gas-liquid chromatographic assay of antipyrine by several groups in 1973 demonstrated that the original spectrophotometric method measured the metabolite -hydroxyantipyrine in plasma as well as the parent compound. However, the amounts of the metabolite appearing in plasma are very low and the result of using this assay is to introduce a small error into the estimation of the volume of distribution without affecting appreciably in most circumstances the plasma half-life. More recently gas-liquid chromatographic methods have been developed for the metabolite -hydroxyantipyrine and this enables its elimination to be followed in urine. Using this approach, a correlation has been demonstrated between the plasma disappearance of antipyrine and the appearance of -hydroxyantipyrine in urine thus justifying what had been a gross assumption in previous studies that the plasma half-life of antipyrine provided a valid measure of its rate of metabolism, (Huffman, Shoeman & Azarnoff, 197). Finally, the most recent development has been in the introduction of saliva sampling as a means of following antipyrine elimination from the body. Several groups of workers have demonstrated that the half-life of antipyrine in plasma is similar to that in saliva and moreover the saliva content of antipyrine is apparently not affected Table Development of the 'antipyrine test' Spectrophotometric assay Gas-liquid chromatographic assay Urinary excretion of -hydroxyantipyrine Elimination of antipyrine from saliva a 0) E xa) a). _ a x r A A I -I ---. I I ----I Time (h) 20 2 Figure 2 Urinary excretion of -hydroxyantipyrine in children ( G.T., age 1.5 years, T7 5.7 h; U W.B., age 6.25 years, T+ 7.1 h; A W.W., age 13.5 years, T,.1 h). by the rate of flow of saliva (Welch, De Angelis, Wingfield & Farmer, 1975). Obviously, urinary and saliva sampling are less invasive than blood sampling and this makes it easier for studies to be carried out in, for example, children, elderly subjects etc. Figure 2 shows some data from this department (Moreland & Stevenson, unpublished observations) on the rate of urinary excretion of - hydroxyantipyrine in children, the metabo}ite being determined by gas-liquid chromatography. The values obtained are lower than those for adults but are similar to those previously reported for children (Alvares, Kapelner, Sassa & Kappas, 1975) using the normal plasma sampling procedure. The original idea of using plasma antipyrine half-life as a general index of microsomal drug metabolizing ability in an individual is no longer a valid concept. Intra-individual variation is considerable, there is a lack of correlation between antipyrine half-life and in vitro enzyme activity, measured in liver biopsy samples from the same individual. In addition there is often a poor correlation with other drug half-lives and with other possible indices of microsomal drug metabolizing enzyme activity, e.g. urinary 6-phydroxycortisol or glucaric acid output (Smith & Rawlins. 197).

3 FACTORS INFLUENCING ANTIPYRINE ELIMINATION 263 -c a1) -c a)._ C. cu _: co E cn co CL ltiliiiiiilillli 1111 II Subjects Figure 3 Plasma antipyrine half-life values in young (0, mean + s.d. T h) and elderly (U, mean ± s.d. Tt h) As far as the individual is concerned, plasma antipyrine half-life determination has little predictive use. The real value of the 'antipyrine test' has been in identifying the situations in which change in antipyrine elimination or metabolism occurs, and in many cases these changes correlate with changes brought about in the elimination of other drugs. The factors influencing antipyrine elimination probably also therefore influence the handling of other drugs metabolized by cytochrome P-50 dependent liver microsomal enzymes. Several groups of workers have measured antipyrine half-life values in groups of healthy volunteers and data from the four largest studies is shown in Table. It is clear that the mean values are very similar for subjects in Pennsylvania, Stockholm and Dundee. In terms of scale, these were eclipsed by the fourth study in which 307 subjects of a wide age range were studied. The first study undertaken in Dundee examined the effect of age and sex on plasma antipyrine half-life (O'Malley, Crooks, Duke & Stevenson, 1971; O'Malley, 1972). The results for 61 controls in the age range years and 19 elderly subjects aged years are Shown in Figure 3 and plasma antipyrine clearance data for a slightly smaller group is given in Table 5. As is clearly evident, the plasma halflife of antipyrine is significantly longer and the clearance significantly lower in the elderly group. This finding of an impaired metabolism of antipyrine in the elderly has since been confirmed in a much larger study (Vestal, Norris, Tobin, Cohen, Shock & Andres, 1975) and subsequently other drugs have been shown to be metabolized more slowly in this age group (Crooks, O'Malley & Stevenson, 1976). It is also evident from Table 5 that there is a small sex difference, the plasma clearance of antipyrine being marginally higher in males than females. Several other studies of the influence of disease and drug treatment on antipyrine elimination have subsequently been carried out in the author's laboratory, the results from which are summarized in Figure. In a study carried out in collaboration with Dr L. Prescott's group in Edinburgh, plasma antipyrine half-life values were found to be particularly long (approximately twice control values) in patients who had developed liver damage following paracetamol overdose (Forrest, Roscoe, Stevenson, Matthew & Prescott, 197). A highly significant correlation existed between the plasma antipyrine halflife and the serum glutamic pyruvate transaminase Table Range of plasma antipyrine half-life values in different populations Number of Range Mean + s.d. Study Place subjects (h) (h) Vesell & Page (1968) Pennsylvania USA ±1.9 Kolmodin et al. (1969) Stockholm, Sweden O'Malley et a. (1971) Dundee, Scotland ± 3.5 Vestal etal. (1975) Maryland, USA ± 5.6 Table 5 Influence of age and sex on antipyrine elimination. Values shown are means + s.d. Group Geriatric Controls p Male Female p n T, (h) ± Vd (/) <0.001 C (I/h) < ± <0.02

4 26 I.H. STEVENSON FH Anticonvulsants * I Barbiturate overdose I*I: Barbiturate dependent Mandrax Hyperthyroid -I-I Amylobarbitone I--+--S 8 Amitriptyline F-1- --e Fe- deficient 1F--f--, IAnaesthetists Controls I ---e +Nitrazepam H-*-s---I Oral contraceptive J * Geriatric -i I, * Hypothyroid z * Paracetamol overdose Plasma antipyrine half-life (h) Figure Summary of factors influencing plasma antipyrine half-life. Results are shown as means ± s.d. for the groups studied..- Significantly different from controls; - -* - - not significantly different from controls. activity measured on the same day. There were also similar but less striking correlations between antipyrine half-life and serum bilirubin and prothrombin time ratio. This relatively simple study therefore showed that there is impairment of antipyrine elimination in the phase immediately following paracetamol overdose and that a significant relationship exists between antipyrine handling and degree of liver damage. Of interest too was the demonstration that antipyrine handling is altered in thyroid disease. Both in terms of half-life and clearance values, the plasma elimination of antipyrine was increased over normal in hyperthyroid patients and was subnormal in hypothyroidism (Crooks, Hedley, Macnee & Stevenson, 1973; Macnee, 197). In both conditions a correlation existed between the extent of alteration in antipyrine handling and the severity of the disease as assessed by standard biochemical parameters. Reversion of the rate of antipyrine metabolism to normal occurred during the course of treatment. In the only other disease state studied-iron deficiency anaemia-no alteration in antipyrine handling ability occurred (Stevenson & O'MaIley, 1973). This may be rather surprising in view of the essential role of cytochrome P50 in microsomal drug hydroxylation and probably reflects lack of alteration in the levels of this cytochrome in liver in all but the most severe state of anaemia. Studies of the effect on antipyrine handling of exposure to several drugs were also carried out and as is evident from Figure, the shortest plasma antipyrine half-life values obtained, in some cases less than 3 h, were in patients on anticonvulsant therapy (Stevenson, O'Malley & Shepherd, 1976), in patients dependent on barbiturates (Ballinger, O'Malley & Stevenson, 1972) and, in collaboration with Dr Prescott's group, in patients following barbiturate overdose (Forrest et al., 197). In the latter two cases values nearer normal were obtained at some time following the barbiturate exposure. If, as is likely, the plasma antipyrine half-life value provides some index of drug metabolism, it is quite clear that these three groups of patients have a markedly increased drug metabolizing ability and are probably particularly at risk with regard to drug interactions or problems associated with the increased breakdown of endogenous substances. With lower (hypnotic doses) of amylobarbitone and with hypnotic doses of methaqualone/diphenhydramine (Mandrax), induction of antipyrine metabolism still occurred although significantly less so that in the three groups of patients described. No alteration in antipyrine handling occurred on the other hand following treatment with nitrazepam (Stevenson, Browning, Crooks & O'Malley, 1972). In these studies, the only drugs which appeared to impair antipyrine handling were the oral contraceptive steroids, the rate of antipyrine metabolism in women taking these being 30% lower than in a control group (O'Malley, Crooks & Stevenson, 1972). Finally, as is evident from Figure, no change was brought about by treatment with the tricyclic antidepressants (O'Malley, Stevenson & Turnbull, 1973) and in the only 'environmental' study carried out plasma antipyrine half-life values were found to be normal in operating-theatre personel (Wood, O'Malley & Stevenson, 197). Conclusions Over the last several years a considerable amount of information has been obtained in the author's laboratory on the factors influencing antipyrine elimination. In the situations studied, the most striking impairment of antipyrine handling occurred following paracetamol overdose and in some elderly patients. Particularly rapid elimination of antipyrine from the plasma was found in patients on anticonvulsant therapy and in patients dependent on or overdosed with barbiturates. Significant alteration in the rate of antipyrine metabolism also occurred in thyroid disease. While such alterations were statistically significant, their clinical importance must await studies of the handling of various therapeutic agents in these situations.

5 FACTORS INFLUENCING ANTIPYRINE ELIMINATION 265 The author wishes to acknowledge the financial support of the Medical Research Council and the Scottish Health Endowment Research Trust in many of these studies and to thank most sincerely his principal collaborators Dr Kevin O'Malley and Dr Christine Macnee. References ALVARES, A.P., KAPELNER, S., SASSA, S. & KAPPAS, A. (197). Drug metabolism in normal children, leadpoisoned children, and normal adults. Clin. Pharmac. Ther., 17, BALLINGER, B., O'MALLEY, K. & STEVENSON, I.H. (1972). Drug metabolism in states of drug dependence and withdrawal. Br. J. Pharmac., 5, BATY, J.D. & PRICE EVANS, D.A. (1973). Norphenazone, a new metabolite of phenazone, in human urine. J. Pharm. Pharmac., 25, BRANCH, R.A., SHAND, D.G., WILKINSON, G.R. & NIES, A.S. (197). Increased clearance of antipyrine and d- propranolol after phenobarbital treatment in the monkey. J. clin. Invest., 53, BRODIE, B.B., AXELROD, J., SOBERMAN, R. & LEVY, B.B. (199). The estimation of antipyrine in biological materials. J. biol. Chem., 179, BRODIE, B.B. & AXELROD, J. (1950). The fate of antipyrine in man. J. Pharmac., 98, CROOKS, J., HEDLEY, AJ., MACNEE, C. & STEVENSON, I.H. (1973). Changes in drug metabolizing ability in thyroid disease. Br. J. Pharmac., 9, 156P. CROOKS, J., O'MALLEY, K. & STEVENSON, I.H. (1967). Pharmacokinetics in the elderly. Clin. Pharmacokin., 1, FORREST, J.A.H., ROSCOE, P., STEVENSON, I.H., MATHEW, H. & PRESCOTT, L.F. (197). Abnormal drug metabolism following barbiturate and paracetamol overdose. Br. med. J.,, GREENBERG, I.A. (1950). Antipyrine. New Haven: Hillhouse Press. HUFFMAN, D.H., SHOEMAN, D.W. & AZARNOFF, D.L. (197). Correlation of plasma elimination of antipyrine and the appearance of -hydroxyantipyrine in the urine of man. Biochem. Pharmac., 23, KOLMODIN, B., AZARNOFF, D.L. & SJOQVIST, F. (1969). Effect of environmental factors on drug metabolism: Decreased plasma half-life of antipyrine in workers exposed to chlorinated hydrocarbon insecticides. Clin. Pharmac. Ther., 10, MACNEE, C (197). Drug metabolism in thyroid disease. Ph.D. Thesis, University of Dundee. O'MALLEY, K. (1972). Some factors affecting drug metabolism. Ph.D. Thesis, University of Dundee. O'MALLEY, K., CROOKS, J., DUKE, E. & STEVENSON, I.H. (1971). Effect of age and sex on human drug metabolism. Br. med. J., 3, O'MALLEY, K., CROOKS, J. & STEVENSON, I.H. 1972). Impairment of drug metabolism by oral contraceptive steroids. Clin. Pharmnac. Ther., 13, O'MALLEY, K., STEVENSON, I.H. & TURNBULL, MJ. (1973). Effects of tricycic antidepressants on drug metabolism in man. Eur. J. clin. Pharmac., 6, SMITH, S.E. & RAWLINS, M.D. (197). Prediction of drug oxidation rates in man: Lack of correlation with serum gamma-glutamyl transpeptidase and urinary excretion of d-glucaric acid and 6,-hydroxycortisol. Eur. J. clin. Pharmac., 7, SOBERMAN, R., BRODIE, B.B., LEVY, B.B., AXELROD, J., HOLLANDER, V. & STEELE, J.M. (199). The use of antipyrine in the measurement of total body water in man. J. biol. Chem., 179, STEVENSON, I.H., BROWNING, M., CROOKS, J. & O'MALLEY, K. (1972). Changes in human drug metabolism following long term exposure to hypnotics. Br. med. J.,, STEVENSON, I.H. & O'MALLEY, K. (1973). Iron deficiency anaemia and drug metabolism. J. Phann. Pharmac., 25, STEVENSON, I.H., O'MALLEY, K. & SHEPHERD, A.M.M. (1976). Relative induction potency of anticonvulsant drugs. In Anticonvulsant drugs and enzyme induction, ed. Richens, A. & Woodford, F.R., pp Amsterdam: Associated Scientific Publishers. VESELL, E.S. & PAGE, J.C. (1968). Genetic control of drug levels in man: Antipyrine. Science, 161, VESTAL, R.E., NORRIS, A.H., TOBIN, J.D., COHEN, B.H., SHOCK, N.W. & ANDRES, R. (1975). Antipyrine metabolism in man: influence of age, alcohol, caffeine, and smoking. Clin. Pharmac. Ther., 18, WELCH, R.M., DeANGELIS, R.L., WINGFIELD, M. & FARMER, T.W. (1975). Elimination of antipyrine from saliva as a measure of metabolism in man. Clin. Pharmac. Ther., 18, WOOD, M., O'MALLEY, K. & STEVENSON, I.H. (197). Drug metabolising ability in operating threatre personnel. Br. J. Anaesth., 6, YOSHIMURA, H., SHIMENO, H. & TSUKAMOTO, H. (1968). Metabolism of drugs. L.I.X. A new metabolite of antipyrine. Biochem. Pharmac., 17,