Intestinal metabolism of ethinyloestradiol and paracetamol

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1 Br. J. clin. Pharmac. (1987), 23, Intestinal metabolism of ethinyloestradiol and paracetamol in vitro: studies using Ussing chambers SILVIA M. ROGERS, D. J. BACK & M. L'E. ORME Department of Pharmacology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX 1 The intestinal mucosal metabolism of ethinyloestradiol (EE2) and paracetamol (P) has been studied in vitro in Ussing chambers. Histologically normal jejunum or ileum was obtained from 19 patients undergoing various resections. 2 The muscularis externa was stripped off the mucosa and the mucosal sheets mounted between two perspex chambers. Tissue viability was routinely assessed by measurement of the transmural potential difference. 3 The percentage of steroid in the serosal chamber, 2 h after addition of EE2 (2,uCi; 80 ng) to the mucosal chamber was 2.3 ± 0.8% (mean ± s.d.) which comprised unconjugated drug (0.4 ± 0.3%), sulphate conjugates (0.7 ± 0.5%) and glucuronides (0.9 ± 0.8%). In the mucosal chamber, 56.6 ± 11.4% was unconjugated steroid, 33.3 ± 12.4% sulphate conjugates and 2.1 ± 2.3% glucuronides. Small amounts of the oxidation products 2- hydroxy and 16-hydroxy-EE2 were present. 4 At 2 h, the percentage of paracetamol in the serosal chamber was 3.2 ± 1.4% (of added P; 2 IiCi; 50 ng) of which 0.5 ± 0.3% was paracetamol sulphate (PS) and 0.1% was paracetamol glucuronide (PG). In the mucosal chamber 2.4 ± 0.8% and 1.0 ± 0.2% was present as PS and PG respectively. The total amount of paracetamol conjugated was approximately 4.0%. When paracetamol in the mucosal chamber was increased to 50,ug (i.e. by a factor of 1000) there was a decrease in the percentage of added drug metabolized to PS and an increase in formation of PG. The glucuronide: sulphate ratio was increased from 0.34 to Competition for sulphation was evident when both paracetamol and EE2 were presented to the intestinal mucosa. This effect was dose-dependent with EE2 sulphation reduced by 32% in the presence of 50,ug paracetamol and by 80% in the presence of 1 mg paracetamol. It is suggested that a decrease in the PAPS (3'-phosphoadenosine-5'- phosphosulphate) pool is responsible for the decrease in steroid conjugation. Keywords gut wall metabolism paracetamol ethinyloestradiol Introduction Drugs which are subject to a first pass effect may tive steroid ethinyloestradiol (EE2) is one of the be removed by the gut wall and/or the liver few drugs undergoing first pass metabolism which before they reach the systemic circulation. We is metabolized to a greater extent by the gut wall have previously shown that the oral contracep- than the liver (Back et al., 1982). Further, in Correspondence: Dr D. J. Back, Department of Pharmacology, University of Liverpool, P.O. Box 147, Liverpool L69 3BX 727

2 728 Silvia M. Rogers et al. vitro studies using small samples of jejunal mucosa obtained from healthy relatives of patients with coeliac disease showed that EE2- sulphate was the main metabolite formed by the mucosa (Back et al., 1981b). Since sulphate formation is a pathway of limited capacity in man (Levy & Matsuzawa, 1967; Houston & Levy, 1975) and is therefore subject to saturation and competitive inhibition, the co-administration of a drug which also forms a sulphate conjugate in the gut wall may be expected to influence the bioavailability of EE2. This has been shown to be so for vitamin C (ascorbic acid). In women given single oral doses of EE2, the bioavailability of the steroid was increased by 60 to 70% when given shortly after 1 g oral doses of vitamin C (Back et al., 1980). In women taking long term oral contraceptive steroids the 24 h plasma EE2 concentrations were increased by 47% when they also received 1 g vitamin C per day (Back et al., 1981a). Paracetamol undergoes extensive conjugation (glucuronylation and sulphation) and is commonly used by women taking oral contraceptive steroids. Recent evidence has indicated that little conjugation occurs in the gastrointestinal tract (Clements et al., 1984). However, this conclusion was based on urinary excretion data following intravenous and oral administration and not a direct assessment of mucosal metabolism. Clearly, if urinary metabolite profiles are comparable following the two routes of administration, mucosal conjugation can only, at most, account for a small percentage of the overall metabolism. However given the relatively large dose of paracetamol normally taken (1000 mg) in comparison with EE2 (30-50 jig) a small amount of mucosal sulphation of paracetamol may significantly influence the bioavailability of the contraceptive steroid. The aim of this study was to assess directly the mucosal metabolism of EE2 and paracetamol adapting a technique (Ussing chambers; Ussing & Zehran, 1951) which has been extensively used for the study of ionic flux, and then to examine the effect of paracetamol on EE2 metabolism. Methods Histologically normal jejunum or terminal ileum was obtained from a total of 19 patients undergoing resections for localized tumour, diverticulosis and Crohn's disease. The tissue was transported from the operating theatre to the laboratory in ice cold oxygenated Krebs-Henseleit buffer solution. While mounted on a glass tube, the muscularis externa was stripped off the mucosa and then following a longitudinal incision, the mucosal sheets were mounted between two perspex chambers (Figure 1). Mounting was Figure 1 Ussing chambers.

3 normally complete within 30 min of resection. Fresh Krebs-Henseleit buffer (2.5 ml, ph 7.4) was placed in each chamber, maintained at 370 C by water jackets, and circulated and aerated by gas lift ports with 95% 02 and 5% CO2. The transmural potential difference (p.d.) was measured on an automatic multimeter (Philips, Model PM 2521) connected to calomel half cells which in turn were connected to the bathing solution by 3M KCl-agar bridges in polyethylene tubing with the bridge tips centred approximately 1 mm from either side of the tissue. The p.d. across the mucosa of the small bowel has a value of about 5-10 mv (Armstrong & Garcia-Diaz, 1984) and we took such a potential as a measure of tissue viability. In some other initial viability experiments we either added D-glucose, which due to active transport augments the p.d. or [14C]-polyethylene glycol which only appears in the serosal chamber if the tissue is damaged in mounting. Histological examination of some tissue was performed before and after the experimental period. Three studies were performed. In study 1, radiolabelled ethinyloestradiol (17a-[6,7-3H]EE2; SSCi mmol-'; 2,uCi; 80 ng) was added to the mucosal chamber with subsequent sampling from both chambers (50 RI aliquots) at 15 min intervals to 2 h for determination of drug concentrations. 1 ml aliquots were removed at the end of the experiment for the determination of metabolites. In study 2, radiolabelled paracetamol (p [3H]-hydroxyacetanilide; 18.9 Ci mmol-1; 2,Ci; 50 ng or 50,g) was added to the mucosal chamber with sampling as above. In study 3, paracetamol, 50,ug or 1 mg was added to the mucosal chamber (a concentration equivalent to 20,ug ml- 1 and 400 jxg ml-' in bathing fluid). After a period of 20 min, [3H]-EE2 (80 ng; 2,uCi) was added to the chamber and sampling commenced. The radioactive content of aliquots was determined by liquid scintillation spectrometry using a Packard Tri-Carb 4640 liquid scintillation spectrometer and scintillant EM299 (Packard). Metabolite separation Gut wall metabolism of ethinyloestradiol and paracetamol Ethinyloestradiol Aliquots of mucosal and serosal fluid (1 ml) were subjected to extraction with diethyl ether (3 ml) for the determination of unconjugated steroid. Aliquots (400 RI1) of the remaining aqueous phase were incubated for 2 h at 370 C with P-glucuronidase/sulphatase enzyme (Helix pomatia, Type H1; 30 units) either in the presence or absence of glucurolactone (24 mg ml-1 in acetate buffer). After incubation, extraction of steroid was performed as above and the proportion of 3H extracted into ether determined. Extracts were evaporated to dryness and the residue dissolved in a small volume of methanol for h.p.l.c. analysis according to the method described by Maggs et al. (1982). A Spectra-Physics SP 8700 solvent delivery pump was used connected to an LKB 2122 Redirac fraction collector. A stainless steel column was used packed with Partisil 10/25 ODS-2 (25 cm x 0.46 cm i.d., Whatman Inc., Clifton, N.J., USA). Samples (10,ul) were eluted at room temperature with a linear gradient of methanol in 0.5% (w/v) ammonium dihydrogen phosphate buffer (ph 3.0) from 50-65% at 2%/ min. The flow rate was 2 ml min-'. The radioactive content of each sample was determined by liquid scintillation spectrometry. Paracetamol A 20,ul sample of fluid from the mucosal chamber collected at the end of the experimental period was mixed with the chromatography markers (paracetamol, paracetamol glucuronide and paracetamol sulphate) and subjected to h.p.l.c. using methodology adapted from Howie etal. (1977). A 1 ml sample from the serosal chamber was evaporated to dryness and resuspended in water (40,ul) containing paracetamol standards. A Spectra-physics SP 8700 solvent delivery system was used connected to an LKB 2112 Redirac fraction collector. The column was a pu- Bondapak Radial-PAK cartridge housed in a radial compression module. 20 pul samples were eluted at room temperature with a mobile phase consisting of 9 parts 0.05 M acetate buffer to 1 part of a mixture of methanol: ethyl acetate (150:1) at a flow rate of 1.8 ml min-'. The retention times of paracetamol glucuronide (PG), paracetamol sulphate (PS) and paracetamol were 3.5, 7.0 and 11.5 min respectively. The radioactive content of each sample was determined by liquid scintillation spectrometry. Results are presented as mean ± s.d. and statistical analysis was by the Student's t-test for unpaired samples. Results 729 An example of the transmural potential difference measured in human ileum is shown in Figure 2. For studies of EE2 metabolism, tissue was obtained from five patients (two ileum, three jejunum). The overall net transport of steroid from the mucosal chamber to the serosal chamber in 2 h was low (2.3 ± 0.8% of added drug; mean ± s.d.) with the presence on the serosal side at that time of unconjugated ('free') steroid (0.4 ±

4 730 Silvia M. Rogers et al. E i1- *._ Time (min) Figure 2 The transmural potential difference (mv) measured in human ileum. The dotted line indicates the initial fall in p.d. when the -tissue is first mounted in the chamber. Experimental procedures commenced at time = %), sulphate conjugates (0.7 ± 0.5%) and glucuronides (0.9 ± 0.8%), (Table 1). In the mucosal chamber, 56.6 ± 11.4% of the added drug was present as unconjugated steroid, 33.3 ± 12.4% as sulphate conjugates and 2.1 ± 2.3% as glucuronides. H.p.l.c. analysis of the various fractions revealed some evidence of trace amounts of phase I hydroxylated metabolites (Figure 3). Identification is only tentative by co-chromatography of the standard material. For studies of paracetamol metabolism (Table 2) tissue was obtained from eight patients. Using 'low dose' paracetamol (i.e. 50 ng), 2.4 ± 0.8% (mean s.d.) of added drug was present as PS and % as PG in the mucosal fluid at 2 h. In the serosal fluid, 0.5 ± 0.3% was PS and 0.1% was PG. Conjugates (PG + PS) accounted for approximately 4.0% of the recovered paracetamol with PS predominant. The overall net transport of drug from the mucosal to the serosal chamber was 3.2 ± 1.4%. Using 'high dose' paracetamol (i.e. 50 Rig), 0.6 ± 0.6% of added drug was present as PS and 2.5 ± 0.8% as PG in the mucosal fluid at 2 h. In the serosal fluid 0.1% was PS and 0.03% was PG. Conjugates accounted for 3.2% of the recovered paracetamol. The glucuronide:sulphate ratio was 0.34 for the low dose and 3.56 for the high dose which represents a relative decrease in the formation of PS and an increase in formation of PG with high dose paracetamol. The effect of paracetamol (50,ug and 1 mg) on EE2 metabolism is shown in Table 3. The data are presented for mucosal samples only, since in the presence of paracetamol the transport of EE2 from the mucosal to serosal side was always less than 1%. In the presence of 50,ug paracetamol, there was a significant decrease in total conjugates resulting from a decrease in formation of EE2-sulphates. With the increase in paracetamol to 1 mg, unconjugated steroid was increased from a mean value of 56.6% (control) to 88.1% (P S 0.001), total conjugates decreased - from 35.3 to 10.0% (P 0.01) and EE2-sulphates - decreased from 33.3 to 6.8% (P 0.001). Discussion The extent of steroid sulphation) seen in this study is in agreement with our previous in vitro work with small (5-20 mg) pieces ofjejunal mucosa (Back et al., 1981b) and in vivo work with hepatic portal vein sampling following oral administration (Back et al., 1982). An interesting finding was that the sulphate conjugates preferentially returned to the mucosal chamber; 33.3 ± 12.4% of the drug in this chamber at 2 h being sulphate conjugates (principally EE2-sulphate). The low and clearly unphysiological extent of transport of drug across the tissue coupled with the appearance of metabolites on the mucosal side is in part a reflection of the nature of the preparation. In all cases the complete removal of the muscularis externa (comprising an outer longitudinal layer conjugation (principally Table 1 Metabolism of ethinyloestradiol (EE2) by human small intestine. EE2 and conjugates were determined in the mucosal and serosal chambers 2 h after addition of [3H]-EE2 (80 ng) to the mucosal chamber % of added drug present as % net Unconjugated transport steroid Sulphates Glucuronides Mucosal 56.6 ± ± ± 2.3 Serosal 2.3 ± ± ± ± 0.8 Results are mean ± s.d. (n = 10) of tissues from five patients.

5 Gut wall metabolism of ethinyloestradiol and paracetamol a Serosal fluid EE2 10 ci) 4) co ai) 0) qj 301- b Mucosal fluid EE t OHEE2 2-OHEE2 - lih I r -I Eluate fractions (ml) 4 5 ) Figure 3 A representative high performance liquid chromatogram of ether extracts of serosal and mucosal fluid in a study with [3H]-EE2. Peak assignments are based on co-chromatography with authentic unlabelled standards. Similar profiles were obtained following enzyme hydrolysis indicating the presence of small amounts of oxidative metabolites in both the unconjugated and conjugated fractions. Table 2 Metabolism of paracetamol by human small intestine. Paracetamol and conjugates (PS and PG) were determined in the mucosal and serosal chambers, 2 h after addition of [3H]-paracetamol (either 50 ng or 50,ug) to the mucosal chamber Net transport Added drug (%) present as (%) P PS PG 'Low dose'-50 ng* Mucosal ± ± ± 0.2 Serosal 3.2 ± ± ± ± 0.0 High dose-50,ug** Mucosal 92.3 ± ± ± 0.8 Serosal 1.0 ± ± ±+0.0 Trace *Results are s.d. patients. mean ± s.d. (n 10) of tissues from three patients. and an inner circular layer) was attempted, but this still leaves a layer of smooth muscle intact, the muscularis mucosae which can act as a physical barrier to the passage of drug/metabolites. Therefore, although transport in no way reflects the in vivo situation, the overall nature and extent of metabolite production appears, on the basis of a comparison with previous in vivo work (Back et al., 1982), to closely parallel what happens in situ. Using a vascularly perfused mouse small intestinal preparation, Wollenberg & Rummel (1984) described the formation of sulphoconjugates of a number of compounds including ethinyloestradiol and paracetamol. They pointed out that conjugates require transport mechanisms to exit from cells and demonstrated that the sulphoconjugate of EE2 appeared only in the luminal perfusion medium whereas that of paracetamol was released exclusively into the vascular medium. They concluded that selective anion transport systems for sulphoconjugates exist in the brush border membrane as well as the basolateral membrane of the enterocyte. Although it is impossible to extrapolate findings from the mouse intestine to human intestine, and noting the previous discussion on the limitations of the

6 732 Silvia M. Rogers et al. Table 3 The effect of paracetamol (either 50 p.g or 1 mg present in the mucosal chamber) on the metabolism of EE2 by human small intestine. EE2 and conjugates were determined 2 h after addition of [3H]-EE2 (80 ng) to the mucosal chamber. Since in the presence of paracetamol less than 1% of steroid was transported to the serosal side, data are presented for drug present in mucosal samples Added drug (%) present in mucosal chamber Paracetamol No paracetamol 50 gg 1 mg Unconjugated EE ± ± ± 10.1*** Conjugates (total) 35.3 ± ± 6.9* 10.0 ± 9.0*** Sulphates 33.3 ± ± 6.3* 6.8 ± 4.6*** Glucuronides 2.1 ± ± ± 5.0 Results are mean ± s.d. of tissues (n = 10) from five patients for the controls, of tissues (n = 7) from three patients for paracetamol (50,ug) and of tissues (n = 10) from three patients for paracetamol (1 mg). Significantly different from control; *P S 0.05; ***P < preparation, the present results are consistent with the release of EE2-sulphate across the brush border membrane. On the basis of urinary excretion data of paracetamol and its glucuronide, sulphate, cysteine and mercapturate conjugates following i.v. and oral administration, Clements et al. (1984) concluded that the gastrointestinal tract is not an important site for paracetamol metabolism. The results of the present study do, however, indicate the presence of both the glucuronide and sulphate conjugates in fluid bathing the mucosa. The extent of conjugation was small with a maximum of 4% of the added paracetamol appearing as either the sulphate (3%) or glucuronic acid (1%) conjugate in the serosal and mucosal chambers. The initial 'dose' of paracetamol used (50 ng; 20 ng ml-1 in the mucosal chamber) is small in comparison with the concentrations of the drug which will be present in the g.i. tract following ingestion of a conventional dosage regimen of 1 g. Hence in subsequent experiments increased concentrations of paracetamol were used. The one thousand fold increase in paracetamol concentration produced a clear shift from sulphation to glucuronidation which is reflected by the increase in glucuronide:sulphate ratio. A dosedependent shift from sulphation to glucuronidation has been described for a number of phenolic substrates, both in vivo and in vitro. In rats (Oehme & Davis, 1970; Weitering et al., 1979; Koster et al., 1981) and in monkeys (Mehta et al., 1978), intravenous and intramuscular injections of phenol were shown to be conjugated increasingly with glucuronic acid as the dose increased. A pronounced shift from sulphation to glucuronidation of paracetamol has also been observed in mice, rats and hamsters when the dose was increased from 0.2 to 8 mmol kg-' (Jollow et al., 1974). A similar effect is evident in man (Clements et al., 1984). The mechanism of the decrease of sulphation at increasing doses in vivo is thought to be depletion of inorganic sulphate (Lin & Levy, 1981; Krijgsheld & Mulder, 1982; Hjelle et al., 1985) although this hypothesis has been questioned by other workers (Weitering et al., 1979; Koster et al., 1981). In vitro, sulphate depletion is a less likely cause of the dose-dependent shift in conjugation since inorganic sulphate is present in the bathing medium (in the present study 0.57 mol l-1). Sundheimer & Brendel (1984) have concluded from hepatocyte studies that the limiting factor in sulphating capacity is the saturation of ATP-sulphurylase rather than saturation of carrier facilitated sulphate uptake. ATPsulphurylase catalyses the reaction between ATP and S042- to give adenyl sulphate (APS) which is then phosphorylated by APS-phosphokinase to yield PAPS (3'-phosphoadenosine-5'-phosphosulphate). Saturation of ATP-sulphurylase thus leads to a depletion of APS and ultimately of PAPS. Clearly the studies described in this paper do not allow any firm conclusions as to the mechanism of the dose-dependent changes in conjugation. The data from the study in which paracetamol and EE2 were present together in the Ussing chamber indicate that paracetamol competes with EE2 for the sulphation reaction but does not appear to affect glucuronidation. The effect seen was dose-dependent, with EE2 sulphation reduced by approximately 32% in the presence of the low dose of paracetamol (50,ug) and by 80% in the presence of the high dose (1 mg). Competition for the sulphation reaction in vivo has been described for a number of drug com-

7 binations. Ascorbic acid was shown to reduce sulphoconjugation of paracetamol (Houston & Levy, 1976) and of EE2 (Back et al., 1981a). Levy & Yamada (1971) have demonstrated reduced sulphation of salicylamide by paracetamol and Ziemniak et al. (1985) reduced sulphation of fenoldopam by paracetamol. There is substantial evidence that paracetamol and EE2 are not substrates for the same sulphotransferase enzyme. The sulphation of paracetamol occurs via a phenol sulphotransferase and oestrogens by steroid sulphotransferase. The two enzymes are quite distinct although each consists of a number of multiple forms having different and in some cases overlapping specificity (Sugiyama etal., 1984). This therefore negates the possibility of substrate competition for the same enzyme. It is probable that, as previously discussed, depletion of the PAPS pool is responsible for the decrease in EE2-sulphation. Miners et al. (1983) and Mitchell et al. (1983) have described the effect of concurrent oral contraceptive steroid therapy on paracetamol metabolism. The major finding is an increase Gut wall metabolism of ethinyloestradiol and paracetamol 733 in paracetamol clearance due to induction of glucuronidation; there was no effect on sulphation. Miners et al. (1983) commented that the latter finding was not surprising since sulphotransferase appears to be a non-inducible enzyme and the low dose of oestrogen in the oral contraceptive steroid would not be expected to result in significant competitive inhibition of paracetamol sulphation. They also speculated that co-administration of paracetamol and OCS would reduce the first pass metabolism of EE2 thereby increasing its bioavailability. Our in vitro findings lend some support to this conjecture, with the site of a potential interaction being in the gastrointestinal mucosa (where first pass loss is greatest for EE2) and also the liver since both drugs are sulphated in these organs. 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