Fundam Clin Pharmacol(199 1) 5,567-582 0 Elsevier. Paris

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Metabolism of digoxin, digoxigenin 'digitoxosides and digoxigenin in human hepatocytes and liver microsomes B Lacarelle, R Rahmani, G de Sousa, A Durand, A4 Placidi, JP Can0 * INSERM U 278, Facult.4 de Pharmacie, 27, boulevard Jean Moulin, 13385Marseille Cedex 5, France

(Received 20 March 1991 ; accepted 20 June 1991)

Summary - In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG,) and digoxigenin bisdigitoxoside (DG,) were not shown to be significantly metabolized in virro in man. Therefore, it appeared that the stepwise cleavage of DG, and DG, sugars was not cytochrome P,,, dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG ,) and digoxigenin (DG,) which are known to be formed after intra-gastric hydrolysis of DG,, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDPglucuronyl transferase (UDPGT) activities responsible for DG I glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation : i), the individual intra-gastric pH which influences the sugar cleavage leading to DG, and DG, ; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation. digoxin / metabolism / human hepatocytes / human liver microsomes

Introduction Digoxin is one of the most widely used drugs in patients with congestive heart failure a n d o r cardiac rhythm disturbances. Biotransformation of digoxin and digoxigenin digitoxosides has been described both in vivo and in vitro in rat (Wirth and Frolich,

* Present address : Sanofi Recherche, rue du Professeur J Blayac, 34082 Montpellier Cedex, France. Abbreviations : UDPGA, UDP glucuronic acid; UDPGT, UDP-glucuronyltransferase; CHAPS, 3-[(3c h o l a m i d o p r o p y l ) d i m e t h y l a m m o n i o ] - 1 - p r o p a n e s u l f o n a t e DG,, d i g o x i n ; DG,, d i g o x i g e n i n bisdigitoxoside; D G I , digoxigenin monodigitoxoside; DG,, digoxigenin.

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1974; Schmoldt and Ahsendorg 1980; Castle 1980), as well as in vivo in man (Gault et al, 1984; Gault et al, 1985). However, routes and extents of digoxin biotransformation in man still remain a matter of controversy. Indeed, it has been shown that there is initially hydrolysis with release of the sugars, either in the stomach or in the liver to produce 3 pdigoxigenin. This is then followed by oxidation to produce 3 keto-digoxigenin, epimerization to give the 3 a-(epi)-digoxigenin and finally conjugation to give polar end metabolites (fig 1) (Gault et al, 1984). The role of the liver in digoxigenin formation has been demonstrated in rat (Schmoldt and Ahsendorg, 1980) but not in humans. Finally, the relative role of the stomach and the liver in digoxin hydrolysis is not known in man. Recently, it has been shown in humans that 6 h after the ingestion of [3H] digoxin-.12a, metabolites accounted for 1 to 99% of the plasma radioactivity (average : 40%) (Gault et al, 1985). Among them, 88% represented polar compounds. Since, digoxin concentrations are routinely and almost exclusively monitored by immunoassays. Such an interindividual biotransformation variability may have great consequences in clinical practice. Indeed, polar digoxin metabolites exhibit an average of 33% cross-reactivity with anti-digoxin antibodies (Gault et al, 1 9 8 3 , whereas they probably have little cardioactivity (Gault et al, 1984; Belz and Heinz, 1977). Therefore, the lack of correlation between digoxin serum concentrations (measured by radioimmunoassay) and therapeutic outcomes (Goldman ef al, 1975) could be due to the fact that anti-digoxin antibodies recognize both unchanged drug and its polar metabolites, and that their ratio exhibits a large inter-individual variability. In order to elucidate the origin of such a variability, it was of interest to investigate the in vitro metabolism of digoxin and its cleavage-related compounds which are absorbed after intra-gastric acid hydrolysis of digoxin (Gault et al, 1981). This study was conducted using both human hepatocytes and liver microsomes obtained from a tissue bank.

Materials and methods Chemicals [3H] digoxin 12 a was purchased from Dupont NEN Research Products. Non-radioactive digoxin was purchased from Fluka. Standard digoxin derivatives (DG,, DG,, DG,) were a kind gift of Dr RA Kaufman (Roche Diagnostic Systems, Nutley), 3-epi-digoxigenin was generously supplied by Dr A Baggioni (Laboratoires Nativelle, Lonjumeau, France). NADPH, UDPGA sodium salt, PD-glucuronidase (Type 3B from bovine liver), crude solution from Helix pomatia (Type H,), 3-[(3-c~olamidopropyl)-dimethylammonio]-l-propanesu~fonate (CHAPS), collagenase (Type.IV) and culture media were obtained from Sigma. PCS scintillation fluid was from Amersham. All other reagents and solvents were of analytical or HPLC grade.

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Tritiated derivatives preparation

Tritiated substrates (DG,, DG,, DGo) were prepared from [3H] digoxin 12 a. [3H] digoxin 12 a (20 Uci) was incubated (37°C for 30 min) in 0.1 HCI N : at the end of incubation, hydrolysis was stopped by adding 0.1 N NaOH. Isolation and purification of all the substrates were

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Fig 1. Structure of digoxin and proposed metabolic pathways. ? : hypothetical pathway.

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performed by HPLC as described below. The radiochemical purity of each substrate was greater than 95%. Human liver specimens

Human liver specimens were obtained under strict ethical conditions shortly after death from kidney and/or heart transplant donors. Patient characteristics (ie, age, sex, relevant drug intake in the last days) have been previously described (Fabre et al, 1988 ; Combalbert et al, 1989). Hepatocytes isolation

Washing and collagenase perfusion procedures of liver have been described elsewhere (Cano et al, 1988a, b ; Fabre et al, 1988). Briefly, the oxygenized perfusate consisted of NaCl (120 mM), KCI (15 mM), HEPES (20 mM, pH 7.5) and glucose (25 mM), at room temperature. After blanching of the majority of the liver, the organ temperature was progressively raised to 37-38°C. Collagenase (0.05% in HEPES buffer) was then added to the perfuate. The collagenase perfusion was stopped after 20 to 30 min. The Glisson’s capsule was then disrupted, and hepatocytes were released by gentle agitation in a washing buffer (HEPES (10 mM), NaCl ( I 20 mM), KCI (6.2 mM), CaCI, (0.9 rnM) and BSA ( 1 % w/v)). The cell suspension was then filtered through a nylon mesh (150 pm) and washed by centrifugation in L,, medium (three times : 50 g , 2 min, 4°C) in order to purify the hepatocyte fraction. The viability of the freshly isolated human hepatocytes assessed by Trypan blue exclusion generally exceeded 85%. Freshly isolated hepatocytes in suspension

Freshly isolated hepatocytes were counted in a hemocyto-meter and then incubated in HAM F,, medium, in specially designed flasks at 37OC (final density: 2.5 x lo6 cells/ml). The pH was maintained at 7.4 by passing a warm and humidified 95% 0,/5% CO, flux over the cell suspension, which was gently stirred by a Teflon paddle throughout the experiment (Fabre et al, 1985). Primary monolayer culture of human hepatocytes

Freshly isolated hepatocytes (2.5 x 106 cells) were plated into 60-mm culture dishes in a total volume of 4 ml of culture medium F12Coon’s modificationDMEM, fetal cal serum (FCS) (lo%), human insulin (0.15 IU/ml), glucagon (2 pg/ml), L-thyroxin (0.02 pg/ml), human transferrin (10 pg/ml), ethanolamine (1 pglml), sodium selenite ( M), penicillin (50 IU/ml), netilmicin (50 pglrnl)). The culture dishes were then placed in a 37°C incubator containing 5% CO, under a water-saturated atmosphere. Four to 6 h after the beginning of the incubation (cell attachment), the medium was renewed by the same initial medium without FCS but supplemented with dexamethasone ( M). For metabolic studies, 24 h after hepatocytes isolation, drugs were added to the culture medium. Incubations were then continued during 48 h. Microsomes preparation and incubation procedures

Microsomal fractions were prepared by differential centrifugation as previously described (Van der Hoven and Coon, 1974) from pieces of human livers stored at -80°C. After prepara-

In virro digoxin metabolism in human

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tion, microsomes were stored at -80°C until use. Microsome (0.3-0.9 mg protein) incubations were performed in a total volume of 0.3 ml of 0. I M KH,PO, (pH 7.4). After preincubation of the mixture (3 min, 37°C) containing the radiolabeled drug, the enzymatic reactions were started by adding NADPH (final concentration 1 mM). When glucuronidation processes had to be specifically studied, MgCl, (5 mM) was added in the incubation medium and UDPGA (3 mM) was used to initiate the reaction. Since various detergents were shown to activate microsomal glucuronidation reactions (Hazelton et al, 1988), the effects of ionic (sodium cholate), zwitterionic (CHAPS) or non-ionic (Triton X- 100) agents, were tested.

Drug metabolism studies Digoxin and its metabolites were dissolved in ethanol (never exceeding a final concentration of I % (v/v)). Metabolic studies on hepatocytes were initiated by adding an isotopic dilution of the drug to the culture medium. At selected time intervals (from 12 to 48 h), the reaction was stopped by removing the medium from the cell monolayer. Samples were immediately stored at -2OOC until analysis. In experiments on microsomal fractions, methanol was added at the end of the incubation period to stop the reaction and precipitate the proteins. In order to evaluate the extent of drug conjugation, extracellular media and microsomal supernatants were incubated after chloroformic extraction, for 12 h at'37"C with P-D-glucuronidase (10 000 units/ml) or with crude solution from Helix pomatia ( p glucuronidase : 10 000 units/ml; arylsulfatase: 500 units/ml) in sodiumsacetate buffer (0.1 M, pH 4.5). Rabbit microsomal fractions were used as positive control of experimental conditions. Protein concentrations were determined by the Bradford method (Bradford, 1976) (Bio-rad protein assay kit) using bovine serum albumin as a standard.

HPLC analysis Culture media or microsomal supernatants were analyzed by reverse phase HPLC on"a :G,,. VBondapak column (Waters Associates, Milford MA). All analyses were performed with a Hewlett-Packard 1084 B or 1090 chromatograph equipped with an automatic injector. The mobile phase was watedmethanol delivered by an isocratic mode for 10 min (70/30) and then by a 5-min linear gradient from 30 to 65%. Flow rate was 1.0 mumin. Standard absorbance was recorded at 220 nm (fig 2). Under these conditions, 3-epi-digoxigenin was eluted ' I .min after DG,. Detection of tritiated compounds was performed using a continuous flow scintillation detector (Radiomatic Instruments).

Results Metabolism of dixogin (DG,)

Digoxin biotransformation by human .hepatoqte.s.-freshly, isolated in suspension :has been studied after cell exposure to 1W M initial drug concentration. In,this specific

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experimental condition, DG, metabolism was very low and, after a 2-h incubation period, extracellular radioactivity represented almost only unchanged DG, (up to 93%). A minor unidentified polar metabolite was however synthetized at this time (5% of the total extracellular radioactivity). 0

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Fig 2. HPLC separation of DG,, DG2, DG, and DG,. Details of chromatographic conditions are given in the test.

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Due to the fact that hepatocytes are not viable more than 2-3 h in suspension, human hepatocytes in primary culture were used to assess DG, metabolism over longer periods of time. Various DG, concentrations (1o-S to 10-6 M) were investigated in this model, confirming that this drug was not significantly metabolized by human hepatocytes even after a 24-h exposure time (fig 3). Similar results were obtained with human liver microsomal fractions. In contrast, under the same experimental conditions, DG, (2 x 10-8 M) was extensively biotransformed by rabbit microsomes, leading to a great proportion of unidentified metabolites (79% of the total radioactivity) after a 30-min incubation period (fig 4). Therefore, it appeared that in man the stepwise cleavage of DG, sugars was not related to the NADPH-dependent cytochrome P450system which probably plays a minor role in explaining the overall inter-individual metabolic variability of this drug in patients. Metabolism of digoxigenin bisdigitoxoside (DG,)

After a 24-h incubation of human hepatocytes in primary culture with DG, (10-7 M), only traces of DG, and polar compounds appeared in the incubation medium: unchanged drug represented.more than 95% of the total radioactivity (fig 3). Metabolism of DG, (10-8 M) by human liver microsomes was also investigated in presence of either NADPH (1 mM) or UDPGA (3 mM), in order to detect the potential phase I (sugar cleavage) or phase I1 (glucuronidation) enzymatic reactions. Neither of these experimental conditions led to metabolite formation in the incubation medium. Metabolism of digoxigenin monodigitoxoside (DG,)

DG, (10-7 M) was extensively metabolized by human hepatocytes in primary culture (fig 3), giving only one metabolite which was more polar than the parent compound. This metabolite was shown to be completely hydrolyzed by P-D-glucuronidase and was therefore identified as being the glucuronide of DG ,. Since glucuronidation appeared to play a major role in the overall metabolism of DG,, we specifically studied glucuronidation of DG, by human liver microsomes. We tested the effects of ionic (sodium cholate), zwitterionic (CHAPS) or non-ionic (Triton X-100) agents on DG, conjugation. No significant increase in enzyme activity was observed after these various treatments. Therefore, all the conjugation events were studied using native microsomes without further activation. In order to compare the kinetic characteristics of the enzyme, which glucuronidates DG,, with those of the well-characterized digitoxigenin monodigitoxoside UDPGT, the parameters of the DG, glucuronidation reaction were determined using a Lineweaver-Burk plot (fig 5). The apparent K, and V , values were respectively 3.1 p M and 23 pmoVmin per mg microsomal protein.

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The inter-individual variability of DG, metabolism was evaluated using human liver microsomal fractions prepared from 13 different subjects (fig 6 ) . This study demonstrated that the amounts of glucuronide synthetized by microsomes varied by a factor of 3 between individuals. Moreover, no correlation was found between this variability and subject characteristics (age, sex), or corticosteroid intake which is known to induce digitoxigenin monodigitoxoside UDPGT (Schuetz, 1986). Metabolism of digoxigenin (DGo)

DG, (10-7 M) was also extensively biotransformed by human hepatocytes in primary culture as illustrated on the radiochromatogram in figure 3. Three main peaks and

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TI ME.(.min.) Fig 3. Metabolism of DG,, DG,, DG, and DG, by human hepatocytes in culture. Initial concentration of each compound was 10-8 M. The figures represent culture media radiochromatograms after 24 h exposure. G : glucuronide ; P: polar metabolites ; 2 : unidentified metabolite ; 3 + 4:predigoxigenin ; 5 : digoxigenin ; 6: 3 epi-digoxigenin.

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three minor ones appeared in the extracellular medium 24 h after incubation. The first peak (labelled P) represented the sum of one or more glucuronides which were hydrolyzed by P-D-glucuronidase (fig 7) or by crude solution from Helix pomatia.

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TIME (Min.1 Fig 4. Inter-species (rabbit v s human) variability of digoxin metabolism by liver microsomes.

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Fig 5. Lineweaver-Burk plot of human liver microsomal UDPGT towards DG, (mean of two determinations with sample 14). The concentration of UDP glucuronic acid was 3 mM.

This peak may also correspond at least in part to one or more unidentified metabolites as previously described (Gault et al, 1982). The 5th peak co-eluted with unchanged DG, and the 6th peak with 3-epi-digoxigenin. Peaks 2 to 4 were not identified due to the lack of the corresponding standards. By analogy with a previous work (Gault et al, 1982), the sum of peaks 3 and 4 has been named predigoxigenin which represents one or more unidentified metabolites. Figure 8 illustrates the kinetics of DG, metabolism in human hepatocytes in primary culture. The decrease of 3-epi-digoxigenin concentration was related to the increased synthesis of polar compounds. It appeared that whereas 3-epi-digoxigenin was retained within the cell, polar compounds effluxed in the extracellular compartment as soon as they were formed. DG, metabolism was also studied using human liver microsomes : in the absence of cofactor (ie NADPH or UDPGA) no metabolite was observed ; in the presence of NADPH, only pre-digoxigenin was observed. Its formation appeared therefore to be cytochrome P,,, dependent and showed a large variability between individuals (fig 9). On the other hand, 3-epi-digoxigenin formation did not depend on microsoma1 enzymes. Indeed, this metabolite was observed after DG, incubation with hepatocyte and did not appear when DG, was incubated with microsomes. In the presence of both NADPH and UDPGA in the incubation medium, only small amounts of polar compounds were observed. This result confirms that prior to polar compounds synthesis, 3-epi-digoxigenin must be formed.

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Fig 6. lnterindividual variability of DG, glucuronidation by human liver microsomes from 13 subjects. Incubations were performed with 2 mg microsomal protein/ml and 3 mM UDPGA. Initial drug concentration was I t 7 M.

Therefore, the main in vitro metabolic pathway for DG, is the formation of 3-epidigoxigenin, a compound which is further conjugated, leading to a glucuronide. These routes are not influenced by the cytochrome P450interindividual variability but seem to depend on UDPGT hepatic levels.

Discussion Our results demonstrate that two main factors can be evoked to explain the interindividual variability of digoxin metabolism. The first is well known and consists of intra-gastric hydrolysis (Gault et al, 1981), a process which plays a determinant role since it leads to the formation of DG, and DG, which can then be conjugated. Hepatic glucuronidation can also be in part responsible for the large variability in the relative amounts of polar metabolites from patient to patient. Indeed, using human microsomes, we observed a large variability in UDPGT activities responsible for DG, glucuronidation. Previous studies indicate that microsomal UDPGT constitutes a family of isozymes exhibiting different aglycone specificities. A new UDPGT activity h as been

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Fig 7. Effect of fi-o-glucuronidase hydrolysis on the polar fraction from an extracellular medium aliquot sampled 24 h after incubation of DG, (10-8 M) with human hepatocytes in culture. P : polar metabolites.

described recently in rat (Schmoldt et al, 1982 ; Schuetz et al, 1986) and characterized (Meyerinck et al, 1985) as glucuronidating the cardiac glycoside digitoxigenin monodigitoxosicie with an apparent K, of 5.8 k 1.6 pM. Moreover, in rat, this enzyme was not shown to be activated by detergents (Hazelton et al, 1988). The human liver UDPGT responsible for DG, glucuronidation exhibits similar properties, with a K,,,estimated at 3.1 pM for this reaction. In addition, this enzyme was not activated by Triton X-100, sodium cholate or CHAPS as previously described in rat. In this context, the variability observed in vivo from patient to patient after digoxin administration is presumably related at least in part to the individual amount of UDPGT. Although an interindividual variability factor of 3 is not very important, for digoxin, which exhibits a narrow therapeutic index, such a variability may have important clinical implications.

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T.IME( t i ) Fig 8. Metabolism of DG, (10-7 M) by human hepatocytes in primary culture. The mean of two determinations with sample 2 2 and 2 3 is shown. Digoxigenin ( 0 ) ; 3-epi-digoxigenin ( m ); pre-digoxigenin ( 0); polar compounds ( 0 ).

Inversely, to rat andmbbit metabolic processes, hepatic enzymes seem to be slightly involved in the stepwise cleavage of DG, sugars. Indeed, only a low amount of polar compounds was found after incubation of digoxin with human hepatocytes in suspension (extra-cellular medium). This fact could imply that DG, or DG, are already formed but in low proportions. These results are in agreement with in vivo data obtained in man after iv injection of [3H] digoxin-12 a,where small amounts of digoxigenin digitoxosides were observed 8 h after administration (Gault et al, 1979). We demonstrated that P,,, cytochromes are involved in the biotransformation of DG, to predigoxigenin. However, this metabolite is not the major precursor of glucuronides. Finally, it seems that the cytochrome P450system is weakly involved in digoxin biotransformation in man. Therefore, this enzymatic system may not be implicated in the overall variability of digoxin metabolism. These results showing the poor implication of P450cytochromes in DG, metabolism are of interest for a better understanding of the origin of some interactions occurring between digoxin and other drugs. For example, it is well known that co-administra-

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HUMAN L I V E R MICROSOMES N ' Fig 9. Interindividual variability of DG, metabolism by human liver microsomes. Initial drug concentration was lCk7 M.

tion of amiodarone increases digoxin serum concentrations (Moyser et al, 1982). On the other hand, Larrey et a1 (1986) showed that amiodarone administration to rats, mice or hamsters resulted in the formation of an inactive cytochrome P450-Fe(II)amiodarone metabolite complex. It was therefore tempting to speculate that this phenomenon could contribute to some known interactions between amiodarone and other drugs. In view of our results, such a hypothesis cannot be evoked to explain the digoxin-amiodarone interaction. In conclusion, cytochrome P,50 isozymes play a minor role in in vitro DG, biotransformation. It is therefore reasonable to consider that they are not involved in metabolic interindividual variability. Two main factors may finally contribute to the overall variability in DG, biotransformation: i) the individual intra-gastric p H ; ii) a variability i n the amount of UDPGT which glucuronidates digitalis compounds.

Acknowledgments W e thank Mr J Covo for his excellent technical assistance. The authors are also very indebted for the collaboration of the clinical staff of the organ transplantation units (Pr Rampal, Pr Bri-

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cot and Pr Di Marino). This work was carried out in the framework of contract No BAP 275 F(CD) of the Biotechnology Action Programme of the Commission of the European Communities.

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