Influence of Gastric Acidity on the Bioavailability of Digoxin Adam F. Cohen, MD, PhD; Ria Kroon, BSc: Rik Schoemaker, MSc; Hans Hoogkamer, MSc; and Anja van Vliet, SRN
• Objective: To study how changes in gastric acidity induced by omeprazole and pentagastrin affect the absorption of unchanged digoxin and its hydrolytic breakdown products. • Design: Double-blind, double-dummy, randomized, crossover study. • Setting: Academic department of clinical pharmacology. • Subjects: Six healthy male volunteers. • Interventions: Subjects received digoxin, 1 mg orally, on three separate occasions: first, after pretreatment with omeprazole; second, after pretreatment with pentagastrin; and third, after "pretreatment" with placebo. • Measurements: The in-vitro decomposition of digoxin was studied using a standard dissolution test. The urinary excretion of digoxin over a 120-hour period was measured using selective high-pressure liquid chromatography (HPLC) and a polarization enzyme immunoassay (EIA). Plasma concentrations were measured at 2 hours with the EIA. • Main Results: Digoxin was rapidly released from the tablets in the in-vitro test. At acid pH, decomposition (as measured with HPLC) was rapid. Pentagastrin reduced the urinary excretion of unchanged digoxin, as measured by HPLC, from 34% to 21.4% of the dose (difference, - 1 2 . 6 % ; 95% CI, - 2 3 . 5 to - 1 . 8 ; P< 0.05), whereas omeprazole increased urinary excretion to 47.4% (difference, 13.4%; 95 CI, 2.5% to 24.4%; P < 0.05). However, such differences were not found with the nonselective polarization EIA. • Conclusions: Our data suggest that gastric acidity causes the breakdown of digoxin to products that cross-react in the assay (EIA) that is commonly used clinically. In patients with reduced gastric acidity, increased plasma concentrations of unchanged digoxin may not be detected because of limitations of the EIA, which may invalidate the quantitative use of the plasma digoxin concentration as a predictor of digoxin toxicity. Omeprazole, and presumably other gastric-acid inhibitors, may increase the bioavailability of unchanged digoxin.
I n the 1970s, after the development of a practical assay for digoxin (1), investigators showed considerable interest in the relation between the plasma digoxin concentration and clinical toxicity (2) as well as in the variable bioavailability of the drug. The studies done during this period, because they increased our knowledge and awareness of the factors influencing the pharmacokinetics of digoxin, are probably responsible for a reduced incidence of digoxin intoxication. However, the predictive value of the plasma digoxin concentration for the occurrence of toxicity has always been poor (2). The many pharmacokinetic studies of digoxin have used nonselective immunoassays almost exclusively because a chromatographic assay for measuring the plasma digoxin concentration has not been developed. Immunoassays might yield misleading results because of the presence of immunoreactive breakdown products of digoxin. It has been recognized that in some patients gut bacteria can metabolize digoxin to dihydrodigoxin (3, 4). In addition, digoxin is hydrolyzed in the stomach to its aglycone digoxigenin (5-7), with the bis-digitoxoside and monodigitoxoside as intermediaries. These hydrolysis products considerably reduce pharmacodynamic activity both in vitro (8, 9) and in vivo (10) and have a plasma half-life that is very short in humans (11). Digoxin and its breakdown products are measured by the immunoassays to a similar extent, and changes in the digoxin-metabolite ratio cannot be detected. However, selective high-pressure liquid chromatography (HPLC) can differentiate between digoxin and its breakdown products. The recent introduction of several potent inhibitors of gastric-acid secretion prompted us to investigate a possible mechanism for drug interactions with digoxin. Using a selective chromatographic assay that can measure unchanged digoxin in urine, we studied how changes in gastric acidity affect the bioavailability of digoxin. Gastric acidity was increased by pentagastrin and reduced by the K + /H + -ATPase inhibitor omeprazole (12). Methods Subjects
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Six male volunteers (21 to 27 years of age and weighing between 69 and 86 kg) were recruited for the study. They were all healthy, as ascertained by a physical examination, a 12-lead electrocardiographic evaluation, and routine biochemical and hematologic tests. Subjects gave written informed consent, and the protocol for the study was approved by the ethics committee of Leiden University Hospital.
From the Center for Human Drug Research, Leiden University Hospital, Leiden, The Netherlands. For current author addresses, see end of text.
Design The study was designed as a double-blind, randomized, three-way crossover trial with double-dummy blinding. The
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order of treatments was determined by two 3 x 3 counterbalanced Latin squares. Treatments were administered every 2 weeks. Subjects received a 1-mg dose of digoxin orally (four 0.25-mg of Lanoxin tablets with 100 mL tap water) on three separate occasions. First, subjects received pretreatment with omeprazole, 40 mg once daily at 0800 hours for 4 days preceding digoxin treatment and then 40 mg at 1 hour before drug administration; a saline infusion was given 30 minutes before digoxin administration; second, subjects received "pretreatment" with placebo and were given a pentagastrin infusion, 6 jug/kg body weight per hour for 90 minutes starting 30 minutes before digoxin administration; third, subjects received "pretreatment" with placebo and were given a saline infusion 30 minutes before digoxin administration. Subjects received the oral pretreatment regimens at home. On the day digoxin was administered, they were transported to the clinical research unit after an overnight fast. After the insertion of an intravenous cannula and application of electrocardiographic electrodes, subjects received their last omeprazole or placebo tablet. Thirty minutes later, the pentagastrin or saline infusion was started, and after another 30 minutes digoxin was administered orally. Urine was collected before treatment, at 2-hour intervals for the 10 hours after treatment, and then again at 24 hours after treatment; for the following four days, urine was collected at 24-hour intervals. The total
amount of urine was determined by weighing, and an aliquot sample was saved for analysis. Subjects returned home at the end of the study day but returned daily with their urine collections. A blood sample for determining digoxin levels was taken by venipuncture 2 hours after digoxin administration. Measurements Digoxin in urine was determined by HPLC with fluorometric detection (13). This method involves the extraction of digoxin and its metabolites by methylene chloride, followed by derivatization by 1-naphtoyl chloride using 4-dimethyl-ammonium chloride as a catalyst. Digitoxin was used as the internal standard. The method has a detection limit for digoxin of 10 ng/mL (0.013 nmol/mL) and a coefficient of variation of 3.9% at a concentration of 0.124 nmol/mL. Digoxin, its bis-digitoxoside and monodigitoxoside, and digoxigenin could be separated and detected by this procedure. The reduced metabolite dihydrodigoxin could also be detected. Plasma and urine samples were also analyzed with a polarization enzyme immunoassay (EIA) (TDx Digoxin II, Abbott Laboratories, Diagnostics Division, North Chicago, Illinois) using a standard automatic analyzer and the standard method for analyzing plasma after appropriate dilution of the urine samples. This polarization EI A uses an antibody that has a
Figure 1. Recovery of digoxin as measured by high-pressure liquid chromatography (top) and enzyme immunoassay (bottom). Individual data for all six subjects are expressed in nanomoles (nmol) of digoxin recovered in urine over a 120-hour period. The administered dose was 1280.4 nmol (1 mg). Digoxin was administered on three occasions: after pretreatment with omeprazole, after pretreatment with pentagastrin, and after placebo. 1 October 1991 • Annals of Internal Medicine • Volume 115 • Number 7 Downloaded from https://annals.org by Tulane University user on 01/18/2019
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Figure 2. Ratio of digoxin concentrations measured by enzyme immunoassay and high-pressure liquid chromatography plotted against collection time of urine. A "treatment x time interaction" was shown by analysis of variance (P = 0.002). Digoxin was administered after pentagastrin pretreatment (O); after placebo (A); and after omeprazole (•). (EIA = enzyme immunoassay; HPLC = high-pressure liquid chromatography.)
cross reactivity of 205% for digoxigenin, of 150% for the monodigitoxoside, and of 115% for the bis-digitoxoside. Urinary excretion as determined by both HPLC and EIA is expressed in nanomoles (nmol). The administered dose of 1 mg corresponds to 1280.4 nmol. For each sample, the ratio of the values measured by EIA and HPLC was calculated. This ratio was expected to be at unity when the two assays were measuring the same substance. Digoxin tablets were subjected to the standard United States Pharmacopoeia (USP) solubility test. The dissolution medium was at pH 1, and, rather than using the USP method, we used HPLC to assess digoxin concentrations. Samples were taken from the dissolution medium and immediately neutralized with borate buffer to prevent further hydrolysis. Data Analysis Urine concentrations were converted to cumulative amounts excreted using the urine volume. In addition, the plasma halflife of digoxin was estimated by plotting the excretion rate of digoxin over time. Calculations were done based on a twocompartment pharmacokinetic model using nonlinear regression. The regression analysis was carried out using the Siphar software package (Simed, Creteil, France). Statistical analysis was done using repeated-measures analysis of variance followed by paired /-tests. The two treatments were compared with placebo when significant overall treatment effects were detected by analysis of variance. P values were corrected for multiple comparisons using Dunnett t values, and 95% confidence intervals (CIs) for the difference from placebo were calculated using the t values. Calculations were carried out using SPSS/PC + statistical software (Version 3, SPSS Inc, Chicago, Illinois). Results All subjects completed the study without experiencing any important side effects. One subject accidentally received 0.75 mg of digoxin instead of 1 mg on one study day. Urinary excretion values for that occasion have been corrected. Digoxin could be detected in urine samples by HPLC (Figure 1, top). The peaks of the other hydrolyzed metabolites could not be positively identified and were generally below the detection limit of the assay. The average recovery of digoxin by HPLC after placebo for 542
the 120-hour period was 435.3 nmol (34% of the administered dose). Recovery after omeprazole increased to 575.2 nmol (difference, 139.9 nmol; CI, 9.2 to 270.6 nmol; P < 0.05), which corresponds to 48% of the administered dose. Recovery after pentagastrin decreased to 273.5 nmol (difference, - 161.8 nmol; CI - 301.0 to - 22.7 nmol; P < 0.05), which corresponds to 21% of the dose. No significant differences in the recovery of digoxin from urine were seen by EIA (Figure 1, bottom). After placebo, recovery for the 120-hour period was 564.5 nmol or 44% of the dose. After omeprazole, recovery was 544.6 nmol (difference, - 19.9 nmol; CI, - 260.5 to 220.8 nmol) or 47% of the dose. Pentagastrin induced a decrease in recovery to 367.9 nmol (difference, - 196.6 nmol; CI, - 549.6 to 156.5 nmol) or 29% of the dose. After omeprazole, the HPLC:EIA ratio was close to unity throughout the sampling period. After placebo, an increase was seen, which became much more marked after the pentagastrin infusion. The differences occurred mainly in the first 12 hours (Figure 2). The difference in the time course of this ratio between the treatments was demonstrated by a ' 'treatment x time interaction" in the analysis of variance (P = 0.002). The mean plasma concentration of digoxin at 2 hours was 2.1 ± 0.6 ng/mL after omeprazole, 2.1 ± 0.7 ng/mL after pentagastrin, and 2.6 ± 1.0 ng/mL after placebo {P > 0.05). The terminal half-life of digoxin could be satisfactorily determined from the slope of the excretion rate as plotted over time. The mean half-life of digoxin was 39.6 ± 4.1 hours after omeprazole, 38.9 ± 6.7 hours after pentagastrin, and 41.5 ± 5.9 hours after placebo (P > 0.05). After the initial complete release of digoxin from the tablets, the digoxin concentration in the medium rapidly decreased, with a corresponding increase in digoxigenin and intermediary breakdown products (Figure 3). The amount of digoxin decreased by 50% after only 20 minutes at this pH. In a separate experiment, no reduction
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in digoxin concentrations could be detected after incubation for 60 minutes at pH 6.8 (data not shown). Discussion Our study has shown the important role that gastric acidity plays in determining the bioavailability of digoxin. Omeprazole, in the relatively high dosage used, probably did produce a longlasting reduction of gastric acidity (14, 15), which persisted beyond the time digoxin was in the stomach. The hydrolysis of digoxin is only possible at pH values under 3 (16), which means that hydrolysis in the stomach must have been almost completely suppressed. This suppression was associated with an increase of almost 50% in the urinary excretion of unchanged digoxin. Conversely, hyperacidity, induced by pentagastrin while the digoxin tablets were present in the stomach, reduced the availability of digoxin by a similar amount. The availability under basal conditions took an intermediate position and showed a marked intersubject variability. Our results strongly suggest that modification of gastric acidity was the mechanism underlying the observed changes in bioavailability. Omeprazole can inhibit the oxidative liver metabolism of diazepam and phenytoin (17, 18), but such inhibition is not known to be an important mechanism for the clearance of digoxin. In addition, if the clearance of digoxin was affected by omeprazole, the change would probably have led to a change in plasma half-life, which was not detected. Finally, the half-life of omeprazole is approximately 2 hours, and this drug would only have been present in the plasma for a small part of the period during which digoxin was eliminated. Pentagastrin only increases gastric acidity, which adequately explains the reduced availability. The quantitative interpretation of the EIA results is difficult because the different metabolites cross-react with the antibody to a different extent. In our study, we
used an antibody that had a greater affinity for the hydrolyzed metabolites than for digoxin. We were unable to detect these metabolites by HPLC, possibly because of the assay's lack of sensitivity. In addition, radioactive studies (19) have shown that some of the digoxin metabolites are excreted as glucuronic acid conjugates. In a study by Magnusson and colleagues (19), recovery of unchanged digoxin over 72 hours was 37.5% of the dose, a finding that is compatible with our data. In that study, concentrations of unspecified metabolites were much higher in the early hours after drug administration, which again confirms our findings. That the increase in the EIA:HPLC ratio was abolished by omeprazole and augmented by pentagastrin in our study indicates that these metabolites were probably further breakdown products of hydrolyzed digoxin. After omeprazole pretreatment, the average EIA:HPLC ratio was at unity throughout the sample period, which could be related to the finding that no immunoreactive metabolites other than digoxin were formed under neutral conditions in the stomach. One study found evidence for extensive further metabolism of digoxigenin; in this study, digoxigenin was administered directly, and only 26% of the dose could be detected as digoxigenin 30 minutes after oral administration (20). In another study, the interaction between omeprazole and digoxin was assessed in the standard manner by immunologic measurement of plasma concentrations, and only minor effects were seen, with an average increase in maximal digoxin concentrations of 6% (21). Based on our results, this increase likely represents an underestimate. It is commonly believed that most of the absorbed dose of digoxin is excreted unchanged in the urine (22). This is evidently untrue. Digoxin undergoes extensive presystemic and systemic metabolism (23), but such metabolism can only be detected if selective assays are used. Data on the urinary clearance of digoxin, which
Figure 3. In-vitro degradation of digoxin at pH 1. A United State Pharmacopeia (USP) dissolution basket was used, and data are the mean ± SD of three separate experiments. Concentrations of digoxin and its metabolites were measured by high-pressure liquid chromatography. The horizontal dashed line indicates the theoretic concentration in the medium if all digoxin had been dissolved without decomposition, (cone = concentration.) 1 October 1991 • Annals of Internal Medicine • Volume 115 • Number 7 Downloaded from https://annals.org by Tulane University user on 01/18/2019
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were obtained with radioimmunoassays, must be considered unreliable as well if the data were obtained after oral administration of the drug (24, 25). As we and others (5) have shown, nonselective assays for the invitro measurement of the dissolution of digoxin tablets conceal the considerable instability of digoxin in acid. The clinical implications of our findings may be considerable. Although immunoassays may give a reasonable indication of unchanged digoxin at later time points, as when EIA:HPLC ratios in the urine were close to unity in our study, this may not be the case in a steady state, when some metabolites may have accumulated. Gibson and Nelson (26) found that digoxin metabolites accumulated in patients with impaired renal function (26); some of these metabolites may have retained cardioactivity, but this is not known. Taken together, these findings may invalidate the quantitative use of the plasma digoxin determination as a predictor of toxicity, especially in patients at risk for the development of intoxication. Elderly patients, in whom achlorhydria is common, might be at risk because, based on our findings after omeprazole administration, they could absorb considerably more unchanged digoxin. The plasma concentration of digoxin measured by EI A has always been a rather poor predictor of toxicity. There has been a large overlap in plasma concentrations between asymptomatic patients and patients with clinical toxicity. The overlap may be explained in part by the presence of hydrolytic cardio-inactive metabolites. The presence of dihydrodigoxin, which is formed in the gut by anaerobic bacteria, may also be a factor (3, 4). These factors give rise to severe doubts about the concept of a "therapeutic range" for digoxin. The HPLC method used in our study was not sufficiently sensitive for use in determining plasma concentrations, but adequate data can be obtained from urine studies, especially in a steady state where timing is less critical. Measurement of digoxin by rubidium-86 methods (27), which relates more closely to cardioactivity, may be preferable. We did not do full plasma pharmacokinetic studies because the chromatographic assay's lack of sensitivity prevented us from comparing HPLC results with EIA results. This emphasizes the need for a practical selective assay for measuring digoxin in plasma. Finally, the increased availability of digoxin seen after the administration of omeprazole may indicate a new mechanism for drug interactions between digoxin and antacids. Studies have found that antacids decrease the bioavailability of digoxin (28), but such results have been based on measurement with nonselective assays and may reflect a change in the relative concentrations of digoxin and its breakdown products. Although cimetidine and digoxin have not been known to interact, similar caution must be exercised. The many drug interactions that have been described for digoxin may have to be restudied. We conclude that the administration of omeprazole may increase the bioavailability of digoxin, probably through a reduction of gastric acidity. Further studies in a steady state are necessary to confirm this finding and to determine its clinical significance. Digoxin is an extensively metabolized drug, a fact that may have impor544
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tant consequences for the widespread "therapeutic monitoring" of the drug with immunoassays. Acknowledgments: The authors thank Astra BV, Rijswijk, The Netherlands, for supplying the omeprazole and the matching placebo. Requests for Reprints: Adam F. Cohen, MD, PhD, Centre for Human Drug Research, Leiden University Hospital, Building 50a, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Current Author Addresses: Dr. Cohen, Ms. Kroon, Ms. van Vliet, and Mr. Schoemaker: Centre for Human Drug Research, Leiden University Hospital, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Mr. Hoogkamer: Hoffman-La Roche Ltd., Department PKF/PD, 71/409, Grenzacherstrasse 124, CH-4002 Basel, Switzerland.
References 1. Smith TW, Haber E. Digoxin intoxication: the relationship of clinical presentation to serum digoxin concentration. J Clin Invest. 1970;49:2377-86. 2. Beller GA, Smith TW, Abelmann WH, Haber E, Hood WB J r . Digitalis intoxication. A prospective clinical study with serum level correlations. N Engl J Med. 1971;284:989-97. 3. Clark DR, Kalman SM. Dihydrodigoxin: a common metabolite of digoxin in man. Drug Metab Dispos. 1974;2:148-50. 4. Lindenbaum J, Rund DG, Butler VP Jr, Tse-Eng D, Saha JR. Inactivation of digoxin by the gut flora: reversal by antibiotic treatment. N Engl J Med. 1981;305:789-94. 5. Sonobe T, Hasumi S, Yoshino T, Kobayashi Y, Kawata H, Nagai T. Digoxin degradation in acidic dissolution medium. J Pharm Sci. 1980;69:410-2. 6. Gault H, Kalra J, Ahmed M, Kepkay D, Barrowman J. Influence of gastric pH on digoxin biotransformation. I. Intragastric hydrolysis. Clin Pharmacol Ther. 1980;27:16-21. 7. Gault H, Kalra J, Ahmed M, Kepkay D, Longerich L, Barrowman J. Influence of gastric pH on digoxin biotransformation. II. Extractable urinary metabolites. Clin Pharmacol Ther. 1981;29:181-90. 8. Marcus FI, Ryan JN, Stafford MG. The reactivity of derivatives of digoxin and digitoxin as measured by the Na-K-ATPase displacement assay and by radioimmunoassay. J Lab Clin Med. 1975;85: 610-20. 9. Mann H, Peters T. The cardioactivity of digitoxin metabolites. Eur J Pharmacol. 1971;14:204-5. 10. Bottcher H, Lullmann H, Proppe D. Comparison of digoxin with some digitoxin metabolites on cat heart lung preparation. Europ J Pharmacol. 1973;22:109-11. 11. Kuhlmann J, Abshagen U, Rietbrock N. Pharmacokinetics and metabolism of digoxigenin-mono-digitoxoside in man. Europ J Clin Pharmacol. 1974;7:87-94. 12. Clissold SP, Campoli-Richards DM. Omeprazole: A preliminary review of its pharmacodynamics and pharmacokinetic properties, and therapeutic potential in peptic ulcer disease and Zollinger-Ellison syndrome. Drugs. 1986;32:15-47. 13. Shepard TA, Hui J, Chandrasekaran A, Sams RA, Reuning RH, Robertson LW, et al. Digoxin and metabolites in urine and feces: a fluorescence derivatization—high-performance liquid chromatography technique. J Chromatogr. 1986;380:89-98. 14. Lind T, Cederberg C, Ekenved G, Haglund U, Olbe L. Effect of omeprazole—a gastric proton pump inhibitor—on pentagastrin stimulated acid secretion in man. Gut. 1983;24:270-6. 15. Prichard PJ, Yeomans ND, Mihaly GW, Jones DB, Buckle PJ, Smallwood RA, Louis WJ. Omeprazole: a study of its inhibition of gastric pH and oral pharmacokinetics after morning or evening dosage. Gastroenterology. 1985;88:64-9. 16. Gault MH, Charles JD, Sugden DL, Kepkay DC. Hydrolysis of digoxin by acid. J Pharm Pharmacol. 1977;29:27-32. 17. Gugler R, Jensen JC. Omeprazole inhibits elimination of diazepam [Letter]. Lancet. 1984; 1:969. 18. Gugler R, Jensen JC. Omeprazole inhibits oxidative drug metabolism. Studies with diazepam and phenytoin in vivo and 7-ethoxycoumarin in vitro. Gastroenterology. 1985;89:1235-41. 19. Magnusson JO, Bergdahl B, Bogentoft C, Jonsson UE, Tekenbergs L. Excretion of digoxin and its metabolites in urine after a single oral dose in healthy subjects. Biopharm Drug Dispos. 1982;3:211-8. 20. Gault H, Kalra J, Longerich L, Dawe M. Digoxigenin biotransformation. Clin Pharmacol Ther. 1982;31:695-704. 21. Oosterhuis B, Jonkman JH, Andersson T, Zuiderwijk PB, Jedema JN. Minor effect of multiple dose omeprazole on the pharmacokinetics of single oral doses of digoxin. Br J Clin Pharmacol. 1991 ;[In press]. 22. Goodman-Gilman A, Goodman LS, Rail TW, Murad F, eds. The Pharmacological Basis of Therapeutics. 7th edition. New York: Macmillan; 1985:735. 23. Gault H, Longerich LL, Loo JC, Ko PT, Fine A, Vasdev SC, et al. Digoxin biotransformation. Clin Pharmacol Ther. 1984;35:74-82.
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24. Steiness E. Renal tubular secretion of digoxin. Circulation. 1974;50: 103-7. 25. Brown DD, Dormois JC, Abraham GN, Lewis K, Dixon K. Effects of furosemide on the renal excretion of digoxin. Clin Pharmacol Ther. 1976;20:395-400. 26. Gibson TP, Nelson HA. The question of cumulation of digoxin
metabolites in renal failure. Clin Pharmacol and Therap. 1980;27: 219-23. 27. Gjerdrum K. Determination of digitalis in blood. Acta Med Scand. 1970;187:371-9. 28. Rodin SM, Johnson BF. Pharmacokinetic interactions with digoxin. Clin Pharmacokinetics. 1988;15:227-44.
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• Objective: To study how changes in gastric acidity induced by omeprazole and pentagastrin affect the absorption of unchanged digoxin and its hydrolytic breakdown products. • Design: Double-blind, double-dummy, randomized, crossover study. • Setting: Academic department of clinical pharmacology. • Subjects: Six healthy male volunteers. • Interventions: Subjects received digoxin, 1 mg orally, on three separate occasions: first, after pretreatment with omeprazole; second, after pretreatment with pentagastrin; and third, after "pretreatment" with placebo. • Measurements: The in-vitro decomposition of digoxin was studied using a standard dissolution test. The urinary excretion of digoxin over a 120-hour period was measured using selective high-pressure liquid chromatography (HPLC) and a polarization enzyme immunoassay (EIA). Plasma concentrations were measured at 2 hours with the EIA. • Main Results: Digoxin was rapidly released from the tablets in the in-vitro test. At acid pH, decomposition (as measured with HPLC) was rapid. Pentagastrin reduced the urinary excretion of unchanged digoxin, as measured by HPLC, from 34% to 21.4% of the dose (difference, - 1 2 . 6 % ; 95% CI, - 2 3 . 5 to - 1 . 8 ; P< 0.05), whereas omeprazole increased urinary excretion to 47.4% (difference, 13.4%; 95 CI, 2.5% to 24.4%; P < 0.05). However, such differences were not found with the nonselective polarization EIA. • Conclusions: Our data suggest that gastric acidity causes the breakdown of digoxin to products that cross-react in the assay (EIA) that is commonly used clinically. In patients with reduced gastric acidity, increased plasma concentrations of unchanged digoxin may not be detected because of limitations of the EIA, which may invalidate the quantitative use of the plasma digoxin concentration as a predictor of digoxin toxicity. Omeprazole, and presumably other gastric-acid inhibitors, may increase the bioavailability of unchanged digoxin.
I n the 1970s, after the development of a practical assay for digoxin (1), investigators showed considerable interest in the relation between the plasma digoxin concentration and clinical toxicity (2) as well as in the variable bioavailability of the drug. The studies done during this period, because they increased our knowledge and awareness of the factors influencing the pharmacokinetics of digoxin, are probably responsible for a reduced incidence of digoxin intoxication. However, the predictive value of the plasma digoxin concentration for the occurrence of toxicity has always been poor (2). The many pharmacokinetic studies of digoxin have used nonselective immunoassays almost exclusively because a chromatographic assay for measuring the plasma digoxin concentration has not been developed. Immunoassays might yield misleading results because of the presence of immunoreactive breakdown products of digoxin. It has been recognized that in some patients gut bacteria can metabolize digoxin to dihydrodigoxin (3, 4). In addition, digoxin is hydrolyzed in the stomach to its aglycone digoxigenin (5-7), with the bis-digitoxoside and monodigitoxoside as intermediaries. These hydrolysis products considerably reduce pharmacodynamic activity both in vitro (8, 9) and in vivo (10) and have a plasma half-life that is very short in humans (11). Digoxin and its breakdown products are measured by the immunoassays to a similar extent, and changes in the digoxin-metabolite ratio cannot be detected. However, selective high-pressure liquid chromatography (HPLC) can differentiate between digoxin and its breakdown products. The recent introduction of several potent inhibitors of gastric-acid secretion prompted us to investigate a possible mechanism for drug interactions with digoxin. Using a selective chromatographic assay that can measure unchanged digoxin in urine, we studied how changes in gastric acidity affect the bioavailability of digoxin. Gastric acidity was increased by pentagastrin and reduced by the K + /H + -ATPase inhibitor omeprazole (12). Methods Subjects
Annals of Internal Medicine. 1991;115:540-545.
Six male volunteers (21 to 27 years of age and weighing between 69 and 86 kg) were recruited for the study. They were all healthy, as ascertained by a physical examination, a 12-lead electrocardiographic evaluation, and routine biochemical and hematologic tests. Subjects gave written informed consent, and the protocol for the study was approved by the ethics committee of Leiden University Hospital.
From the Center for Human Drug Research, Leiden University Hospital, Leiden, The Netherlands. For current author addresses, see end of text.
Design The study was designed as a double-blind, randomized, three-way crossover trial with double-dummy blinding. The
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order of treatments was determined by two 3 x 3 counterbalanced Latin squares. Treatments were administered every 2 weeks. Subjects received a 1-mg dose of digoxin orally (four 0.25-mg of Lanoxin tablets with 100 mL tap water) on three separate occasions. First, subjects received pretreatment with omeprazole, 40 mg once daily at 0800 hours for 4 days preceding digoxin treatment and then 40 mg at 1 hour before drug administration; a saline infusion was given 30 minutes before digoxin administration; second, subjects received "pretreatment" with placebo and were given a pentagastrin infusion, 6 jug/kg body weight per hour for 90 minutes starting 30 minutes before digoxin administration; third, subjects received "pretreatment" with placebo and were given a saline infusion 30 minutes before digoxin administration. Subjects received the oral pretreatment regimens at home. On the day digoxin was administered, they were transported to the clinical research unit after an overnight fast. After the insertion of an intravenous cannula and application of electrocardiographic electrodes, subjects received their last omeprazole or placebo tablet. Thirty minutes later, the pentagastrin or saline infusion was started, and after another 30 minutes digoxin was administered orally. Urine was collected before treatment, at 2-hour intervals for the 10 hours after treatment, and then again at 24 hours after treatment; for the following four days, urine was collected at 24-hour intervals. The total
amount of urine was determined by weighing, and an aliquot sample was saved for analysis. Subjects returned home at the end of the study day but returned daily with their urine collections. A blood sample for determining digoxin levels was taken by venipuncture 2 hours after digoxin administration. Measurements Digoxin in urine was determined by HPLC with fluorometric detection (13). This method involves the extraction of digoxin and its metabolites by methylene chloride, followed by derivatization by 1-naphtoyl chloride using 4-dimethyl-ammonium chloride as a catalyst. Digitoxin was used as the internal standard. The method has a detection limit for digoxin of 10 ng/mL (0.013 nmol/mL) and a coefficient of variation of 3.9% at a concentration of 0.124 nmol/mL. Digoxin, its bis-digitoxoside and monodigitoxoside, and digoxigenin could be separated and detected by this procedure. The reduced metabolite dihydrodigoxin could also be detected. Plasma and urine samples were also analyzed with a polarization enzyme immunoassay (EIA) (TDx Digoxin II, Abbott Laboratories, Diagnostics Division, North Chicago, Illinois) using a standard automatic analyzer and the standard method for analyzing plasma after appropriate dilution of the urine samples. This polarization EI A uses an antibody that has a
Figure 1. Recovery of digoxin as measured by high-pressure liquid chromatography (top) and enzyme immunoassay (bottom). Individual data for all six subjects are expressed in nanomoles (nmol) of digoxin recovered in urine over a 120-hour period. The administered dose was 1280.4 nmol (1 mg). Digoxin was administered on three occasions: after pretreatment with omeprazole, after pretreatment with pentagastrin, and after placebo. 1 October 1991 • Annals of Internal Medicine • Volume 115 • Number 7 Downloaded from https://annals.org by Tulane University user on 01/18/2019
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Figure 2. Ratio of digoxin concentrations measured by enzyme immunoassay and high-pressure liquid chromatography plotted against collection time of urine. A "treatment x time interaction" was shown by analysis of variance (P = 0.002). Digoxin was administered after pentagastrin pretreatment (O); after placebo (A); and after omeprazole (•). (EIA = enzyme immunoassay; HPLC = high-pressure liquid chromatography.)
cross reactivity of 205% for digoxigenin, of 150% for the monodigitoxoside, and of 115% for the bis-digitoxoside. Urinary excretion as determined by both HPLC and EIA is expressed in nanomoles (nmol). The administered dose of 1 mg corresponds to 1280.4 nmol. For each sample, the ratio of the values measured by EIA and HPLC was calculated. This ratio was expected to be at unity when the two assays were measuring the same substance. Digoxin tablets were subjected to the standard United States Pharmacopoeia (USP) solubility test. The dissolution medium was at pH 1, and, rather than using the USP method, we used HPLC to assess digoxin concentrations. Samples were taken from the dissolution medium and immediately neutralized with borate buffer to prevent further hydrolysis. Data Analysis Urine concentrations were converted to cumulative amounts excreted using the urine volume. In addition, the plasma halflife of digoxin was estimated by plotting the excretion rate of digoxin over time. Calculations were done based on a twocompartment pharmacokinetic model using nonlinear regression. The regression analysis was carried out using the Siphar software package (Simed, Creteil, France). Statistical analysis was done using repeated-measures analysis of variance followed by paired /-tests. The two treatments were compared with placebo when significant overall treatment effects were detected by analysis of variance. P values were corrected for multiple comparisons using Dunnett t values, and 95% confidence intervals (CIs) for the difference from placebo were calculated using the t values. Calculations were carried out using SPSS/PC + statistical software (Version 3, SPSS Inc, Chicago, Illinois). Results All subjects completed the study without experiencing any important side effects. One subject accidentally received 0.75 mg of digoxin instead of 1 mg on one study day. Urinary excretion values for that occasion have been corrected. Digoxin could be detected in urine samples by HPLC (Figure 1, top). The peaks of the other hydrolyzed metabolites could not be positively identified and were generally below the detection limit of the assay. The average recovery of digoxin by HPLC after placebo for 542
the 120-hour period was 435.3 nmol (34% of the administered dose). Recovery after omeprazole increased to 575.2 nmol (difference, 139.9 nmol; CI, 9.2 to 270.6 nmol; P < 0.05), which corresponds to 48% of the administered dose. Recovery after pentagastrin decreased to 273.5 nmol (difference, - 161.8 nmol; CI - 301.0 to - 22.7 nmol; P < 0.05), which corresponds to 21% of the dose. No significant differences in the recovery of digoxin from urine were seen by EIA (Figure 1, bottom). After placebo, recovery for the 120-hour period was 564.5 nmol or 44% of the dose. After omeprazole, recovery was 544.6 nmol (difference, - 19.9 nmol; CI, - 260.5 to 220.8 nmol) or 47% of the dose. Pentagastrin induced a decrease in recovery to 367.9 nmol (difference, - 196.6 nmol; CI, - 549.6 to 156.5 nmol) or 29% of the dose. After omeprazole, the HPLC:EIA ratio was close to unity throughout the sampling period. After placebo, an increase was seen, which became much more marked after the pentagastrin infusion. The differences occurred mainly in the first 12 hours (Figure 2). The difference in the time course of this ratio between the treatments was demonstrated by a ' 'treatment x time interaction" in the analysis of variance (P = 0.002). The mean plasma concentration of digoxin at 2 hours was 2.1 ± 0.6 ng/mL after omeprazole, 2.1 ± 0.7 ng/mL after pentagastrin, and 2.6 ± 1.0 ng/mL after placebo {P > 0.05). The terminal half-life of digoxin could be satisfactorily determined from the slope of the excretion rate as plotted over time. The mean half-life of digoxin was 39.6 ± 4.1 hours after omeprazole, 38.9 ± 6.7 hours after pentagastrin, and 41.5 ± 5.9 hours after placebo (P > 0.05). After the initial complete release of digoxin from the tablets, the digoxin concentration in the medium rapidly decreased, with a corresponding increase in digoxigenin and intermediary breakdown products (Figure 3). The amount of digoxin decreased by 50% after only 20 minutes at this pH. In a separate experiment, no reduction
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in digoxin concentrations could be detected after incubation for 60 minutes at pH 6.8 (data not shown). Discussion Our study has shown the important role that gastric acidity plays in determining the bioavailability of digoxin. Omeprazole, in the relatively high dosage used, probably did produce a longlasting reduction of gastric acidity (14, 15), which persisted beyond the time digoxin was in the stomach. The hydrolysis of digoxin is only possible at pH values under 3 (16), which means that hydrolysis in the stomach must have been almost completely suppressed. This suppression was associated with an increase of almost 50% in the urinary excretion of unchanged digoxin. Conversely, hyperacidity, induced by pentagastrin while the digoxin tablets were present in the stomach, reduced the availability of digoxin by a similar amount. The availability under basal conditions took an intermediate position and showed a marked intersubject variability. Our results strongly suggest that modification of gastric acidity was the mechanism underlying the observed changes in bioavailability. Omeprazole can inhibit the oxidative liver metabolism of diazepam and phenytoin (17, 18), but such inhibition is not known to be an important mechanism for the clearance of digoxin. In addition, if the clearance of digoxin was affected by omeprazole, the change would probably have led to a change in plasma half-life, which was not detected. Finally, the half-life of omeprazole is approximately 2 hours, and this drug would only have been present in the plasma for a small part of the period during which digoxin was eliminated. Pentagastrin only increases gastric acidity, which adequately explains the reduced availability. The quantitative interpretation of the EIA results is difficult because the different metabolites cross-react with the antibody to a different extent. In our study, we
used an antibody that had a greater affinity for the hydrolyzed metabolites than for digoxin. We were unable to detect these metabolites by HPLC, possibly because of the assay's lack of sensitivity. In addition, radioactive studies (19) have shown that some of the digoxin metabolites are excreted as glucuronic acid conjugates. In a study by Magnusson and colleagues (19), recovery of unchanged digoxin over 72 hours was 37.5% of the dose, a finding that is compatible with our data. In that study, concentrations of unspecified metabolites were much higher in the early hours after drug administration, which again confirms our findings. That the increase in the EIA:HPLC ratio was abolished by omeprazole and augmented by pentagastrin in our study indicates that these metabolites were probably further breakdown products of hydrolyzed digoxin. After omeprazole pretreatment, the average EIA:HPLC ratio was at unity throughout the sample period, which could be related to the finding that no immunoreactive metabolites other than digoxin were formed under neutral conditions in the stomach. One study found evidence for extensive further metabolism of digoxigenin; in this study, digoxigenin was administered directly, and only 26% of the dose could be detected as digoxigenin 30 minutes after oral administration (20). In another study, the interaction between omeprazole and digoxin was assessed in the standard manner by immunologic measurement of plasma concentrations, and only minor effects were seen, with an average increase in maximal digoxin concentrations of 6% (21). Based on our results, this increase likely represents an underestimate. It is commonly believed that most of the absorbed dose of digoxin is excreted unchanged in the urine (22). This is evidently untrue. Digoxin undergoes extensive presystemic and systemic metabolism (23), but such metabolism can only be detected if selective assays are used. Data on the urinary clearance of digoxin, which
Figure 3. In-vitro degradation of digoxin at pH 1. A United State Pharmacopeia (USP) dissolution basket was used, and data are the mean ± SD of three separate experiments. Concentrations of digoxin and its metabolites were measured by high-pressure liquid chromatography. The horizontal dashed line indicates the theoretic concentration in the medium if all digoxin had been dissolved without decomposition, (cone = concentration.) 1 October 1991 • Annals of Internal Medicine • Volume 115 • Number 7 Downloaded from https://annals.org by Tulane University user on 01/18/2019
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were obtained with radioimmunoassays, must be considered unreliable as well if the data were obtained after oral administration of the drug (24, 25). As we and others (5) have shown, nonselective assays for the invitro measurement of the dissolution of digoxin tablets conceal the considerable instability of digoxin in acid. The clinical implications of our findings may be considerable. Although immunoassays may give a reasonable indication of unchanged digoxin at later time points, as when EIA:HPLC ratios in the urine were close to unity in our study, this may not be the case in a steady state, when some metabolites may have accumulated. Gibson and Nelson (26) found that digoxin metabolites accumulated in patients with impaired renal function (26); some of these metabolites may have retained cardioactivity, but this is not known. Taken together, these findings may invalidate the quantitative use of the plasma digoxin determination as a predictor of toxicity, especially in patients at risk for the development of intoxication. Elderly patients, in whom achlorhydria is common, might be at risk because, based on our findings after omeprazole administration, they could absorb considerably more unchanged digoxin. The plasma concentration of digoxin measured by EI A has always been a rather poor predictor of toxicity. There has been a large overlap in plasma concentrations between asymptomatic patients and patients with clinical toxicity. The overlap may be explained in part by the presence of hydrolytic cardio-inactive metabolites. The presence of dihydrodigoxin, which is formed in the gut by anaerobic bacteria, may also be a factor (3, 4). These factors give rise to severe doubts about the concept of a "therapeutic range" for digoxin. The HPLC method used in our study was not sufficiently sensitive for use in determining plasma concentrations, but adequate data can be obtained from urine studies, especially in a steady state where timing is less critical. Measurement of digoxin by rubidium-86 methods (27), which relates more closely to cardioactivity, may be preferable. We did not do full plasma pharmacokinetic studies because the chromatographic assay's lack of sensitivity prevented us from comparing HPLC results with EIA results. This emphasizes the need for a practical selective assay for measuring digoxin in plasma. Finally, the increased availability of digoxin seen after the administration of omeprazole may indicate a new mechanism for drug interactions between digoxin and antacids. Studies have found that antacids decrease the bioavailability of digoxin (28), but such results have been based on measurement with nonselective assays and may reflect a change in the relative concentrations of digoxin and its breakdown products. Although cimetidine and digoxin have not been known to interact, similar caution must be exercised. The many drug interactions that have been described for digoxin may have to be restudied. We conclude that the administration of omeprazole may increase the bioavailability of digoxin, probably through a reduction of gastric acidity. Further studies in a steady state are necessary to confirm this finding and to determine its clinical significance. Digoxin is an extensively metabolized drug, a fact that may have impor544
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tant consequences for the widespread "therapeutic monitoring" of the drug with immunoassays. Acknowledgments: The authors thank Astra BV, Rijswijk, The Netherlands, for supplying the omeprazole and the matching placebo. Requests for Reprints: Adam F. Cohen, MD, PhD, Centre for Human Drug Research, Leiden University Hospital, Building 50a, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Current Author Addresses: Dr. Cohen, Ms. Kroon, Ms. van Vliet, and Mr. Schoemaker: Centre for Human Drug Research, Leiden University Hospital, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Mr. Hoogkamer: Hoffman-La Roche Ltd., Department PKF/PD, 71/409, Grenzacherstrasse 124, CH-4002 Basel, Switzerland.
References 1. Smith TW, Haber E. Digoxin intoxication: the relationship of clinical presentation to serum digoxin concentration. J Clin Invest. 1970;49:2377-86. 2. Beller GA, Smith TW, Abelmann WH, Haber E, Hood WB J r . Digitalis intoxication. A prospective clinical study with serum level correlations. N Engl J Med. 1971;284:989-97. 3. Clark DR, Kalman SM. Dihydrodigoxin: a common metabolite of digoxin in man. Drug Metab Dispos. 1974;2:148-50. 4. Lindenbaum J, Rund DG, Butler VP Jr, Tse-Eng D, Saha JR. Inactivation of digoxin by the gut flora: reversal by antibiotic treatment. N Engl J Med. 1981;305:789-94. 5. Sonobe T, Hasumi S, Yoshino T, Kobayashi Y, Kawata H, Nagai T. Digoxin degradation in acidic dissolution medium. J Pharm Sci. 1980;69:410-2. 6. Gault H, Kalra J, Ahmed M, Kepkay D, Barrowman J. Influence of gastric pH on digoxin biotransformation. I. Intragastric hydrolysis. Clin Pharmacol Ther. 1980;27:16-21. 7. Gault H, Kalra J, Ahmed M, Kepkay D, Longerich L, Barrowman J. Influence of gastric pH on digoxin biotransformation. II. Extractable urinary metabolites. Clin Pharmacol Ther. 1981;29:181-90. 8. Marcus FI, Ryan JN, Stafford MG. The reactivity of derivatives of digoxin and digitoxin as measured by the Na-K-ATPase displacement assay and by radioimmunoassay. J Lab Clin Med. 1975;85: 610-20. 9. Mann H, Peters T. The cardioactivity of digitoxin metabolites. Eur J Pharmacol. 1971;14:204-5. 10. Bottcher H, Lullmann H, Proppe D. Comparison of digoxin with some digitoxin metabolites on cat heart lung preparation. Europ J Pharmacol. 1973;22:109-11. 11. Kuhlmann J, Abshagen U, Rietbrock N. Pharmacokinetics and metabolism of digoxigenin-mono-digitoxoside in man. Europ J Clin Pharmacol. 1974;7:87-94. 12. Clissold SP, Campoli-Richards DM. Omeprazole: A preliminary review of its pharmacodynamics and pharmacokinetic properties, and therapeutic potential in peptic ulcer disease and Zollinger-Ellison syndrome. Drugs. 1986;32:15-47. 13. Shepard TA, Hui J, Chandrasekaran A, Sams RA, Reuning RH, Robertson LW, et al. Digoxin and metabolites in urine and feces: a fluorescence derivatization—high-performance liquid chromatography technique. J Chromatogr. 1986;380:89-98. 14. Lind T, Cederberg C, Ekenved G, Haglund U, Olbe L. Effect of omeprazole—a gastric proton pump inhibitor—on pentagastrin stimulated acid secretion in man. Gut. 1983;24:270-6. 15. Prichard PJ, Yeomans ND, Mihaly GW, Jones DB, Buckle PJ, Smallwood RA, Louis WJ. Omeprazole: a study of its inhibition of gastric pH and oral pharmacokinetics after morning or evening dosage. Gastroenterology. 1985;88:64-9. 16. Gault MH, Charles JD, Sugden DL, Kepkay DC. Hydrolysis of digoxin by acid. J Pharm Pharmacol. 1977;29:27-32. 17. Gugler R, Jensen JC. Omeprazole inhibits elimination of diazepam [Letter]. Lancet. 1984; 1:969. 18. Gugler R, Jensen JC. Omeprazole inhibits oxidative drug metabolism. Studies with diazepam and phenytoin in vivo and 7-ethoxycoumarin in vitro. Gastroenterology. 1985;89:1235-41. 19. Magnusson JO, Bergdahl B, Bogentoft C, Jonsson UE, Tekenbergs L. Excretion of digoxin and its metabolites in urine after a single oral dose in healthy subjects. Biopharm Drug Dispos. 1982;3:211-8. 20. Gault H, Kalra J, Longerich L, Dawe M. Digoxigenin biotransformation. Clin Pharmacol Ther. 1982;31:695-704. 21. Oosterhuis B, Jonkman JH, Andersson T, Zuiderwijk PB, Jedema JN. Minor effect of multiple dose omeprazole on the pharmacokinetics of single oral doses of digoxin. Br J Clin Pharmacol. 1991 ;[In press]. 22. Goodman-Gilman A, Goodman LS, Rail TW, Murad F, eds. The Pharmacological Basis of Therapeutics. 7th edition. New York: Macmillan; 1985:735. 23. Gault H, Longerich LL, Loo JC, Ko PT, Fine A, Vasdev SC, et al. Digoxin biotransformation. Clin Pharmacol Ther. 1984;35:74-82.
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24. Steiness E. Renal tubular secretion of digoxin. Circulation. 1974;50: 103-7. 25. Brown DD, Dormois JC, Abraham GN, Lewis K, Dixon K. Effects of furosemide on the renal excretion of digoxin. Clin Pharmacol Ther. 1976;20:395-400. 26. Gibson TP, Nelson HA. The question of cumulation of digoxin
metabolites in renal failure. Clin Pharmacol and Therap. 1980;27: 219-23. 27. Gjerdrum K. Determination of digitalis in blood. Acta Med Scand. 1970;187:371-9. 28. Rodin SM, Johnson BF. Pharmacokinetic interactions with digoxin. Clin Pharmacokinetics. 1988;15:227-44.
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