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DRUG INTERACTIONS |
From Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey (Mr Bifano, Dr Smith, Dr Zhang, Dr Grasela, Dr LaCreta); and Novartis Pharmaceuticals Corporation, East Hanover, New Jersey (Dr Yan).
Address for correspondence: Marc Bifano, MS, Bristol-Myers Squibb, PO Box 4000, Princeton, NJ 08543-4000.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: Entecavir • adefovir • pharmacokinetics • drug interactions • hepatitis B virus
Although entecavir and adefovir are both marketed as stand-alone therapy for hepatitis B infection, coadministration of both a nucleoside analog and a nucleotide analog may result in an unfavorable pharmacokinetic interaction because both entecavir and adefovir are excreted by tubular secretion and glomerular filtration. The potential for an interaction may lie in the nucleoside transporter system, a highly specialized family of transport proteins that mediates the cellular uptake and salvage of nucleosides, an example being those that have been described for the purine nucleoside analog, adenosine.7 Nucleoside transporters are expressed by a wide variety of healthy mammalian tissues. There is evidence suggesting that renal tubular epithelial cells express a similar transport system.8-10 Nucleoside transporters function either by transporting their substrates bidirectionally across a biological membrane (equilibrative transporters) or by accumulating compounds in the cellular cytoplasm (concentrative transporters).9 Equilibrative transport mechanisms have been primarily observed in basolateral membrane vesicle preparations, whereas concentrative mechanisms have been observed in apical membrane vesicles, suggesting that the effects of these transporters in humans are primarily in the direction of reabsorption (accumulation).11
Therefore, a study to assess the potential for a pharmacokinetic interaction between entecavir and adefovir was warranted. The objectives of the current study were to assess the effect of adefovir on the pharmacokinetics of entecavir and to assess the effect of entecavir on the pharmacokinetics of adefovir in healthy adult subjects. In addition, the study evaluated the safety and tolerability of the combination of these 2 agents in healthy adult subjects.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Study Design
This was an open-label, fixed-sequence crossover design study in healthy adult subjects. Subjects were admitted to the clinical unit on day -1. Fasting subjects received 10 mg of adefovir once daily (QD) on days 1 to 4, 1 mg of entecavir QD on days 5 to 14, and 1 mg of entecavir QD plus 10 mg of adefovir QD on days 15 to 24. Study medication was administered at the same time each morning at approximately 8 AM. Subjects were discharged from the study on day 25 after completing discharge procedures. Seven-milliliter blood samples (predose, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, and 24 hours postdose) were collected on days 4 and 24 for adefovir, and 5-mL blood samples were collected on days 14 and 24 for entecavir. Trough blood samples were also collected immediately before dosing on the mornings of days 3, 4, 5, 6, 9, 10, 11, 12, and 13. Each blood sample was collected into a labeled tube containing tripotassium EDTA as the anticoagulant. Urine (24-hour collection) was collected on days 4 to 5, 14 to 15, and 24 to 25 for the analysis of entecavir and/or adefovir. Urine predose "spot" samples were collected on days 1 and 5 for adefovir and entecavir, respectively. Within 60 minutes of collection, blood samples were centrifuged, and the plasma was transferred to screw-cap polypropylene tubes and stored at -20°C until assayed. One 10- to 14-mL aliquot of each urine sample was transferred to labeled screw-capped polypropylene tubes and stored at -20°C until analysis. Stability of entecavir was tested in the plasma and urine samples under the storage condition used in the study. Entecavir was found to be stable in plasma and urine during storage at -20°C for the entire storage period and following 3 freezethaw cycles.
The subjects were not permitted to consume alcohol-containing beverages or grapefruit-containing products within 3 days of dosing and until discharge from the study. In addition, they were not permitted to take any medications (including over-the-counter medications and complementary and alternative medications), smoke, or engage in strenuous exercise, contact sports, or sunbathing for the duration of the study.
The study protocol was approved by the Independent Investigational Institutional Review Board Inc and conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Freely given written informed consent was obtained from every subject prior to clinical study participation, including informed consent for any screening procedures conducted to establish subject eligibility for the study.
Biological Specimen Analysis
Plasma and urine samples from the study were assayed for entecavir at Advion Biosciences (Ithaca, NY) using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method. Plasma samples (0.5 mL each) were processed using an Oasis HLB 96-well extraction plate (30 mg, Waters, Milford, Mass). The dried extracts were reconstituted with 100 µL of 0.1% formic acid in water and centrifuged before sample injection. Chromatographic separation was achieved with a gradient elution on a Waters Xterra MS C18 analytical column (2.1 x 50 mm, 5 µ, Waters). The mobile phases contained 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The following gradient was used with a flow rate of 300 µL/min: linearly increase B from 0 to 100% from 0 to 2.5 minutes, hold B at 100% for 1 minute, and ramp to 0% B in 0.1 minute with a total run time of 5 minutes. The injection volume was 10 µL. The analytes were detected by positive ion electrospray tandem mass spectrometry on a Sciex API 4000 LC/MS instrument (Applied Biosystems Inc, Foster City, Calif). The selected reaction monitoring transitions were m/z 278 > m/z 152 for entecavir and m/z 266 > m/z 152 for lobucavir (internal standard). The standard curves, which ranged from 0.010 to 25 ng/mL for entecavir, were fitted to a 1/x weighted quadratic regression model. Plasma samples were analyzed for entecavir in a total of 7 analytical runs. Values for between-run and within-run precision for analytical quality control samples were no greater than 7.6% coefficient of variation (CV), with deviations from the nominal concentrations of no more than ±3.6%.
Urine samples for entecavir (0.5 mL each) were processed using a solid-phase extraction procedure similar to that described above for plasma. The dried residues were reconstituted with 250 µL of 0.1% formic acid in water and centrifuged before injection of 10 µL of sample. The high-performance liquid chromatography and mass spectrometric conditions were the same as those of the plasma assay described above. The standard curves, which ranged from 0.25 to 500 ng/mL for entecavir, were fitted to a 1/x weighted quadratic regression model. Urine samples were analyzed for entecavir in 2 analytical runs. Values for the between-run and within-run precision for analytical quality control samples were no greater than 5.3% CV, with deviations from the nominal concentrations of no more than ±2.2%.
Plasma and urine samples from the study were analyzed for adefovir by Covance Bioanalytical Services LLC (Indianapolis, Ind) using a validated LC/MS/MS method. Briefly, plasma samples, 100 µL each, were processed by protein precipitation after the samples were first treated with acid. After evaporation under nitrogen flow, the residue was reconstituted and analyzed by LC/MS/MS. Chromatographic separation was achieved with a gradient elution on a BioBasic AX analytical column (3 x 50 mm, 5 µ, Thermo Hypersil-Keystone, Thermo Scientific, Bellefonte, Pa). The mobile phases were 10 mM ammonium acetate in acetonitrile/water (30:70, v:v, pH 6.0) as mobile phase A and 10 mM ammonium acetate in acetronitrile/water (30:70, v:v, pH 9.5) as mobile phase B. The following gradient was used: B at 15% from 0 to 1.0 minute, linearly increase B from 15% to 100% from 1.0 to 2.6 minutes, hold B at 100% for 1.4 minutes, and ramp to 15% B in 0.01 minute with a total run time of 5.1 minutes. The flow rate was 1.0 mL/min except for 4.01 minutes to 5.0 minutes, where the flow rate was 1.5 mL/min. The injection volume was 10 µL. The analytes were detected by positive ion electrospray tandem mass spectrometry on a Sciex API 3000 LC/MS instrument. The multiple reaction monitoring transitions were m/z 274 > m/z 162 for adefovir and m/z 280 > m/z 112 for cidofovir (internal standard). Plasma samples were analyzed for adefovir in a total of 14 analytical runs. The standard curves, which ranged from 1.00 to 200 ng/mL, were well fitted by a 1/x2-weighted linear regression model. Values for the between-run and within-run precision for analytical quality control samples were no greater than 11.3% CV, with deviations from the nominal concentrations of no more than ±1.7%.
For the human urine assay, adefovir along with the internal standard, cidofovir, were processed using dilution and filtration. After addition of internal standard to 50 µL of urine, the samples were further diluted with mobile phase resulting in a dilution factor of 10-fold. The diluted samples were then filtered using a plate filter to remove particulates for preparation for LC/MS/MS analysis. The chromatographic and mass spectrometric conditions were identical to those of the plasma assay as described above except for variations on the gradient method as noted below. The gradient used was 15% B from 0 to 0.5 minutes, linearly increase B from 15% to 90% from 0.5 to 2.5 minutes, hold B at 90% for 0.5 minutes, and ramp to 15% B in 0.01 minute with a total run time of 4.5 minutes. The flow rate was 1.0 mL/min except for the time interval from 3.01 to 4.25 minutes, when the flow rate was 1.5 mL/min. Urine samples were analyzed in 2 analytical runs. The standard curves were fitted by a 1/x2-weighted linear equation over the concentration range of 50.0 to 5000 ng/mL. Values for the between-run and within-run precision for analytical quality control samples were no greater than 2.8% CV, with deviations from the nominal concentrations of no more than ±1.3%.
Pharmacokinetic Analysis
The plasma concentration-time data for entecavir and adefovir were analyzed by noncompartmental methods.12,13 The peak plasma concentration, Cmax, the 24-hour postdose plasma concentration, Cmin, and the time to reach the peak concentration, Tmax, were obtained from experimental observations. The area under the plasma concentration-time curve in 1 dosing interval, AUC
, was determined by summing the areas from time zero to the time of last measured concentration of the interval, calculated by using a trapezoidal method. The amount of entecavir and adefovir excreted in urine during each collection interval was calculated by multiplying the concentration by the volume of urine collected over that interval. The total urinary recovery (UR) was calculated as the cumulative amount excreted over 1 dosing interval [UR(0-T)] and expressed as a percentage of the administered dose (%UR). Renal clearance was estimated by dividing UR(0-T) by AUC(0-T), where T = 24 hours for both entecavir and adefovir.
Safety and Tolerability Assessments
Safety assessments were based on medical review of adverse event reports and the results of vital sign measurements (body temperature, respiratory rate, seated blood pressure, and heart rate), electrocardiograms, physical examinations, and clinical laboratory tests. Vital signs were recorded at screening, prior to dosing on day 1, and at study discharge on day 25. Twelve-lead electrocardiograms were recorded at screening and at discharge from the study on day 25. Physical examinations were performed at screening, prior to dosing on day 1, and at discharge from the study on day 25. Blood and urine samples for clinical laboratory testing were collected at screening, prior to dosing on days 1, 5, and 15, and at discharge from the study on day 25. Subjects were closely monitored for adverse events from the time of enrollment until discharge from the study. Adverse events included any illness, sign, symptom, or clinically significant laboratory test abnormality that appeared or worsened during the course of the study regardless of any causal relationship to the study drug.
Statistical Analysis
To assess the effect of concomitant administration of adefovir on the multiple-dose pharmacokinetics of entecavir, an analysis of variance was performed on entecavir log (Cmax), log (AUC), and log (Cmin). The factors in the analysis were subject and treatment (1 mg of entecavir alone or 1 mg of entecavir with 10 mg of adefovir). Point estimates and 90% confidence intervals (CIs) for treatment differences in Cmax, AUC, and Cmin on the log scale were exponentiated to obtain estimates for geometric means and ratios of geometric means on the original scale. Absence of an effect of coadministration of adefovir on entecavir Cmax, AUC, and Cmin was concluded if the 90% CIs for the ratios of population geometric means with and without adefovir were contained within 80% to 125% for entecavir Cmax, AUC, and Cmin. The data analysis performed to assess the effect of concomitant administration of entecavir on adefovir was identical to that outlined above.
Summary statistics were presented by treatment for all pharmacokinetic parameters of all analytes. Geometric means and CVs were reported for Cmax, AUC, and Cmin. Medians and ranges were presented for Tmax. Means and SDs were provided for %UR and renal clearance. No adjustment was made for multiplicity. All statistical analyses were carried out using SAS/STAT® (version 8.2, SAS Institute Inc, Cary, NC).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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, Cmax, and Cmin of entecavir are summarized in Table I. There was no statistically significant effect of coadministration of adefovir on entecavir pharmacokinetics. The geometric means for entecavir AUC, Cmax, and Cmin on day 24 were only 3%, 7%, and 8% higher than those on day 14, respectively. The 90% CIs for day 24 versus day 14 AUC, Cmax, and Cmin ratios were well within the prespecified no-effect interval of 0.80 to 1.25.
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Adefovir Pharmacokinetics
The mean plasma concentration-time profiles of adefovir in subjects on days 4 and 24 are depicted in Figure 2. The adefovir concentration-time profile appeared to be similar when adefovir was administered in combination with entecavir or alone. Summary statistics for adefovir pharmacokinetic parameters for days 4 and 24 and the statistical analysis for AUC, Cmax, and Cmin for adefovir are summarized in Table II. There was no statistically significant effect of coadministration of entecavir on adefovir pharmacokinetics. The geometric means for adefovir AUC, Cmax, and Cmin on day 24 were only 3% and 14% lower and 5% higher than those on day 4, respectively. The 90% CIs for day 24 versus day 4 AUC, Cmax, and Cmin ratios were within the prespecified no-effect interval of 0.80 to 1.25.
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Safety and Tolerability
There were no serious adverse events (AEs) or deaths in this study. Safety data were available for all 26 enrolled subjects, with 22 subjects completing the study. Overall, 16 AEs were reported for 12 of the 26 subjects who received entecavir alone, 10 AEs were reported for 8 of the 26 subjects who received adefovir alone, and 7 AEs were reported for 6 of the 23 subjects who received entecavir plus adefovir. Most AEs (26/33, 79%) were mild in intensity; the remaining 7 AEs (21%) were moderate in intensity. Of the 33 AEs, the investigator considered the relationship of the AE to study drug to be unrelated for 14 AEs (42%), not likely for 10 AEs (30%), possible for 3 AEs (9%), probable for 3 AEs (9%), and certain for 3 AEs (9%). All AEs resolved before discharge from the study.
The most frequently reported AE was headache, which was reported in 4 subjects while receiving adefovir alone and in 9 subjects receiving entecavir alone; no headaches were reported after concomitant administration of the 2 drugs. Other frequently reported AEs included dizziness, which occurred in 3 subjects while receiving adefovir alone, and dysmenorrhea, which occurred in 2 subjects while receiving entecavir alone. All other AEs were reported in no more than 1 subject per treatment group. Overall, the incidence of AEs following entecavir alone (16 AEs) was greater than that following adefovir alone (10 AEs) and concomitant entecavir and adefovir (7AEs). These results suggest that the administration of adefovir concomitantly with entecavir does not adversely affect the safety profile of entecavir or adefovir.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Entecavir renal tubular secretion may be mediated by nucleoside transporters, whereas adefovir renal tubular secretion seems to occur via a combination of organic anion transporters, P-gp, and possibly nucleoside transporters as well. It is also possible that the systemic concentration of each drug is not high enough to inhibit the transport of the other drug. It has been shown that adefovir is efficiently transported by human renal OAT 1 (hOAT1), a membrane transport protein localized in the kidney, that presumably mediates the accumulation of adefovir in renal proximal tubules.15 It has also been reported that this hOAT1-mediated accumulation of adefovir plays an active role in the mechanism of nephrotoxicity caused by adefovir administration.16 Entecavir renal secretion is not anticipated to be mediated by organic anion or cation transporters because it is a neutral compound under physiological pH conditions. In addition, entecavir is not transported by P-gp; therefore, an interaction at P-gp would not be anticipated. It is likely that the lack of a drug-drug interaction between entecavir and adefovir is attributable to a combination of these factors.
In addition to the lack of a pharmacokinetic interaction between adefovir and entecavir shown in this study, there is also evidence suggesting that a pharmacodynamic interaction between the 2 drugs is unlikely. In an in vitro study, the anti-HBV activity of entecavir was not antagonized by the coadministration of the nucleoside reverse transcriptase inhibitors (NRTIs) stavudine, didanosine, abacavir, zidovudine, lamivudine, and tenofovir.17 The lack of antagonism was seen at concentrations of the selected NRTIs meeting or exceeding the maximum plasma concentration likely to be seen in human subjects given those NRTIs for the treatment of HBV and/or HIV infections.18 Like NRTIs in general, inhibition of viral replication by entecavir occurs through its phosphorylation to the triphosphate form and subsequent binding to the viral reverse transcriptase, preventing viral DNA synthesis. Although adefovir was not included in the in vitro evaluation, no interaction was observed with tenofovir, which is a nucleotide like adefovir. In addition, no interaction was observed with didanosine and entecavir, which are both purine nucleosides.
These results also suggest that the administration of adefovir concomitantly with entecavir does not adversely affect the adverse event profile of entecavir or adefovir. The higher incidence of AEs with entecavir may be attributable to the longer duration of that treatment segment, which resulted in AEs being collected for a longer period of time (4 days for treatment with adefovir alone vs 10 days for treatment with entecavir alone). On an AE per day basis, the incidence of AEs was comparable for both treatment groups. It was interesting to note that when entecavir and adefovir were coadministered, the incidence of headache was less than when either drug was administered alone (no events vs 4 and 9 events for adefovir and entecavir, respectively).
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CONCLUSION |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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Financial disclosure: Mr Bifano, Dr Smith, Dr Zhang, Dr Grasela, and Dr LaCreta are employees and stockholders of Bristol-Myers Squibb.
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REFERENCES |
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12. Gibaldi M. Non-compartmental analysis based on statistical moment theory. In: Gibaldi M, Perrier D, eds. Pharmacokinetics. 2nd ed. New York, NY: Marcel Dekker; 1982: 409-418.
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15. Mulato AS, Ho ES, Cihlar T, et al. Nonsteroidal anti-inflammatory drugs efficiently reduce the transport and cytotoxicity of adefovir medicated by the human renal organic anion transporter 1. J Pharmacol Exp Ther. 2000;295: 10-15.
16. Gihlar T, Lin DC, Pritchard JB, et al. The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1. Mol Pharmacol. 1999; 56: 570-580.
17. Colonno R, Discotto L, Gong Y-F, Kimmel B, Weinheimer S. Combination studies involving entecavir and HIV nucleoside reverse transcriptase inhibitors showed no antagonistic effects on HBV or HIV activity in vitro. Paper presented at: 39th Annual Meeting of the European Association for the Study of the Liver; April 14-18, 2004; Berlin, Germany.
18. Wolf DL, Rodriguez CA, Mucci M, Ingrosso A, Duncan BA, Nickens DJ. Pharmacokinetics and renal effects with a reduced dose of probenicid in HIV-infected patients with cytomegalovirus retinitis. J Clin Pharmacol. 2003;43: 43-51.
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