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PHARMACOKINETICS |
From the Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland (Dr Ndovi, Dr Cao, Mr Fuchs, Ms Guidos, Dr Hendrix), and the Department of Clinical Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado (Dr Fletcher).
Address for correspondence: Craig W. Hendrix, MD, Harvey 502, 600 North Wolfe Street, Baltimore, MD 21287.
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ABSTRACT |
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Key Words: Zidovudine • food effect • HIV • hepatic blood flow • pharmacokinetics
During a study in which we dosed the antiretroviral drug zidovudine (ZDV) by continuous infusion for 5 days to maintain steady-state blood levels,6,7 we observed a reduction in ZDV blood plasma concentration several hours after a meal. It has been reported that eating a meal before oral ZDV dosing results in decreased peak concentration8-10 and prolonged time to maximum concentration (Tmax),8,9 lag time, and terminal half-life,8 although these results were not seen by other researchers.10 Because the changes that we observed were with intravenous administration, they could not be explained by food-induced changes in absorption. Rather, we reasoned that a food-induced increase of hepatic blood flow11 was the cause because ZDV is primarily metabolized by uridine-5'-diphospho-glucuronosyltransferase 2B7 into 5'-glucuronyl zidovudine (GZDV) in the liver12 and has an estimated hepatic extraction ratio of 0.62,13 making the metabolism sensitive to hepatic blood flow. To formally test the hypothesis that a meal decreases ZDV systemic exposure independent of an effect on drug absorption, we dosed 7 healthy male volunteers with ZDV via continuous intravenous infusion (as a nested substudy of a larger protocol) and evaluated the blood plasma concentrations of both the parent drug (ZDV) and the metabolite (GZDV) for two 8-hour periods, one following a standardized meal and the other in the fasting state.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Subjects
Written informed consent was obtained from all subjects prior to screening. Healthy adult male subjects free of active medical conditions were recruited into the study. The Johns Hopkins Medicine Institutional Review Board approved the study.
Study Design
Subjects were admitted to the inpatient General Clinical Research Center at The Johns Hopkins Hospital on the day prior to dosing (day 0). Subjects were not allowed to use any medications while in the study unless deemed necessary by the study investigators. Subjects received ZDV (10 mg/mL water; Retrovir) by continuous infusion at a rate of approximately 10 mg/h via portable programmable infusion pumps beginning at 8 AM of day 1 continuing through 11 AM of day 5. The target ZDV concentration of 0.14 mg/mL was selected so that steady-state concentrations in plasma exceeded the assay limit of quantitation and, for reasons of safety in these healthy subjects, fall below the pseudo steady-state concentrations associated with oral dosing. (The 5-day infusion was part of a larger study with different objectives within which we nested this food effect substudy.) The effect of food on pharmacokinetics was tested on day 2 (fed) and day 3 (fast). During these 2 days, subjects were on bed rest, with activity limited to bathroom privileges from 5:30 AM until after the 4:00 PM blood draw.
Subjects maintained a fast after midnight of day 1. The following morning, day 2, the baseline blood sample for the fed observation period was drawn at 7:55 AM. At 8:00 AM, the subject was served a study-specific breakfast, consisting of 2 eggs, 2 slices of bacon, 2 slices of white toast, 16 oz of orange juice, 2 pats of margarine, and salt and pepper for taste. The subject consumed the breakfast within 15 minutes and was then restricted to nothing by mouth until the 8-hour blood samples were completed. The blood samples were collected at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, and 8 hours after the start of the breakfast. Subjects received an early dinner at 4:00 PM (after the 8-hour sample) and a snack at 8:00 PM. Subjects were then fasted after midnight until 4:00 PM of day 3. On day 3, the protocol followed exactly as on day 2, except that no breakfast was served.
All blood samples were collected in a 7-mL heparinized green-top Vacutainer tube through an indwelling cannula in the arm contralateral to the ZDV infusion. Blood samples were delivered to the processing laboratory on ice immediately after being drawn. The samples were centrifuged at 1500g for 10 minutes at 4°C. The plasma was collected and stored at -70°C for later analysis by high-performance liquid chromatography (HPLC).
ZDV and GZDV Assays
A validated, reversed-phase HPLC assay was used for quantitation of ZDV and GZDV in human plasma. The HPLC system was composed of a Waters Alliance 2690 separations module with a Waters 2487 dual-wavelength ultraviolet (UV) absorbance detector (Waters Corporation, Milford, Massachusetts). After addition of internal standards, dideoxyuridine, and 8-azidoadenosine, a solid-phase extraction (SPE) extraction procedure with a Waters OASIS HLB cartridge (30 mg, 1 mL, Waters Corporation) was used to prepare the samples. The chromatographic separation was performed on a Waters YMC HPLC, 3.0 x 100 mm, reversed-phase octyl column with a particle size of 3 µm. The mobile phase consisted of a gradient mixture (by volume) that ranged from 1.3% to 8.0% acetonitrile and 98.7% to 92.0% 20 mM ammonium formate buffer (pH 3.80) at a flow rate of 0.5 mL/min. Detection and quantification of ZDV was achieved by UV detection at 266 nm. The assay is linear in the range of 25 ng/mL to 10 000 ng/mL, with a minimum quantifiable lower limit of quantitation (LLOQ) of 25 ng/mL using 0.20 mL of human plasma. Interday and intraday accuracy and precision were within ±20% at the LLOQ and ±15% at all other concentrations.
For quantitation of GZDV, heparinized plasma was subjected to an SPE extraction with Waters OASIS MAX (3 mL-60 mg) SPE cartridges. The chromatographic separation was performed on a Phenomenex Synergi, 4.6 x 250 mm, polar-RP column with a particle size of 4 µm. The isocratic mobile phase consists of 9% acetonitrile:91% 20 mM ammonium formate buffer (pH 3.80). Detection and quantitation of GZDV was achieved by UV detection at 270 nm at approximately an 8-minute retention time. The assay is linear in the range of 25 ng/mL to 10 000 ng/mL and has an LLOQ of 25 ng/mL using 0.200 mL of human plasma. Interday and intraday accuracy and precision were within ±20% at the LLOQ and ±15% at all other concentrations. This method is validated to quantify GZDV from heparinized or EDTA patient plasma based on EDTA plasma standard curves. The performance is equivalent to the specifications outlined above.
Data Analysis
For analysis purposes, the pharmacokinetic time clock was set to 0 when the subject started the study-specific breakfast on day 2 (fed; around 8:00 AM). The time clock was reset to 0 at the corresponding time on day 3 (fast). The concentrations of ZDV and GZDV and the GZDV-to-ZDV molar concentration ratios were summarized for each pharmacokinetic time point after being logarithmically transformed and calculated with the following equation:
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where X represents either [ZDV], [GZDV], or the GZDV-to-ZDV concentration ratio. 
is the mean of the natural log of X, and SD indicates the standard deviation of 's.
The effect of food was determined by comparing the time course of ZDV and its metabolite GZDV between fed and fasted periods. To minimize the potential effect of circadian rhythm on distribution, glucuronidation, and excretion, the concentrations of ZDV and GZDV at the same time of the day on both pharmacokinetic days were compared. The potential food-induced changes were calculated for a given pharmacokinetic time point with the following equation:
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where X represents [ZDV], [GZDV], and the GZDV-to-ZDV molar concentration ratio, clearance estimate, and cumulated area under the concentration-time curve (AUC); 
represents the mean of the natural log of the ratio of X in fed status to X in the fasted status; and SD indicates the standard deviation of 's. Clearance estimates were calculated by dividing the ZDV infusion rate by the average of ZDV concentrations at the beginning and the end of a sampling interval. The cumulative AUC from 0.5-hour to each subsequent sampling time was calculated using the linear-trapezoidal method. The initial 30-minute period was excluded from the cumulative AUC calculation as the subjects were eating breakfast throughout this 30-minute period, and their body positions and activities were not well controlled, which might have resulted in an observed larger variation of AUC0-30min. A Wilcoxon signed rank test (SPSS version 9.0.1; SPSS Inc, Chicago, Illinois) on logarithmically transformed data was used for hypothesis testing wherever a statistical significance was stated and the test statistic agreed with the inference from the confidence interval. The multiple comparisons were not adjusted because the observations were not independent.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The GZDV concentrations were more variable than the ZDV concentrations (Figure 2A). Two subjects appeared to have distinctly higher GZDV concentrations when compared with the other 5 subjects. The mean GZDV concentration decreased by about 6.0% postprandial, although this was not statistically significant except at the 4-hour point (Figure 2B). The overall time course of the GZDV downward trend was similar to that of ZDV for both concentration and cumulative AUC (Figure 2C).
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The median molar ratio of GZDV to ZDV concentrations was 1.6 during the fed period and 1.5 during the fasting period (Figure 3A). There was no statistically significant difference in molar ratio between the fasted and fed states (Figure 3B).
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DISCUSSION |
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In HIV-infected patients, 18% of intravenous ZDV is excreted unchanged; 60% is excreted as GZDV in urine.14 A small amount of intravenously administered ZDV can also be metabolized into 3'-amino-3'-deoxythymidine.14 Oral ZDV clearance was reduced by two-thirds in patients with Child-Pugh scores of 6 to 1215 and by >50% in patients with creatinine clearance in the range of 6 to 31 mL/min.16 The kidney may conjugate ZDV with glucuronide also17; however, the glucuronidation of ZDV occurs primarily in the liver.
On average, the systemic and renal clearance of ZDV following intravenous administration was 1.6 L/h/kg and 0.34 L/h/kg, respectively.14 Thus, the nonrenal clearance of ZDV is about 1.26 L/h/kg, which is close to the estimated hepatic blood flow (about 1.2 L/h/kg) for healthy adults.18 Assuming that the hepatic metabolism can be described with the well-stirred model, the metabolism of ZDV is perfusion-rate limited. Because metabolism is the major clearance mechanism of ZDV, factors affecting hepatic blood flow will affect the systematic clearance and the steady-state concentration of intravenously administered ZDV.
Food increases hepatic blood flow. In one study, the hepatic flow estimated with indocyanine green was reported to be increased by 69% 40 minutes following a high-protein meal.11 The flow level did not come back to baseline until 4.7 hours postprandial. If similar hepatic blood flow change occurred in this study, the breakfast would decrease ZDV concentration up to 35%. Thus, a 14% decrease seen 1 hour postprandial would be reasonable. The insignificant change of ZDV concentration at 1.5 hours postprandial was unexpected. It is not clear whether ZDV and/or its metabolites undergo enterohepatic recirculation and, if so, whether a food-induced increase in portal flow could transiently increase the reabsorption of ZDV and/or its metabolites excreted via bile, thus briefly negating the ZDV reduction because of increased hepatic clearance.
Food might increase renal blood flow as well. Using inulin and para-aminohippurate, Simon et al19 conducted a crossover randomized study in normal subjects and found that food increased renal blood flow at 1, 2, and 3 hours after the meal. The peak increase, occurring at 2 hours postprandial, was about 20% for both glomerular filtration rate and renal plasma flow. Because only 18% of ZDV appears in urine unchanged and because the renal extraction ratio of ZDV is likely low in healthy volunteers, we think that the renal contribution to the decreased ZDV concentration was small. Further study would be necessary to quantify the potential effect of the meal-induced change in renal blood flow on the glucuronidation of ZDV in kidney or the excretion of ZDV and GZDV.
Circadian change has been observed in enzyme activity, regional blood flow, drug protein binding, gastric function, and drug penetration.20 One study examined the hepatic blood flow in healthy subjects after a 6-hour fast in the supine position on 2 consecutive days (at 8 AM and 8 PM on day 1 and at 2 AM and 2 PM on day 2). It was found that hepatic blood flow was highest at 8 AM.21 Our study compared the drug concentration at the same time of day and should have minimized the influence of circadian rhythm.
The molar ratio of GZDV concentration to ZDV in our study, 1.6, is higher than 1.2 found in children on continuous infusion,22 suggesting either a stronger glucuronidation in adults or just large variation as is commonly seen with this drug. Because the half-lives of terminal elimination of GZDV and ZDV are similar,22,23 GZDV follows a formation rate-limited disposition with a distribution volume smaller than ZDV. In other words, GZDV itself is cleared faster than ZDV, resulting in a GZDV concentration-time profile similar to that of ZDV at steady state. Our data indicate that the GZDV-ZDV ratio did not change after the meal, even though both GZDV and ZDV decreased, which could happen when the ZDV metabolism to GZDV increased. Consider the following relationship for formation clearance24:
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where fm is the fraction of GZDV converted to ZDV in the liver. Because the disposition of GZDV is dependent on the rate of its formation, increasing CLZDV (and fm) results in the increase of GZDV formation and thus the increase of CLGZDV. Therefore, AUCGZDV may be proportional to AUCZDV, and the molar ratios of GZDV to ZDV may be constant even if the metabolism of ZDV to GZDV is enhanced.
Because our end points were blood ZDV and GZDV concentrations, which were relatively objective, and because the study was nested in a larger study with a limited number of subjects, we chose a single-sequence, 2-period design instead of randomized, concurrent control, double-masked, parallel-groups design. The known factors affecting liver blood flow such as body position and daily activities were well controlled, and we think that this design is an efficient way to assess the food effect. However, because the study was not randomized, unknown factors might have contributed to the observed food-induced effect. Such factors may include the selection of study population and tobacco and medication use of subjects before enrollment. Thus, caution is needed when the food effect observed in our study is extrapolated to other subjects. In addition, because the fed period preceded the fasting period for each subject, we could not test for sequence effects. In addition to study design, the assay variation of ZDV and GZDV may make the potential underlying true effect difficult to detect.
In summary, we have confirmed that food decreases the concentration of ZDV in blood plasma independent of any effect on absorption. The time course of the change and the parallel decrease in GZDV concentration suggests that the food-induced increase in hepatic blood flow and thus hepatic metabolism is likely the underlying mechanism. Because intravenous ZDV is currently used for intrapartum prophylaxis of mother-to-child HIV transmission, feeding may affect the blood ZDV level in this clinical setting, although the magnitude of the food effect is small and absorption enhancement is dominant with the clinically relevant oral ZDV dosing. Our study provides an example of an important physiological effect relating food, increased hepatic blood flow, and enhanced metabolism of a high-extraction drug. This food effect may have a clinically relevant impact in other settings where drugs with a high hepatic extraction ratio are administered parenterally when food may also be consumed. Common examples may include intravenous morphine or fentanyl when used for patient-controlled analgesia or inhaled nicotine. In these and similar settings, food consumption may significantly lower drug levels and reduce pharmacologic effects.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Financial disclosure: This work was supported in part by a Mid-career Investigator Award in Patient-Oriented Research (NIH K24 AI01825), the ASCPT Young Investigator Award, General Clinical Research Center (NIH M01RR000052-430919, 5M01RR000052-430852), HIV Prevention Trials Network Central Laboratory (NIH U01 AI46745 and AI068613), and NIH R01 AI33835 and P30-AI054907. None of the authors identify a conflict of interest relevant to this article.
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REFERENCES |
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