in Healthy Volunteers Plasma, Venous Blood, and ...

Comparison of Oseltamivir and Oseltamivir Carboxylate Concentrations in Venous Plasma, Venous Blood, and Capillary Blood in Healthy Volunteers Insti Instiaty, Niklas Lindegardh, Podjanee Jittmala, Warunee Hanpithakpong, Daniel Blessborn, Sasithon Pukrittayakamee, Nicholas J. White and Joel Tarning Antimicrob. Agents Chemother. 2013, 57(6):2858. DOI: 10.1128/AAC.02408-12. Published Ahead of Print 18 March 2013.

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Comparison of Oseltamivir and Oseltamivir Carboxylate Concentrations in Venous Plasma, Venous Blood, and Capillary Blood in Healthy Volunteers Insti Instiaty,a,b Niklas Lindegardh,b,c† Podjanee Jittmala,b Warunee Hanpithakpong,b Daniel Blessborn,b,c Sasithon Pukrittayakamee,a Nicholas J. White,b,c Joel Tarningb,c Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailanda; Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailandb; Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdomc

O

seltamivir (OS) is an orally bioavailable neuraminidase inhibitor active against most strains of influenza viruses and licensed for the prevention and treatment of influenza infections (1). OS is rapidly absorbed after oral administration and is extensively converted by hepatic and plasma esterases to the bioactive metabolite oseltamivir carboxylate (OC) (2). OS and OC exhibit dose-linear pharmacokinetics (3), with peak plasma concentrations observed after approximately 1 and 4 h, respectively. OS has an elimination half-life of between 1 and 1.5 h, while the OC half-life is longer, ranging from 5 to 8 h (2, 3). The fraction of OC bound to plasma proteins is negligible (⬍3%) compared to that of OS (approximately 42%) (4). OS and OC are usually quantified in venous plasma. Simpler sampling procedures using venous blood or capillary blood taken by finger prick would be advantageous for field studies, particularly in young children and infants. We report a comparison of the concentrations of OS and OC in samples of venous plasma, venous blood, and capillary blood taken simultaneously from healthy volunteers. This study was nested into pharmacokinetic investigations in 24 healthy volunteers conducted at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, in 2010. It was approved by the ethics committee of the Faculty of Tropical Medicine. Venous blood samples (2.0 ml) taken through an indwelling catheter and capillary blood (50 ␮l) taken by finger prick into a heparinized capillary tube were obtained from subjects at 1, 4, and 24 h after dosing with either 75 or 150 mg of oseltamivir (F. Hoffmann-La Roche Ltd., Basel, Switzerland) and transferred immediately into fluoride oxalate tubes. A 50-␮l venous blood aliquot was stored, and the remainder was centrifuged to obtain plasma. All samples were stored at ⫺80°C until analysis. Plasma OS and OC concentrations were quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (5). The lower limits of quantification (LLOQ) of OS and OC were set at 1 ng/ml and 10 ng/ml, respectively. The sample preparation and drug quantification in venous and capillary blood were identical to those described previously for plasma

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(5) except that the sample volume was set to 20 ␮l and the injection volume was set to 5 ␮l. Comparisons were performed using the Wilcoxon matchedpairs signed-rank test for paired observations and the Friedman test for groups. Pairs were excluded from analysis if the analyte concentrations in one or both samples were below the LLOQ. Agreement between matrices and potential concentration-dependent differences were also assessed using Bland-Altman plots. The matrices were considered bioanalytically equivalent if ⱖ67% of data pairs were within ⫾20% (6). Statistical analyses were performed using GraphPad Prism 5 (San Diego, CA). The concentration measurements of OS and OC are summarized in Table 1 and Fig. 1. All OS concentrations were below the LLOQ at 24 h after dosing. OS concentrations in venous plasma were significantly lower than those in venous blood (median [range], 27.5 [3.52 to 171] ng/ml versus 37.5 [5.18 to 250] ng/ml, respectively; n ⫽ 96 pairs; P ⬍ 0.0001). Bland-Altman plots showed that 14.6% (14/96) of data pairs were within the ⫾20% limit of agreement. OS venous plasma concentrations were also significantly lower than those in capillary blood (25.4 [3.52 to 171] versus 31.1 [4.41 to 258] ng/ml, respectively; n ⫽ 89 pairs; P ⬍ 0.0001). A total of 33.7% (30/89) of data pairs were within the ⫾20% limit of agreement. OS concentrations in venous blood were slightly higher than those in capillary blood (33.6 [5.18 to 250] versus 31.1 [4.41 to 258] ng/ml, respectively; n ⫽ 89 pairs; P ⫽ 0.03), although 70.8% (63/89) of data pairs were within the ⫾20% limit of agreement. OS did not show time-

Antimicrobial Agents and Chemotherapy

Received 5 December 2012 Returned for modification 22 January 2013 Accepted 12 March 2013 Published ahead of print 18 March 2013 Address correspondence to Joel Tarning, [email protected]. † Deceased. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02408-12 The authors have paid a fee to allow immediate free access to this article.

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Oseltamivir and oseltamivir carboxylate concentrations were measured in venous plasma, venous blood, and capillary blood taken simultaneously from 24 healthy volunteers. Median (range) venous-blood-to-plasma ratios were 1.42 (0.920 to 1.97) for oseltamivir and 0.673 (0.564 to 0.814) for oseltamivir carboxylate. Capillary blood/venous plasma ratios were 1.32 (0.737 to 3.16) for oseltamivir and 0.685 (0.502 to 1.34) for oseltamivir carboxylate. Oseltamivir concentrations in venous and capillary blood were similar. Oseltamivir carboxylate showed a time-dependent distribution between venous and capillary blood.

Oseltamivir Concentrations in Different Matrices

TABLE 1 Measured oseltamivir and oseltamivir carboxylate concentration ratios in venous plasma, venous blood, and capillary blood P value by each statistical test

Median (range) concn ratio Drug and ratio

1 h after dosing

4 h after dosing

24 h after dosinga

Pooled

Wilcoxonb

Oseltamivir Venous blood/venous plasma Venous blood/capillary blood Venous plasma/capillary blood

1.40 (0.925–1.69) 1.04 (0.456–1.67) 0.760 (0.320–1.36)

1.44 (1.04–1.97) 1.06 (0.760–1.68) 0.760 (0.450–1.21)

— — —

1.42 (0.920–1.97) 1.05 (0.460–1.68) 0.759 (0.317–1.36)

0.162 0.065 0.693

Oseltamivir carboxylate Venous blood/venous plasma Venous blood/capillary blood Venous plasma/capillary blood

0.683 (0.564–0.814) 0.760 (0.520–1.02) 1.14 (0.746–1.68)

0.664 (0.575–0.755) 0.985 (0.901–1.10) 1.47 (1.31–1.81)

0.685 (0.578–0.794) 1.07 (0.847–1.29) 1.55 (1.30–1.99)

0.673 (0.564–0.814) 0.992 (0.525–1.20) 1.46 (0.746–1.99)

Friedmanc

0.139 ⬍0.0001d ⬍0.0001e

a

Oseltamivir concentrations measured in all matrices were all below the lower limit of quantification at 24 h after dosing (indicated by dashes). Comparison of oseltamivir concentration ratios between matrices at 1 and 4 h after dosing. c Comparison of oseltamivir carboxylate concentration ratios between matrices at 1, 4, and 24 h after dosing. d By Dunn’s multiple-comparison analysis, the ratios for 1 versus 4 h, 1 versus 24 h, and 4 versus 24 h after dosing were all significantly different (P ⬍ 0.05). e By Dunn’s multiple-comparison analysis, the ratios for 1 versus 4 h and 1 versus 24 h were significantly different (P ⬍ 0.05), but the ratios at 4 versus 24 h were not significantly different (P ⬎ 0.05). b

agreement (Fig. 2). OC concentrations in venous blood were significantly lower than those in capillary blood (66.8 [10.4 to 562] versus 78.5 [10.3 to 556] ng/ml, respectively; n ⫽ 129 pairs; P ⫽ 0.0014). This was because venous blood OC concentrations were significantly lower at 1 h (56.2 [13.6 to 335] versus 76.3 [16.4 to 329] ng/ml, respectively; P ⬍ 0.001), whereas the concentrations were similar at 4 h (209 [94.6 to 562] versus 216 [98.3 to 556] ng/ml, respectively; P ⫽ 0.070) and OC concentrations were slightly higher at 24 h (29.0 [10.4 to 92.5] versus 27.5 [10.3 to 92.1] ng/ml, respectively; P ⬍ 0.001) after dosing. The OC venous-blood-to-capillaryblood ratios at 1, 4, and 24 h after dosing were therefore significantly different, indicating a time-dependent distribution between venous and capillary blood (P ⬍ 0.0001) (Table 1). A total of 100% and 97.9% of data pairs were within the limits of agreement at 4 and 24 h

FIG 1 Oseltamivir (OS) concentrations measured in venous plasma, venous blood, and capillary blood at 1 h (A) and 4 h (B) and oseltamivir carboxylate (OC) concentrations measured at 1 h (C), 4 h (D), and 24 h (E) after dosing. Error bars indicate median and interquartile ranges.


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dependent or concentration-dependent differences between matrices (Table 1). OC concentrations in venous plasma were significantly higher than those in venous blood (101 [16.0 to 888] versus 73.0 [10.4 to 562] ng/ml, respectively; n ⫽ 137 pairs; P ⬍ 0.0001). The venousblood-to-plasma ratios were similar at each time point (Table 1 and Fig. 1). All data pairs were outside the ⫾20% limit of agreement. OC concentrations in venous plasma were also significantly higher than those in capillary blood (92.9 [9.85 to 888] versus 78.2 [10.3 to 556] ng/ml, respectively; n ⫽ 130 pairs; P ⬍ 0.0001). The OC venous-plasma-to-capillary-blood ratios at 1, 4, and 24 h after dosing were significantly different (P ⬍ 0.0001), indicating a timedependent distribution between the two matrices (Table 1). All data pairs at 4 and 24 h after dosing were outside the limits of

Instiaty et al.

Percent (%) difference on the y axis is the measured venous plasma OC concentration minus the capillary blood OC concentration divided by the mean of the capillary blood and venous plasma concentrations. (D) Venous-plasma-to-capillary-blood oseltamivir carboxylate concentration ratios at 1 h, 4 h, and 24 h after dosing, with error bars indicating median and interquartile ranges.

after dosing, respectively (Fig. 3). There was no evidence of concentration-dependent distribution between the matrices. Total intersubject variability (percent coefficient of variation [%CV]) measurements in venous plasma, venous blood, and capillary blood were 100%, 100%, and 107%, respectively, for OS and 105%, 114%, and 97%, respectively, for OC. There was no correlation between the hematocrit values (median, 38.8%; range, 31.6% to 46.8%) and OS blood/plasma ratios (P ⫽ 0.071), but there was a very weak but significant negative correlation between hematocrits and the OC blood/plasma ratios (r2 ⫽ 0.069; P ⫽ 0.002). In this study, OS concentrations in venous blood were approximately 42% higher than those in venous plasma whereas concentrations of the active metabolite OC were 33% higher in plasma than in venous blood (Table 1). Similar results were found comparing venous plasma and capillary blood concentrations. Compound lipophilicity is an important determinant of distribution into cells (7). OC is a highly hydrophilic compound with a log P value of ⫺2.1 (P is the octanol-water partition coefficient), which is much more polar than that for OS (log P value of 0.36) (8). Blood taken from a finger prick is often referred to as capillary blood, although it is a mixture of arterial, venous, and capillary blood and may also contain interstitial and intracel-

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lular fluids (9). The greater pressure in arterioles and in the arterial limb of capillaries results in a greater ratio of arterial to venous blood in finger prick blood (9). Significant arteriovenous concentration differences have been identified in a number of drugs, and for these compounds, venous/capillary differences would also be expected (10). Venous/capillary differences in drug concentrations have been reported for piperaquine (11), paracetamol (12), and artemisinin (13), while venous/capillary concentrations of cyclosporine (9) and tobramycin (14) are reported to be similar. In this study, there was a reasonable agreement between venous blood and capillary blood for OS measurements (Table 1). However, a time-dependent distribution of OC between the two matrices was evident. At 1 h after dosing, venous blood OC concentrations were significantly lower than those in capillary blood, indicating incomplete equilibration. By 4 h, concentrations were similar (Fig. 3). The overall variabilities of OS and OC were comparable in the different matrices, even though venous and capillary blood sample volumes (20 ␮l) for OS and OC measurements were lower than that used for quantification in venous plasma (50 ␮l). Sampling capillary blood taken by finger prick is simple and should facilitate pharmacokinetic sampling in field studies, especially in pediatric studies. The next step will be to determine whether ac-



FIG 2 Bland-Altman plots of oseltamivir carboxylate (OC) concentrations in venous plasma and capillary blood at 1 h (A), 4 h (B), and 24 h (C) after dosing.

Oseltamivir Concentrations in Different Matrices

curate and reliable assays can be performed on dried blood spot samples taken onto filter paper. In conclusion, measurements of OS and OC in venous plasma and venous blood are not readily interchangeable, whereas venous blood and capillary blood sampling can be used interchangeably provided that the time-dependent distribution of OC is taken into consideration.

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ACKNOWLEDGMENTS This study was supported by the Southeast Asia Influenza Clinical Research Network with funding provided by the U.S. National Institute of Allergy and Infectious Diseases. This study was part of the Mahidol-Oxford Tropical Medicine Research Unit funded by the Wellcome Trust of Great Britain. The funders had no role in the design, conduct, analysis, or interpretation of this study.

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FIG 3 Bland-Altman plots of oseltamivir carboxylate (OC) concentrations in venous blood and capillary blood at 1 h (A), 4 h (B), and 24 h (C) after dosing. Percent (%) difference on the y axis is the measured venous blood OC concentration minus the capillary blood OC concentration divided by the mean of the venous blood and capillary blood concentrations. (D) Venous-blood-to-capillary-blood oseltamivir carboxylate concentration ratios at 1 h, 4 h, and 24 h after dosing, with error bars indicating median and interquartile ranges.

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