Women were recruited from 3 clinical research centers in the Eunice Kennedy Shriver National Institutes of Child Health and Human Development (NICHD) sponsored Obstetric-Fetal Pharmacology Research Units Network (OPRU). Sites include Magee-Womens Hospital of the University of Pittsburgh Medical Center, Pittsburgh, PA, University of Texas Medical Branch, Galveston, TX, and the University of Washington Medical Center, Seattle, WA. Pregnant subjects were recruited and enrolled during the 2009 influenza season from May 1, 2009 until December 31, 2010. All pregnant women were receiving oseltamivir either for prophylaxis or for treatment of proven 2009 H1N1 influenza or influenza-like-illness (ILI). The standard oral doses suggested for these indications are 75 mg once daily and 75 mg orally twice daily, respectively. Non-pregnant reproductive-age women took oseltamivir only for the study and served as control subjects. All protocols were approved by each site's respective institutional review board (IRB) and all participants underwent the informed consent process using IRB-approved consent documents.
For all participants, demographic details as well as medical history and any concomitant medications used during the study period were collected. For pregnant women, gestational age, obstetric history, and pregnancy outcomes also were collected. All pregnant subjects were already on oseltamivir as prescribed for clinical purposes by their respective caregivers. Eligibility criteria for these women included: a) pregnant and on oseltamivir, b) 16 years of age or older, c) able to perform study procedures, and d) having a hematocrit of greater than 28%. For the non-pregnant cohort eligibility criteria included: a) age 18-50 years, b) willingness to participate, and c) lacking evidence of kidney or liver dysfunction (no elevations of either serum creatinine or AST/ALT levels) or significant anemia (hematocrit less than 28%) detected at the screening visit which took place within 5 working days of enrollment.
The pharmacokinetic study was performed in pregnant subjects after they had already been on oseltamivir for at least 48 hours. On the day of the study, serial blood samples were obtained just prior to the next scheduled dose and again over one dosing interval at 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours (depending on the scheduled dosing regimen) after the dose. The non-pregnant women took a 48 hour course of 75 mg oseltamivir (either QD or BID) at home prior to reporting for study procedures. On the third day of therapy, the participants arrived at the clinical research site in the early morning after a 6 hour fast, underwent a pre-dose blood draw (trough), then took another 75 mg oral dose under direct observation by study staff. They subsequently underwent the same blood sampling protocol as the pregnant cohort.
Oseltamivir, oseltamivir carboxylate (the active mebolite), and the internal standards were extracted from K3-EDTA/NaF or K3-EDTA plasma samples by protein precipitation and the concentrations were determined by HPLC with tandem mass spectrometric detection. Detection was accomplished utilizing ion spray MS/MS in positive ion multiple reaction monitoring mode (MRM). The lower limit of quantification was 1.00 ng/mL with a calibration range up to 250 ng/mL for oseltamivir (parent drug) and the lower limit of quantification was 10.0 ng/mL with a calibration range up to 10,000 ng/mL for oseltamivir carboxylate (active metabolite). For the analysis of urine samples, the analytes and the internal standards were isolated from urine samples by dilution and determined by HPLC with tandem mass spectrometric detection. Detection was accomplished utilizing ion spray MS/MS in positive ion multiple reaction monitoring mode (MRM). The lower limit of quantification was 5.0 ng/mL with a calibration range up to 1000 ng/mL for oseltamivir and the lower limit of quantification was 30.0 ng/mL with a calibration range up to 30,000 ng/mL for oseltamivir carboxylate. All the samples were assayed by PRA International (Early Development Services, Westerbrink 3, 9405 BJ Assen, The Netherlands).
Non-compartmental analysis was performed and various pharmacokinetic parameters were generated using WinNonlin software (version 4.1; Pharsight Corporation, Mountainview, CA). The half life (t1/2) of oseltamivir and oseltamivir carboxylate was calculated from the terminal disposition phase using at least three data points. In each of the participating subjects the area under the plasma concentration-time curve (AUC) for oseltamivir and oseltamivir carboxylate for the study dose (0-end of dosing interval) was calculated using reverse superposition principle. The apparent clearance of the parent drug was calculated as dose/AUC. The apparent clearance of oseltamivir carboxylate was calculated as dose of oseltamivir × 0.91 (corrected for molecular weight) / AUC of oseltamivir carboxylate. The apparent volume of distribution of oseltamivir (Vz/F) during the terminal disposition phase was calculated from apparent clearance and half life.
The pharmacokinetic parameters and demographic data were compared between pregnant and non-pregnant subjects using a two-tailed Student t-test or non-parametric analytical methods where appropriate (depending on normality of distribution). A p value of <0.05 was considered statistically significant.