Antiretroviral agents are frequently administered to neonates born to HIV-infected mothers. With HIV-1 DNA polymerase chain reaction (PCR) testing, HIV infection can be detected by 2 weeks of life in more than 90% of formula-fed infants perinatally infected with HIV.1 Current guidelines suggest that combination therapy with at least 3 antiretroviral agents should be initiated in all HIV-infected newborns with clinical symptoms or immune deficiency and should be considered in the rest.2 The development and licensure of rapid HIV antibody assays now allow for identification of HIV infection among women presenting for delivery without prior testing or therapy.3 If an HIV-infected woman in labor has not received antiretroviral treatment before delivery, treatment of the infant with antiretrovirals after birth has been shown to provide some protection against transmission of HIV.4-6 Investigations of several antiretroviral regimens, some including protease inhibitors, for postexposure prophylaxis of infants born to untreated mothers are now underway.
Nelfinavir mesylate is a potent HIV protease inhibitor available in powder and tablet forms suitable for administration to infants.7 Pharmacokinetic studies of nelfinavir in children older than the age of 2 years have demonstrated the need for doses from 60 to 90 mg/kg/d to achieve plasma concentrations equivalent to those seen in adults receiving the standard 1250-mg twice-daily dose (approximately 36 mg/kg/d in adults).8 The need for these relatively higher doses has been attributed to developmental changes in drug absorption and/or metabolic pathways. Although the few studies of nelfinavir pharmacokinetics that have been performed in infants less than 2 years of age have suggested that even larger doses may be needed in this age range, the number of infants included in the first weeks of life has been small.9-12 The aim of this study was to describe the safety and pharmacokinetics of nelfinavir in infants during the first 6 weeks of life.
Pediatric AIDS Clinical Trials Group (PACTG) protocol 353 was a multicenter phase 1 study of the safety, tolerance, and pharmacokinetics of nelfinavir coadministered with zidovudine and lamivudine in HIV-infected pregnant women and their infants. The protocol was approved by the institutional review board at each participating site, and informed consent was obtained from each woman included in the study before enrollment. This report contains only the infant data from the protocol; the maternal data are to be reported separately. All study mothers were infected with HIV and received the combination of zidovudine, lamivudine, and nelfinavir during pregnancy. After birth, study infants received combination therapy with zidovudine (2.6 mg/kg 3 times daily), lamivudine (2 mg/kg 2 times daily), and nelfinavir until 6 weeks of age. Study infants were followed for toxicity until the age of 24 weeks. At the time this study was initiated, there had been only limited experience with nelfinavir in neonates; thus, nelfinavir dosing of infants in cohort 1 was initiated at lower doses, 10 mg/kg of nelfinavir 2 times a day, than had been administered in older children. The pharmacokinetic target was an extrapolated 24-hour area under the curve (AUC) greater than 30 μg·h/mL, equivalent to the 10th percentile of the AUC in nonpregnant adults receiving standard dosing. After the data from cohort 1 had been analyzed, the nelfinavir dose for cohort 2 was increased to 40 mg/kg twice a day. Nelfinavir was administered as a crushed tablet mixed in water or as the powder formulation mixed with infant formula.
Infants were followed through the age of 6 months for clinical or laboratory signs of toxicity. Toxicities were graded according to the National Institutes of Health Division of AIDS Toxicity Grading Table for pediatric adverse events. Toxicities were classified as follows: mild, grade 1; moderate, grade 2; severe, grade 3; and life threatening, grade 4. The site investigators managed adverse events and assessed their relation to the study medications. The protocol team subsequently reviewed all adverse events. All study drugs (zidovudine, lamivudine, and nelfinavir) were permanently discontinued for confirmed grade 4 toxicity. All study drugs were temporarily discontinued for confirmed grade 3 toxicity but could be restarted within 72 hours if the toxicity resolved.
Pharmacokinetic Methods and Analysis
Serial blood samples were obtained over a dosing interval at 5 to 8 days after birth and, again, at 5 to 6 weeks after birth. Samples were obtained before the dose was administered and at 1, 2, 4, 6, and 8 hours after the dose was administered from infants in cohort 1 and at 2, 6, 8, and 12 hours after the dose was administered from infants in cohort 2. Plasma concentrations of nelfinavir and its active hydroxyl-tert-butylamide metabolite (M8) were measured by high-performance liquid chromatography (HPLC).
Samples were frozen at −20°C or colder, shipped to the central analytic laboratory (Pediatric Clinical Pharmacology Laboratory, University of California, San Diego), and assayed, on average, within 2 weeks of collection. Serum nelfinavir and M8 concentrations were determined simultaneously by HPLC. The method used was similar to that described by Wu et al13 with the following modifications. Serum proteins were precipitated using acetonitrile, and the supernatant was injected directly onto a C18 reversed-phase HPLC column (150 mm × 4.6 mm). The drugs were separated isocratically using 10 mM of potassium phosphate buffer, pH 4.0 (62%), and acetonitrile (38%), with UV detection at 206 nm. Detection was by ultraviolet radiation (UV) at 206 nm. The mean recovery from spiked samples was 97% for nelfinavir and 89% for M8. The lower limit of quantification was 0.148 μg/mL for nelfinavir and 0.0625 μg/mL for M8. The mean (±standard deviation) interassay and intra-assay coefficients of variation based on other controls were 10.56 ± 2.04% and 4.01 ± 2.38% for nelfinavir and 11.6 ± 1.12% and 5.96 ± 1.69% for M8.
Pharmacokinetic calculations were performed using 2 commercial computer programs (WinNonlin, Pharsight Corporation, Palo Alto, CA; Microsoft Excel, Redmond, WA). Trough concentration (Cmin), peak concentration (Cmax), and time of peak concentration (Tmax) were determined by inspection of concentration time curves. Area under the serum concentration-time curve during the dosing interval (8- or 12-hour AUC) was calculated by the trapezoidal rule and then extrapolated to a 24-hour AUC for comparison purposes. Oral clearance (Cl/F) was calculated from the formula Cl/F = Dose/AUC. The ratio of M8 to nelfinavir was calculated for each plasma sample. These ratios were then averaged for each sampling period, excluding samples with nelfinavir concentrations less than 1.0 μg/mL.
Demographic and pharmacokinetic parameters are presented as medians (range). The Wilcoxon signed rank test was used to compare pharmacokinetic parameters between the sampling periods.
Twenty-six infants were enrolled in the trial and received study drugs: 7 in cohort 1 and 19 in cohort 2. The median gestational age at birth was 38.5 weeks (range: 36-41 weeks), and the median birth weight was 3040 g (range: 2050-4210 g). The ethnicity of the infants was 17 Hispanic, 5 black non-Hispanic, and 4 white non-Hispanic.
Pharmacokinetic sampling was successfully completed for 7 cohort 1 infants at week 1 and for 5 cohort 1 infants at week 6. Four of the 19 infants in cohort 2 stopped study treatment early and had no pharmacokinetic sampling performed at the request of their parent or pediatrician. Two of the remaining 15 cohort 2 infants received 6 weeks of study drugs but did not participate in pharmacokinetic sampling at parental request. Sampling was inadequate for 2 cohort 2 infants at weeks 1 and 6, leaving 11 cohort 2 infants who successfully completed pharmacokinetic sampling at weeks 1 and 6.
Nelfinavir concentration time plots for weeks 1 and 6 for each cohort are presented in Figures 1 and 2. Summaries of the pharmacokinetic parameters for each cohort are presented in Table 1. Of the infants in cohort 1, who received nelfinavir doses of 10 mg/kg 3 times a day, only 2 infants at week 1 and none at week 6 exceeded the 24-hour AUC target of 30 μg·h/mL. Infants in cohort 2, who received 40 mg/kg twice a day, had a median 24-hour AUC of 38.4 μg·h/mL at week 1 and 38.8 μg·h/mL at week 6. There was considerable variability in AUC among the cohort 2 infants, and 3 of 11 infants at week 1 and 4 of 11 infants at week 6 did not meet the 24-hour AUC pharmacokinetic target. The median nelfinavir Cl/F was 2.1 L/h/kg at week 1 and week 6.
Nelfinavir concentrations in cohort 1 were generally low, preventing reliable determination of the M8-to-nelfinavir ratio in cohort 1 infants. The M8-to-nelfinavir ratio could be calculated at both sampling times for 9 infants in cohort 2, as presented in Figure 3. The median M8-to-nelfinavir ratio increased from 0.16 (range: 0-0.38) during week 1 to 0.56 (range: 0.4-1.47) during week 6 (P < 0.01). There were no significant differences in any of the other pharmacokinetic parameters when week 1 and week 6 results were compared.
Twenty-two of the 27 infants completed the scheduled 24 weeks of follow-up. Of the remaining 5, 1 infant (in cohort 1) moved out of the country, 1 infant (in cohort 2) was lost to follow-up, and the mother declined further contact for 3 infants (in cohort 2). Including all follow-up, there were 10 adverse clinical events reported in 7 infants in cohort 1 (Table 2). Only 1 (intrauterine growth retardation) was considered possibly related to study treatment. There were 41 adverse clinical events reported in 13 infants in cohort 2. Only 3 were considered possibly related to study treatment: diarrhea, intrauterine growth retardation, and peripheral pulmonic stenosis in 1 infant each.
The frequency of grade 3 or 4 laboratory adverse events is listed in Table 3. Hematologic adverse events were the most common. There were 4 occurrences of grade 3 anemia and 1 occurrence of grade 3 neutropenia in 5 cohort 1 infants and 7 occurrences of grade 3 anemia, 2 occurrences of grade 3 neutropenia, and 1 occurrence of grade 4 neutropenia in 9 cohort 2 infants. These hematologic toxicities were attributed to zidovudine and lamivudine exposure by site investigators and the protocol team.
Study drugs were temporarily held in 7 infants (2 in cohort 1 and 5 in cohort 2). Zidovudine was temporarily held in 4 infants because of anemia and in 1 infant because of neutropenia. Nelfinavir was temporarily held in 1 infant because of vomiting. All study drugs (zidovudine, lamivudine, and nelfinavir) were temporarily held in 1 infant because of bronchiolitis. No infant in cohort 1 permanently discontinued study drugs before week 6. Four cohort 2 infants were permanently taken off all study drugs early for the following reasons: unspecified parent request, refusal of infant blood sampling by parent, at parent request because of diarrhea, and at site investigator request because of recurrent anemia. One infant was infected with HIV as determined by a positive HIV DNA PCR assay at delivery.
These results are consistent with previous studies of nelfinavir pharmacokinetics demonstrating that neonates need relatively large nelfinavir doses on a per kilogram basis to achieve serum exposures equivalent to those seen with standard dosing in older children and adults. In studies of infants less than 2 years of age receiving 75 to 135 mg/kg/d of nelfinavir, nelfinavir plasma concentrations were highly variable and were lower than those seen in older children and adults receiving labeled dosing.9,10 In a study of infants between 2 and 8 months of age, an average nelfinavir dose of 136 mg/kg/d was needed to achieve a median extrapolated 24-hour AUC of 30.6 μg·h/mL, and when dosing was normalized to 150 mg/kg/d, 28% of infants failed to meet the same AUC target used in our study, an extrapolated 24-hour AUC greater than 30 μg·h/mL.11
The only previous study of infants as young as those in the current study was performed in Thai infants receiving 45 mg/kg twice a day for the first 4 weeks after birth. These infants had a median extrapolated 24-hour AUC of 46.8 μg·h/mL at the age of 2 weeks and 37.0 μg·h/mL at 4 weeks. There was a significant decrease in AUC at 4 weeks of age compared with 2 weeks of age, and 3 (27%) of 11 infants in that study would have failed to meet our pharmacokinetic target at 4 weeks.12 These results are similar to our observations of a median 24-hour AUC of approximately 38 μg·h/mL in infants receiving 40 mg/kg twice a day at 1 and 6 weeks of life, with 4 (36%) of 11 infants at 6 weeks failing to meet the AUC target. In contrast to the Thai study, we observed no difference in AUC in infants between the ages of 1 week and 6 weeks.
The pharmacokinetic target used in this study, an extrapolated 24-hour AUC of 30 μg·h/mL, is equivalent to an AUC of 10 μg·h/mL over 8 hours or 15 μg·h/mL over 12 hours. This target is based on the 10th percentile value for adults receiving 750 mg 3 times a day and has been used in previous pediatric and adult studies.7,11,14,15 Although other pediatric studies have used Cmin as a target, there is a close correlation between AUC and Cmin.16,17 The goal of most pediatric nelfinavir pharmacokinetic studies has been to devise a dose that can achieve a plasma exposure equivalent to that seen in adults receiving standard dosing, whose 24-hour AUC generally ranges between 45 and 60 μg·h/mL.18,19 Although increased plasma protease inhibitor concentrations are generally related to improved virologic suppression, no studies have demonstrated clear nelfinavir concentration standards.20-22 Other factors such as initial viral load, change in viral load during initial period of treatment, time to achieve full virologic suppression, and adherence play a role in determining individual response to antiretroviral agents.19-21,23 One proposed use of nelfinavir in neonates is for postexposure prophylaxis against perinatal HIV exposure when no antenatal antiretroviral therapy was used. No data are available establishing minimum nelfinavir plasma concentrations effective for prophylaxis against HIV infection.
The predominant pathways for nelfinavir elimination are metabolism in the liver by cytochrome P450 enzymes (CYP), including 3A4, 2C9, 2C19, and 2D6.24 Nelfinavir metabolism by CYP2C19 leads to the formation of M8, which is the most common metabolite of nelfinavir found in plasma and has nearly equal activity against HIV as nelfinavir.25 Nelfinavir and M8 are both eliminated via metabolism by CYP 3A4.26 The median M8-to-nelfinavir ratio in adults receiving nelfinavir is 0.29, with a range of 0 to just greater than 1.2, and the ratio is lower in non-Hispanic black and Asian patients compared with non-Hispanic white patients.27 In the current study, the M8-to-nelfinavir ratio increased from a median of 0.16 at the age of 1 week to 0.56 at the age of 6 weeks. The only other study to investigate M8 concentrations in infants receiving nelfinavir found M8-to-nelfinavir ratios ranging from 0.12 to 0.89 in infants between 2 and 7 months old, with no relation between M8-to-nelfinavir ratio and age.11 Our data are consistent with previous observations that the concentration of other CYP2C enzymes is low in infants immediately after birth and rises during the first weeks of life.28 Although CYP3A4 activity is also low immediately after birth and rises over the first months of life, the activity of CYP3A7 is high in the fetus and at birth and declines gradually over the first months of life.29 One explanation for our observation that nelfinavir Cl/F does not change between 1 and 6 weeks of life is that CYP3A7 may play a major role in nelfinavir and M8 metabolism in the absence of CYP3A4.
In summary, nelfinavir seemed to be well tolerated by the infants in this study, with few adverse events attributed to the drug. We found inadequate nelfinavir plasma concentrations in infants receiving 10 mg/kg 3 times a day during the first 6 weeks of life. Nelfinavir exposure was improved with 40-mg/kg twice-daily dosing; however, approximately one third of infants receiving this dose failed to meet our pharmacokinetic target. As in other populations, these infants demonstrated considerable intersubject variability in nelfinavir pharmacokinetic parameters. Further investigations of larger doses, such as 75 mg/kg twice a day, should be undertaken.
The authors acknowledge the contributions of the following members of the PACTG 353 protocol team: Betsy Smith, MD, Yette Asfaw, Kimberly Hughes, Bethann Cunningham-Schrader, MS, Maureen Shannon, CNM, FNP, MS, and Lynette Purdue, PharmD.
The following investigators and institutions participated in this study: Boston Medical Center, Boston, MA: Laureen Kaye, RN, Anne Marie Regan, MEd, PNP, MSN, Ellen Cooper, MD, and Stephen Pelton, MD; Children's Hospital and Medical Center, Seattle, WA: Jane Hitti, MD, Deborah Goldman, ARNP, MPH, Michele Acker, and Kathleen Mohan, ARNP, MN; San Juan City Hospital, San Juan, PR: Jorge Gandia, MD, Rodrigo Diaz-Velasco, MD, and Milagros Gonzalez, MD; Bronx/Lebanon Hospital, Bronx, NY: Andrew Wiznia, MD, Wanda Biernick, RN, Karen Dorio RN, and Joanna Dobroszycki, MD; Los Angeles County Medical Center, Los Angeles, CA: Alice Stek, MD, Andrea Kovacs, MD, and James Homans, MD; Howard University Hospital, Washington, DC: Sohail Rana, MD, Deepika Darbari, MD, Patricia Houston Yu, MS, and Jhoanna Roa, MD; Harbor-UCLA Medical Center, Los Angeles, CA: Nasser Redjal, MD, Marie Beall, MD, Judy Hayes, RN, and Arceli Gagajena, RN; University of California Medical Center, Los Angeles, CA: Maryanne Dillon, BSN, NP, Karin Nielsen, MD, MPH, and Jaime Deville, MD; and University of California, San Diego, CA: Andrew D. Hull, BMedSci, BMBS, Patricia Franklin, RN, NP, and Stephen A. Spector, MD.
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