Initiation of HAART has been recommended in the United States for all HIV-infected infants under 12 months of age . High viral loads found in the initial months of life appear to correlate with disease progression, and control of viral replication by early HAART may allow preservation of immune function and prevention of dissemination to/seeding of sanctuary sites such as the central nervous system [2,3]. However, few studies have demonstrated that HAART before 6 months of age has had clinical, immunological and/or virological benefits [4,5]. One of the most potent protease inhibitors, lopinavir/ritonavir (LPV/r), has been shown to be safe and effective in infants and children over 6 months of age; however, its pharmacokinetic profile in younger infants has not been studied. Considering that young infants often have higher apparent clearance of medications and altered absorption, extrapolating doses from older children may result in lower exposure in young infants and incomplete virological suppression. This study was initiated to evaluate the pharmacokinetics as well as the safety, tolerability, and efficacy of LPV/r in infants < 6 months of age; this report includes results from the first 24 weeks of treatment.
The Pediatric AIDS Clinical Trials Group (PACTG) protocol P1030 is a prospective multicenter, phase I/II open-label trial performed in the United States and Brazil. The study is ongoing with a planned duration of follow-up to 48 weeks after enrollment of the last subject; the 24-week evaluation was a planned interim evaluation. The study treatment consisted of the liquid formulation of LPV/r in combination with two nucleoside reverse transcriptase inhibitors (NRTI) for HIV-infected infants enrolled between the ages of 6 weeks and 6 months. Infants weighing ≥ 2.5 kg with confirmed HIV infection and plasma HIV-1 RNA > 10 000 copies/ml within 30 days of study entry were eligible for enrollment. Prior treatment with LPV/r and/or concurrent nonnucleoside reverse transcription inhibitor or protease inhibitor therapy was not allowed. Infants were not enrolled if they had any of the following: laboratory values consistent with grade 2 or higher toxicity as measured by the Division of AIDS (DAIDS) Toxicity Tables for Grading Severity of Pediatric Adverse Experiences (in which a score of 0 indicates no abnormality, 1 indicates mild toxicity, 2 indicates moderate toxicity, 3 indicates severe toxicity, and 4 indicates potentially life-threatening events), a newly diagnosed acute opportunistic infection or serious bacterial infection at the time of enrollment, chemotherapy for active malignancy, or gestational age < 32 weeks at birth.
Infants were treated with LPV/r at a starting dose of LPV 300 mg/m2 plus ritonavir (RTV) 75 mg/m2 by mouth twice a day in combination with two NRTI's chosen by the principal investigator in consultation with the protocol team. A 12 h intensive pharmacokinetic study was performed at study week 2. If the trough LPV concentration (Ctrough) was < 1 μg/ml in a subject whose adherence was assessed to be adequate, the LPV/r dose was increased to 450/112.5 mg/m2 twice daily. If the subject's lopinavir area under the concentration–time curve 0–12 h (AUC0-12) was > 170 μg.h/ml, the LPV/r dose was reduced to 230/57.5 mg/m2 twice daily. In subjects requiring dose adjustments, a repeat pharmacokinetic study was performed 2 weeks following the dose change. All subjects in this report completed at least 24 weeks of follow-up or had permanently discontinued study therapy.
Criteria for permanent discontinuation of study treatment included any of the following: (i) grade 4 or recurrent/persistent grade 3 toxicity, (ii) confirmed deterioration of CD4 cell percentage to less than half of the baseline level, (iii) confirmed plasma HIV RNA increase to >50 000 copies/ml following initial suppression, (iv) parent/guardian request, or (v) investigator discretion owing to noncompliance with the protocol.
Subjects were evaluated at the screening visit and at study entry prior to receipt of any treatment, at 2 and 4 weeks, then every 4 weeks through week 24. Physical examination and nonfasting laboratory evaluations (including electrolytes, glucose, blood urea nitrogen, creatinine, total bilirubin, alanine aminotransferase, aspartate aminotransferase, calcium, phosphorus, triglycerides, cholesterol, total amylase, complete blood count with differential and platelets, and HIV RNA) were performed during each visit. Lymphocyte surface markers were analyzed every 12 weeks.
The study was approved by the Institutional Review Board for each participating site and written informed consent was obtained from each child's legal guardian before performance of any study-specific procedure. Guidelines of the Department of Health and Human Services governing experimentation in human subjects were followed.
The intensive pharmacokinetic study at week 2 of LPV/r therapy consisted of blood samples obtained prior to the morning dose and at 2, 4, 8, and 12 h following an observed dose. In subjects requiring dose adjustment, a repeat pharmacokinetic study was carried out 2 weeks after the dosage change, with blood samples obtained predose and at 4 and 12 h postdose. Predose concentration samples were also drawn at weeks 8, 12, 16, and 24, stored at −70°C and batched for analysis, as no further dose adjustments were made after the intensive pharmacokinetic study. A multianalyte high-pressure liquid chromatography (HPLC) assay using reverse-phase HPLC separation measured LPV and RTV and was performed at St. Jude's pharmacology laboratory. This method used a YMC 100 mm × 4.6 mm C-8 column for separation followed by ultraviolet detection. Internal standard (A-86093 provided by Abbott Laboratories, Abbott Park, Illinois, USA) and 125 μl of 0.05 mol/l NaOH was added to125 μl samples of plasma and extracted with 1 ml of tert-butylmethylether. The samples were centrifuged and the organic layer removed, evaporated to dryness, and reconstituted with 100 μl of mobile phase (54% 20 mmol/l sodium acetate at pH 4.88 plus 46% acetonitrile). The samples were transferred to HPLC autosampler vials and 50 μl from each vial was injected into the HPLC; peaks were monitored at 212 nm. The range for the LPV standard curve was 0.1–40 μg/ml with coefficients of variation of 4% at 0.1 μg/ml and 4% at 40 μg/ml.
Noncompartmental pharmacokinetic parameters were calculated using standard methods to determine the AUC0-12, maximum concentration (Cmax), time to maximum concentration (tmax), and predose concentration (Cpre). The lowest observed concentration (typically Cpre or C12h) was defined as Ctrough and used to determine if a dose increase was necessary for a specific subject. A one-compartment model with a first-order absorption (Ka) and lag time, if necessary, was fitted to each subject's data using a maximum likelihood estimation and ADAPTII version 4 . The modeling was performed parameterized as apparent clearance (CL/F) and apparent volume of distribution (V/F).
Chemistries and urinalyses were performed in local clinical laboratories. Complete blood and differential and platelet counts were performed at central laboratories (either the University of Medicine and Dentistry of New Jersey or the University of Massachusetts) using standard methods. Lymphocyte subset analysis was performed in the immunology laboratory of the University of Massachusetts using standard flow cytometry according to standard procedures.
The Amplicor HIV-1 Monitor test, version 1.5 (Roche Molecular Systems, Branchburg, New Jersey, USA) was used to determine quantitative plasma HIV-1 RNA levels (lower limit of quantification, 400 copies/ml). All assays were performed at the Core Virology Laboratory of the University of Medicine and Dentistry of New Jersey, which is DAIDS-certified, with incorporated quantitative standards supplied by the DAIDS Virology Quality Assurance Program . Plasma samples were stored at −70°C until the assay was performed. Samples from the first 28 days of the study were assayed in batch format to avoid interassay variability, while subsequent samples were assayed in real time. Samples at study entry and in the event of early termination from study were collected and stored for genotypic resistance assays.
Toxicity was defined as the development of diagnoses or signs and symptoms at grade 3 or more. Laboratory abnormalities that occurred while on study treatment were evaluated using the DAIDS' toxicity table and considered related or possibly related to study treatment. Criteria for virological endpoints used in the protocol-defined statistical analyses were more restrictive compared with the criteria for permanent discontinuation of study treatment for virological failure (described above). Criteria for virological endpoints included: (i) < 1 log10 copies/ml decrease in HIV-1 RNA from baseline to week 8, (ii) HIV-1 RNA > 400 copies/ml at week 16 of treatment, or (iii) rebound of HIV RNA to > 4000 copies/ml after week 16. All endpoints required confirmation with a repeat sample. Virological endpoints were analyzed using a modified ‘intent-to-treat’ approach in which children who discontinued study treatment for any reason were considered to be failures (that is off-treatment was equated to failure) and an ‘as-treated’ approach that only included measurements obtained while a child was on study treatment. In the modified intent-to-treat analysis of quantitative changes in HIV-1 RNA, children who went off study treatment early were considered to have a decrease smaller than any children who remained on treatment. Comparisons of children achieving versus not achieving virological suppression at week 16 were undertaken using Wilcoxon's rank sum test.
Twenty one infants [14 (67%) females, 10 (48%) African American, 9 (43%) Hispanic and 2 (10%) Caucasian] were enrolled between August 2002 and March 2005; 16 were from the United States and 5 from Brazil. All infants acquired HIV-1 perinatally, with the median age at enrollment of 14.7 weeks (range, 6.9–25.7). The Centers for Disease Control and Prevention clinical categories of study participants at entry were class N, 11; class A, 2; class B, 6; and class C, 2. Perinatal prophylaxis had been given to 15 of the 21 patients: zidovudine monotherapy (12), zidovudine plus nevirapine (2), and zidovudine, lamivudine, and nevirapine (1); the three regimens that included nevirapine prophylaxis were completed at least 2 months prior to starting study drugs.
Twenty one infants initiated LPV/r with background NRTI therapy that included zidovudine and lamivudine (12), stavudine and lamivudine (7) or stavudine and abacavir (2). Two infants discontinued study medications prior to 24 weeks: one at the parent's request because of grade 1 vomiting and soft stools at week 2 and the second owing to progression of disseminated cytomegalovirus at week 7; this infant died at week 8.
Pharmacokinetic parameters from the intensive study at week 2 are available for 18/21 enrolled subjects; one patient discontinued study prior to week 2, the second was given an incorrect dose of LPV/r for the intensive pharmacokinetic study, and the third was nonadherent to study medications at the time of two intensive pharmacokinetic studies, as evidenced by undetectable predose LPV/r levels. The ages and weights at study entry and median starting dose for the 18 patients are summarized in Table 1. The starting dose of LPV/r 300/75 mg/m2 every 12 h initiated at study entry was not changed during the first 2 weeks on study regardless of changes in body surface area; therefore, the median dose at the time of the pharmacokinetic study was 276 mg/m2 (range, 235–306), which was equivalent to 15.2 mg/kg (range, 12.2–16.3). The median concentration–time profile is shown in Fig. 1 and pharmacokinetic parameters are summarized in Table 1. There was an approximately sevenfold range (24–164 μg.h/ml) for AUC0-12. The RTV AUC0-12 was correlated with LPV AUC0-12 (r = 0.67) but not half-life, suggesting a preferential impact on bioavailability. This is consistent with the correlation between LPV CL/F and V/F (r = 0.42).
One subject met the study criteria for dose escalation based on the week 2 pharmacokinetic evaluation. The repeat pharmacokinetic study at the increased dose exhibited LPV concentrations in the acceptable range.
The values of Cpre were highly variable, having within-subject coefficients of variation ranging from 51 to 95%. Overall the median Cpre values at week 2 were lower than the average of weeks 8, 16, and 24 (2.4 and 5.3 μg/ml, respectively) and week 2 Cpre also had the highest intersubject variability.
The median entry plasma HIV RNA for the 21 infants was 5.8 log10 copies/ml (range, 3.7–6.9). No infant had baseline resistance mutations in the protease gene. Details regarding baseline NRTI resistance mutations are reported elsewhere . Nineteen infants continued therapy through week 8, at which time 15 had > 1 log10 copies/ml decline in HIV RNA. Eight infants remained on study treatment and achieved HIV RNA < 400 copies/ml by week 16 [38% by intent-to-treat) analysis; exact 95% confidence interval (CI), 18–62], representing a median 3.0 log10 copies/ml reduction in HIV RNA; all eight maintained plasma HIV RNA < 400 copies/ml through week 24. Eleven infants with detectable virus at week 16 experienced a median 1.7 log10 copies/ml fall in HIV RNA from baseline to week 16. Comparing those who did achieve < 400 copies at week 16 with those who not achieve this, there was no difference in median age at start of treatment (0.24 and 0.29 months, respectively; P = 0.90) or median baseline HIV RNA (5.9 and 5.8 log10 copies/ml, respectively; P = 0.80). By week 24, two additional subjects experienced a decrease to < 400 copies/ml (48% by intent-to-treat analysis) and one infant reached a viral load < 400 copies at week 20 but had 498 copies/ml at week 24. Using an as-treated analysis for patients taking study medications at week 24, 10/19 (53%) had HIV RNA < 400 copies/ml. Figure 2 shows the median plasma HIV RNA over time. At week 24, the median HIV-1 RNA decrease was 3.13 log10 copies/ml by intent-to-treat analysis (95% CI, 1.71–3.46) and 3.33 log10 copies/ml by as-treated analysis (95% CI, 2.86–3.75). The early virological response (HIV RNA < 400 copies/ml by week 16) was not predicted by LPV exposure at week 2. Among 17 patients with evaluable pharmacokinetic and virology data at week 16, the median pharmacokinetic data for the eight infants with viral suppression at week 16 compared with the nine who did not achieve suppression were as follows: AUC, 75.48 μg.h/ml (range, 23.66–133.06) and 58.13μg.h/ml (range, 27.76–95.66), respectively (P = 0.28); Cpre, 2.28 μg/ml (range, 0.49–8.01) and 1.37μg/ml (range, 0.13–5.98), respectively (P = 0.37); and Cmax 10.9 μg/ml (range, 3.02–18.60) and 6.88 μg/ml (range, 3.62–11.90), respectively (P = 0.20).
Data analysis was restricted to measurements obtained in subjects taking study medications. The median entry CD4 cell percentage was 32% (range, 11–54) with a median increase of 4% (95% CI, −1 to +9) from baseline to week 24 in 19 subjects. The median entry CD8 cell percentage was 24% (range, 14–57): at week 24, the median CD8 cell percentage declined by 4% (95% CI, 1–7).
One infant discontinued therapy prior to week 2 owing to grade 1 vomiting. Six adverse events of grade 3 or higher occurring in three infants (14.3%) were deemed to be possibly related to study treatment: asymptomatic grade 3 serum sodium and/or potassium disturbances in two infants that resolved with disruption in study therapy of 0–2 weeks and did not recur when therapy was resumed; and grade 3 elevation of serum glutamate–pyuvate transaminase (alanine aminotransferase) in one infant that did not recur after treatment suspension for 3 days. There were no new symptoms, signs, or diagnoses that were considered related or possibly related to study treatment. A total of 15 adverse events at grade 3 or higher occurring in nine (42.8%) subjects were deemed not related to study treatment. Seven were transient grade 3 events: creatinine and sodium associated with urosepsis (1), anemia after interruption of erythropoetin therapy (1), perianal ulcers secondary to Clostridium difficile diarrhea (1), potassium disturbance in a hemolyzed sample (1), and fevers owing to intercurrent illness (2). There was one grade 3 psychosocial failure to thrive, and two grade 4 events owing to laboratory error: hypoglycaemia (1) and thrombocytopenia (1). One infant had grade 3 respiratory acidosis, shortness of breath, anemia (twice) and died (grade 5) from cytomegalovirus sepsis. Lipid samples were drawn fasting only if the nonfasting sample was grade ≥ 2, (triglycerides ≥ 7.5 mg/l and cholesterol ≥ 5.0 mg/l) and were collected from 19/21 infants; two infants who discontinued study before 16 weeks had insufficient samples for analysis. Seventeen infants had triglycerides < 5.0 mg/l from entry through 24 weeks; two infants with baseline triglycerides < 3.0 mg/l had a single level value in the range 6.0–7.0 mg/l that spontaneously decreased to < 3.0 mg/l during the study period. Seventeen infants had a baseline cholesterol of < 1.70 mg/l; four maintained levels ≤ 1.70 mg/l, seven had cholesterol of 1.71–2.00 mg/l and six had two or more cholesterol samples confirmed over the observation period that were between 2.01 and 2.64 mg/l. Two had cholesterol of 1.80 mg/l at baseline that increased to 2.22–2.28 mg/l by 24 weeks.
This is the first prospective report on the pharmacokinetics and 24-week safety and efficacy of LPV/r therapy in very young HIV-infected infants. The apparent clearance of LPV/r was slightly higher than that previously seen in older children, but by using a dose of 300/75 mg/m2, the median AUC0-12 achieved by these young infants was in a similar range to that reported for older children taking the recommended dose of 230/57.5 mg/m2 . Predose concentrations stabilized at a higher level after the first 2 weeks of study, suggesting improved absorption, dietary changes, variation in RTV oral clearance, and/or more consistent administration of the study medications over time. Similar Cpre and C12h concentrations after observed administration of the week 2 dose suggests that poor adherence is unlikely to be the primary cause for the lower Cpre compared with Cpre at later weeks.
High oral clearance leading to increased dose requirements has also been seen in infants < 24 months of age with other protease inhibitors such as RTV and nelfinavir [10–12]. In addition to the high median oral LPV clearance in this age group, intersubject variability of LPV concentrations was great, with nearly a 10-fold range in oral clearance, even after adjusting for subjects' body surface area. High variability has also been seen with nelfinavir and RTV in this age group [10,12].
While, as a group, infants tolerated the liquid suspension of LPV/r, some caregivers reported that it took time to develop an administration technique that was amenable to the child. Many reported success by adding the dose of LPV/r to a small amount of infant formula, or administering small increments of the solution to the rear of the buccal mucosa using a 1 ml syringe.
Grade 3 or higher adverse events considered possibly or definitely related to study treatment occurred in 14% of infants and were asymptomatic and transient. Lipid changes were difficult to assess; while nonfasting cholesterol showed a modest increase in most infants, fasting lipids, which correlate better with cardiovascular risk, were not collected. Of interest, triglyceride levels did not increase substantially over 24 weeks.
Although only 53% of infants taking LPV/r achieved a viral load < 400 copies/ml at 24 weeks, the percentage with viral suppression continued to improve over time. Five of the nine infants who did not reach < 400 copies/ml by 24 weeks had suboptimal adherence, three with Cpre below the limit of detection at week 8 and/or week 16, and two with nonadherence by caretaker report. In all five, the primary provider initiated interventions (intensive adherence training or transfer of care to new caregivers) that resulted in achievement of viral suppression. Successful administration of chronic antiretroviral medications in this population is challenging but critical to the success of therapy.
Among the few published studies of ART in young infants, many have reported difficulty in achieving long-term viral suppression. In a multicenter study of 20 infants using nelfinavir-containing HAART initiated under 3 months of age, The Pediatric European Network for Treatment of AIDS found that 11/16 experienced virologic failure by 72 weeks of follow-up and 30% had developed new resistance mutations . Investigators for PACTG Protocol 345 found that only 46% of infants between 1 and 24 months of age receiving therapeutic doses of RTV sustained plasma HIV-1 RNA at < 400 copies/ml through 104 weeks . Although half of our subjects demonstrated delayed achievement of virological suppression, most were able to sustain HIV-1 RNA at < 400 copies/ml with longer follow up; this implies that treatment-limiting resistance to the study medications did not occur. In addition, all infants remained clinically and immunologically stable, with preservation or improvement of CD4 cell percentages at an age when CD4 cell values should undergo a natural decline.
In conclusion, despite the higher clearance of LPV/r in young infants, a twice daily dose of 300/75 mg/m2 LPV/r provides similar exposure to that seen in older children, albeit slightly less than seen in adults . Furthermore, LPV/r appears to provide favorable virological and clinical efficacy in this age range even when virological decay is prolonged.
The P1030 team wishes to dedicate this study to the memory of our colleague, John H. Rodman, Pharm.D. We also would like to thank Katherine Luzuriaga, Jennifer Gardella, Carmelita Alvaro, Barbara Heckman, and Marisol Martinez for their valued contributions to the study. Special thanks also go to the patients, families, and study personnel who participated in this trial.
Investigators participating in P1030: Denise Ferraro, Jane Perillo, Jennifer Griffin (SUNY Stony Brook); Janet Chen, Jill Foster, Roberta Laguerre, Daniel Conway (St. Christopher's Hospital for Children); Hans M L Spiegel, Diana Robbins, Tracy Perron, Deidre Wimbley (Children's Medical Center, George Washington University); Emily Barr, Lisa Mauro, Jody Maes, Elizabeth McFarland (The Children's Hospital, Denver); Ruth Williams, Katherine Kabat (Children's Memorial Hospital); Mobeen H. Rathore, Ayesha Mirza, Michelle Tucker, Chas Griggs (University of Florida, Jacksonville); Douglas Watson (University of Maryland School of Medicine); Aditya Gaur, Katherine Knapp, Nehali Patel, Marion Donohoe (St. Jude Children's Research Hospital); Ann Petru, Teresa Courville (Children's Hospital Oakland); Barbara W. Stechenberg, Donna J. Fisher, Alicia M. Johnston, Maripat Toye (Baystate Medical Center).
Supported by a grant from the Pediatric AIDS Clinical Trials Group (PACTG)/International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) Group of the National Institute of Allergy and Infectious Diseases and the National Institute of Child Health and Development, National Institutes of Health; and by Abbott Laboratories.
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