Infection with HIV during childhood is associated with challenging virologic and clinical features that interfere with response to antiretroviral (ARV) therapy. Among these are very high plasma viral loads, the presence of a developing immune system with limited reaction capacity and the lack of suitable, tolerable and palatable ARV therapies for pediatric subjects. As a result HIV-infected children are more likely to have rapid disease progression and theoretically develop substantial HIV resistance to existing ARV therapies. Limited published data indicate that fewer than 65% of children achieve and maintain plasma viral loads of <400 copies/ml while remaining on study-defined ARV regimens. 1–6
Lopinavir (ABT-378) is a novel HIV protease inhibitor (PI) that is coformulated with a small quantity of ritonavir, an inhibitor of cytochrome P-450 CYP3A. In the presence of 50% human serum, lopinavir is 10-fold more potent than ritonavir (50% effective concentration, 0.102 μM vs. 1.040 μM). When lopinavir is given in combination with ritonavir, lopinavir is very sensitive to pharmacokinetic enhancement by ritonavir, resulting in substantially increased plasma lopinavir concentrations, even at ritonavir doses not associated with antiviral activity. The high lopinavir plasma concentrations relative to the protein binding-adjusted 50% inhibitory concentration for wild-type HIV achieved with this coformulation potentially provides a pharmacologic barrier to the emergence of viral resistance, while also providing antiviral activity against some resistant HIV. 7
The purpose of this study was to evaluate the safety, tolerability, pharmacokinetics and antiviral activity of a liquid coformulation of lopinavir/ritonavir (Kaletra) at two dosage levels in combination with reverse transcriptase inhibitors in ARV-naive and ARV-experienced pediatric subjects.
This was a Phase I/II, open label, parallel arm, multicenter clinical trial of lopinavir/ritonavir in combination with reverse transcriptase inhibitors in HIV-infected children.
One hundred HIV-infected children, ages 6 months to 12 years, with plasma viral load >400 copies/ml and no prior nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy were recruited for study participation from several sites in the United States, Canada, Argentina, Bahamas, Panama and South Africa. Subjects were excluded if they had a life expectancy of <1 year, had had prior treatment with an investigational agent within 30 days before enrollment, an active opportunistic infection within the last 90 days, required systemic chemotherapy or had had symptoms of encephalopathy or developmental delay that would have reduced the ability of the subject to comply with protocol requirements. The institutional review board or independent ethics committee for each site approved the protocol before participation of the site in this clinical trial.
The investigator or his/her representative explained the nature of the study to the parent, guardian or legally acceptable representative (hereafter referred to as parent) and answered all questions regarding this study. Before the conduct of any study-related procedures, the informed consent statement was reviewed, signed and dated by the parent and the person who administered the informed consent.
The children were stratified according to age (3 months to 2 years and 2 to 12 years) and prior antiretroviral treatment experience (ARV-naive and ARV-experienced) at the time of their enrollment in this study. Subjects were considered ARV-naive if they had received <3 months of prior therapy and <1 week of treatment with lamivudine (3TC) or ARV-experienced if they had received >3 months of prior therapy or >1 week of treatment with 3TC. Subjects were randomized in a 1:1 ratio within each stratum to receive either 230/57.5 mg/m2 or 300/75 mg/m2 lopinavir/ritonavir twice daily (BID) taken with food. The 230/57.5 mg/m2 regimen is an estimate of the adult-equivalent (400/100 mg BID) pediatric dosage on the basis of body surface area and the 300/75 mg/m2 regimen is a 30% increase over the body surface area-equivalent adult dose. These dosage levels were chosen in an attempt to find a dose that approximates the adult drug exposure at 400/100 mg twice daily. In addition to lopinavir/ritonavir, ARV-naive subjects received treatment with stavudine and 3TC. ARV-experienced subjects received treatment with nevirapine and one or two nucleoside reverse transcriptase inhibitors (NRTIs) of the investigator’s choice in addition to lopinavir/ritonavir. Safety and efficacy results from Phase II adult studies supported the use of these combinations. 8, 9
Based on results from an interim evaluation of safety and antiviral activity, as well as the pharmacokinetics (PK) in a subset of subjects after 3 weeks of treatment at the two dosage levels, subjects were to be assigned to the same specific dosage of lopinavir/ritonavir for the remaining study period. The final dose was selected based on the following criteria: <20% of subjects with Grade 3 or higher clinical or laboratory adverse events; >75% of subjects with at least a 0.5-log reduction in viral load during the first 21 days after enrollment; and the mean values of the PK parameters within 80 to 130% of the corresponding adult PK parameters.
Lopinavir/ritonavir was supplied as a coformulated liquid in 90-ml bottles, with 400 mg of lopinavir and 100 mg of ritonavir per 5 ml. All other ARV therapies were administered in dosages recommended by their manufacturer’s package inserts. Medications highly dependent on the hepatic cytochrome P-450 CYP3A isoform for their metabolism, for which elevated plasma concentrations may be (or have been) associated with serious or life-threatening adverse events, were not allowed. These medications included ergot derivatives, astemizole, terfenadine, midazolam, triazolam, cisapride and pimozide.
A thorough medical history was taken, and physical examination was performed during the screening period. The physical examination, including evaluation for the development of HIV-related clinical events, was repeated on enrollment in the study and at each study visit (Days 1, 10 and 21; Weeks 8, 12, 16, 20, 24, 32, 40 and 48; and every 3 months thereafter). A complete set of laboratory analyses (hematology, chemistry and urinalysis) was obtained at each study visit with the exception of the Day 10 and Week 20 visits. Blood samples for the determination of HIV RNA levels [Roche Amplicor HIV-1 assay; limit of quantitation (LOQ) of 400 copies/ml; Roche Diagnostics, Branchburg, NJ] and CD4+ T lymphocyte counts [absolute (cells/mm3) and relative (percent)] were obtained at each study visit with the exception of the Day 10 visit. In addition the Roche Amplicor HIV-1 Monitor Ultrasensitive assay (LOQ, 50 copies/ml; Roche Diagnostics) was used at Weeks 32, 40 and 48. Archive blood samples for the determination of viral phenotype and genotype were obtained from all subjects at baseline and from subjects who did not experience virologic response (viral load >400 copies/ml) at Weeks 24 or 48. Viral phenotype was determined by the PhenoSense HIV assay (ViroLogic, Inc., San Francisco, CA). 10 Viral genotype was determined by population sequencing.
A full PK evaluation was to be performed on the first 20 subjects between 2 and 12 years of age and the first 8 subjects between 3 months and 2 years of age in each dosing arm. After 3 weeks of dosing, plasma samples were to be collected during a 12-h interval (predose; 2, 4, 6, 8 and 12 h after the morning dose; and before the evening dose) for steady state PK analyses of lopinavir and ritonavir. Plasma concentrations of lopinavir and ritonavir were determined by a validated liquid chromatographic method with tandem mass spectrometry detection. The lower limits of quantitation were 5.0 and 1.0 ng/ml for lopinavir and ritonavir, respectively. For the remaining subjects only predose plasma concentrations of lopinavir/ritonavir were measured. The following PK parameters were evaluated: area under the plasma concentration-time curve (AUC), minimum and maximum concentrations throughout a dosing interval (Cmin and Cmax), morning predose plasma concentrations (Ctrough or Cpredose), half-life within a 12-h dosing interval (t1/2) and time to the maximum concentration (Tmax).
Subjects who exhibited inadequate response to therapy could elect to continue study medications; however, the investigator could consider the discontinuation or alteration of the antiretroviral regimen. Criteria for altering antiretroviral therapy included: an HIV RNA level that did not decrease at least 1.0 log10 copies/ml from baseline by Week 12; an HIV RNA level that increased at least 0.5 log10 copies/ml above the nadir at two consecutive visits; an HIV RNA level that never decreased below 400 copies/ml by Week 24; and an HIV RNA level that rose above the LOQ of the viral load assay for two consecutive visits. Subjects discontinuing study therapy were considered to have HIV RNA levels >400 copies/ml at all subsequent time points.
Each subject was closely monitored for the development of any clinical and/or laboratory evidence of an adverse event throughout the course of the study. The investigator rated the adverse event as mild, moderate or severe and judged the relationship of the adverse event to lopinavir/ritonavir as probable, possible, probably not or not related. Reduction of drug doses and temporal or permanent discontinuations followed strict protocol guidelines.
The number of subjects with plasma HIV RNA levels below the LOQ of the viral load assay was used as the primary measure of antiviral activity, whereas the number of subjects who experienced adverse events was used as the primary measure of safety. Assuming a type I error rate of 5% for a two tailed test, the planned sample size of 100 subjects (50 for each treatment arm) allowed for the detection of a 30% difference between treatment arms with 80% power. In addition the number of subjects included in the 12-h PK assessment was determined to provide adequate estimates of the population means for the PK parameters of interest.
Baseline demographic and clinical characteristics were compared between the ARV-naive and ARV-experienced groups with Fisher’s exact test or a one way analysis of variance model with ARV treatment experience as the only effect. Fisher’s exact test was also used to evaluate between-group differences in the proportion of subjects with plasma HIV RNA below the LOQ at each visit. In addition the one way analysis of variance model was used to evaluate between-group differences in the change from baseline to each visit, with respect to both plasma HIV RNA (log10 copies/ml) and CD4 cell counts [absolute (cells/mm3) and relative (percent)].
Noncompartmental methods were used to provide estimates of the PK parameters for individual subjects included in the initial interim PK analysis. For each lopinavir/ritonavir dose level, the (mean) point estimate and 90% confidence interval (CI) was obtained for Cmax, Cmin and AUC for both lopinavir and ritonavir. For each dose level, point estimates were compared with historical PK results obtained from adult subjects.
Subsequent to the interim PK analysis, an analysis of covariance (ANCOVA) model was used to investigate the relationship between lopinavir/ritonavir dose level, age, gender, race and nevirapine coadministration. Because nevirapine coadministration was a significant factor in the ANCOVA model, the point estimate and 90% CI of the bioavailability of subjects receiving concurrent nevirapine relative to those not receiving concurrent nevirapine were computed for each lopinavir/ritonavir dose level.
All statistical tests were two tailed and performed at the overall 0.05 level of significance. Efficacy data have been analyzed using both on-treatment (HIV RNA and CD4 cell counts) and intent-to-treat (HIV RNA only; proportion of subjects with plasma HIV RNA below the LOQ) approaches. Safety data have been summarized with an intent-to-treat approach.
A total of 168 subjects were screened for participation in this study after informed consent was obtained, and 100 subjects were subsequently enrolled into the study from July to September 1999. Of the 68 subjects that were not enrolled, 44 did not meet study entry criteria, 14 were not randomized before enrollment completion, 3 were lost to follow-up before randomization and 1 withdrew consent before randomization. The remaining 6 subjects were not enrolled for other reasons [nevirapine pediatric solution unavailable 9 (n = 3), plasma samples received in ambient condition (n = 2) and investigator decision to discontinue study participation with only 1 eligible subject (n = 1)]. A summary of study subject disposition is presented in Figure 1.
Of 100 subjects enrolled, 49 were randomized to the 230/57.5 mg/m2 BID group and 51 were randomized to the 300/75 mg/m2 BID group. After an initial PK/safety assessment, all eligible subjects were escalated to the 300/75 mg/m2 BID dose for the rest of the study. The mean age of the subjects was 5.3 years, with an age range of 6 months to 12.6 years. Of the 100 subjects 14 were <2 years old. There were 44 ARV-naive subjects and 56 ARV-experienced subjects. Of the 56 ARV-experienced subjects, 32 were PI-naive/NRTI-experienced and 24 were PI-experienced/NRTI-experienced, including 7 who had received multiple PIs. Baseline characteristics of the two ARV experience groups are presented in Table 1. Higher mean viral loads were observed in the ARV-naive group. Age, gender and baseline CD4 cell counts were comparable between the two ARV experience groups. Prior ARV use is summarized in Table 2.
Initial analysis of the steady state 12-h lopinavir PK results from 53 subjects (26 in the 230/57.5 mg/m2 dose group and 27 in the 300/75 mg/m2 dose group) obtained after 3 weeks of dosing showed that the lower lopinavir/ritonavir dosage level resulted in mean lopinavir AUC and Cmin values that were ∼77 and 68% of the adult references, respectively, whereas the higher lopinavir/ritonavir dosage level resulted in AUC and Cmin values that were ∼118 and 127% of the adult references, respectively. Based on results from an initial assessment of the PK, safety and efficacy data (see Methods, Study Design), all eligible subjects were escalated to the 300/75 mg/m2 lopinavir/ritonavir BID dose between Study Days 82 and 141.
In the final analysis of the complete PK data with the use of a statistical model that included additional covariates, including the potential nevirapine effect, it was apparent that nevirapine has a PK interaction with lopinavir/ritonavir in children. Table 3 displays a summary (mean ± sd) of the lopinavir PK parameters for the two dose regimens, further stratified by concurrent nevirapine use. For the lopinavir/ritonavir 300/75 mg/m2 BID regimen, nevirapine coadministration reduced the lopinavir AUC by 22% (90% CI −44%, +9%), Cmax by 14% (90% CI −36%, +16%), Cmin by 55% (90% CI −75%, −18%) and Cpredose by 38% (90% CI −58%, −9%). Ritonavir AUC, Cmax, Cmin and Cpredose were affected in a similar manner (data not shown). When coadministered with nevirapine, the mean lopinavir AUC, Cmax and Cmin at the 300/75 mg/m2 BID dose are very similar to those observed in adults receiving 400/100 mg BID. However, in the absence of nevirapine, the 230/57.5 mg/m2 BID dose produces lopinavir concentrations that are very similar to those observed in adults. Figure 2 presents mean concentration-time profiles for lopinavir concentrations in pediatric subjects at the 300/75 mg/m2 dose during nevirapine coadministration and 230/57.5 mg/m2 dose in the absence of nevirapine, as well as mean concentrations (with interquartile ranges) observed in a Phase II study in adults. 8, 11
Within the range of 6 months to 12 years, the PK of lopinavir did not appear to be dependent on age (P > 0.10 for AUC, Cmax, Cmin and Cpredose from ANCOVA). Further there did not appear to be a significant relationship between lopinavir PK and race or gender (P > 0.10; data not shown). No difference in the proportion of subjects with HIV RNA <400 copies/ml was observed between ARV-naive subjects taking the 230/57.5 or 300/75 mg/m2 lopinavir/ritonavir BID dose at Week 12 [82% (18 of 22) vs. 82% (18 of 22);P > 0.999], around the time of the dose escalation of all subjects to the 300/75 mg/m2 lopinavir/ritonavir BID dose. However, ARV-experienced subjects receiving the 230/57.5 mg/m2 lopinavir/ritonavir BID dose tended to have a lower virologic response rate at Week 12 compared with subjects receiving the 300/75 mg/m2 lopinavir/ritonavir BID dose [63% (17 of 27) vs. 72% (21 of 29);P = 0.570]. At Week 24, after all subjects were dose-escalated to 300/75 mg/m2 lopinavir/ritonavir BID, no significant difference was detected between either the ARV-naive subjects [86% (19 of 22) vs. 77% (17 of 22);P = 0.698] or the ARV-experienced subjects [63% (17 of 27) vs. 69% (20 of 29);P = 0.779] based on the originally randomized dose group.
HIV RNA response
The proportions of subjects with HIV RNA <400 copies/ml at Week 48 using an intent-to-treat (ITT, missing = failure) analysis were 79% (79 of 100) for all subjects enrolled, 84% (37 of 44) for ARV-naive subjects and 75% (42 of 56) for ARV-experienced subjects (Table 4). Because only two subjects discontinued the study before Week 48, results from the on-treatment analysis were similar to those from the ITT analysis [86% (37 of 43) and 76% (42 of 55) for ARV-naive and ARV-experienced subjects, respectively]. When ARV-experienced subjects were further stratified by prior PI use, the proportion of subjects with HIV RNA <400 copies/ml at Week 48 was 88% (28 of 32) for PI-naive/NRTI-experienced subjects and 58% (14 of 24) for PI-experienced/NRTI-experienced subjects by ITT analysis (Fig. 3). Based on results from this stratified analysis, subjects in the ARV-naive group and the PI-naive/NRTI-experienced group had significantly higher percentages with plasma HIV RNA <400 copies/ml compared with subjects in the PI-experienced/NRTI-experienced group (P = 0.038 and P = 0.027, respectively).
The proportions of subjects with HIV RNA <50 copies/ml at Week 48 according to ITT analysis were 71% (31 of 44) for ARV-naive subjects and 63% (35 of 56) for ARV-experienced subjects. When ARV-experienced subjects were further stratified by prior PI use, the proportions of subjects with HIV RNA <50 copies/ml were 69% (22 of 32) for PI-naive/NRTI-experienced subjects and 54% (13 of 24) for PI-experienced/NRTI-experienced subjects (not statistically significantly different).
For those subjects with plasma HIV RNA >400 copies/ml at Week 48, the corresponding mean decreases from baseline were 1.60 log10 copies/ml for ARV-naive subjects and 0.64 log10 copies/ml for ARV-experienced subjects (P = 0.035;Table 4).
Samples from ARV-naive and PI-naive/NRTI-experienced subjects who did not have virologic response (viral load >400 copies/ml) at Weeks 24 or 48 were analyzed for evidence of genotypic or phenotypic resistance. No detectable change in phenotypic susceptibility to lopinavir was apparent in any of the rebound isolates (n = 14) compared with the corresponding baseline isolates. Similarly no new primary or active site mutation associated with PI resistance 12 occurred in any of the rebound isolates (n = 13; one rebound isolate did not have genotype available) compared with the corresponding baseline isolates. The M184V mutation in reverse transcriptase, which confers resistance to 3TC, was observed in the rebound isolates of all 13 ARV-naive and PI-naive/NRTI-experienced subjects. In the PI-naive/NRTI-experienced subjects who received nevirapine, new mutations associated with resistance to the NNRTI class were observed in six of eight rebound isolates.
CD4 cell response
Figure 4 shows the relative (percent) CD4 cell count response over time for the ARV-naive and ARV-experienced subjects. ARV-naive subjects had a larger mean relative (percent) CD4 cell count increase from baseline to Week 48 when compared with ARV-experienced subjects (10.3%vs. 5.9%;P < 0.001). ARV-naive subjects also had a larger mean relative CD4 cell count increase from baseline to Week 48 than did either PI-naive/NRTI-experienced subjects (5.3%;P < 0.001) or PI-experienced/NRTI-experienced subjects (6.7%;P = 0.037); however, no significant difference was detected between the PI-naive/NRTI-experienced and PI-experienced/NRTI-experienced subjects (P = 0.419). Mean absolute CD4 cell count increases from baseline to Week 48 of 404 cells/mm3 and 284 cells/mm3 were observed for ARV-naive and ARV-experienced subjects, respectively. Further, 38 of 43 (88%) subjects with a baseline CD4 cell count ≤24% experienced an improvement of at least one CDC immunologic category 13 through Week 48, whereas only 1 of 57 (2%) subjects with baseline CD4 cell count >24% experienced a decline of at least 1 CDC immunologic category.
Few adverse events of at least moderate severity and of probable or possible relationship to the study drug were reported (Table 5). Rash was reported in two subjects, one of whom also received nevirapine; in both cases rash resolved after transient interruption of the study drugs and did not recur on rechallenge. No other adverse event meeting these criteria was reported for more than one pediatric subject participating in this study. Similarly few subjects exhibited Grade 3 or 4 laboratory abnormalities (Table 5). Dosing was interrupted intermittently for one subject with increased aspartate transaminase and one subject with increased amylase. In most cases the investigator did not consider laboratory alterations to be related to study drug; alternative etiologies included development of Burkitt’s lymphoma, potential liver toxicity to nevirapine (which did not recur after rechallenge with lopinavir/ritonavir) and reactivation of chronic Epstein-Barr virus. All six subjects who developed Grade 3 or 4 amylase elevations while participating in this study also had elevated serum amylase values at baseline.
Discontinuation of study drug
Of the 100 subjects enrolled in this study, 2 prematurely discontinued treatment through the Week 48 visit. One discontinuation was because of development of Burkitt’s lymphoma (HIV-related) and complications of its chemotherapy. This subject developed Burkitt’s lymphoma on Day 33 and died on Day 110 (17 days after discontinuation of study treatment). The second discontinuation was because pancreatitis developed, reported as possibly related to study drug. This subject had a Grade 2 elevation in serum amylase at baseline and discontinued study treatment on Day 239.
Despite receiving ARV therapy at the time of their enrollment in this clinical trial, 29% of ARV-experienced subjects had viral loads of >100 000 copies/ml, and 13% had CD4 cell counts <15% at baseline. Significant reductions in viral loads and increases in CD4 counts were observed as early as Week 8 and continued to improve or were maintained for the entire 48-week study period. Previous studies in predominantly ARV-naive pediatric subjects have reported virologic response rates (HIV RNA <400 copies/ml) ranging from 0 to 65%. 1–6 However, the rates observed in this study for the ARV-naive, PI-naive/NRTI-experienced and PI-experienced/NRTI-experienced subjects were 84, 88 and 58%, respectively. In addition sustained increases in CD4 percentages were observed throughout the study period.
The lack of genotypic or phenotypic PI resistance demonstrated by viral isolates obtained from the ARV-naive subjects with plasma HIV RNA >400 copies/ml at Week 24 and/or Week 48 is consistent with results from a larger Phase III study in adults in which no PI resistance was observed in 37 subjects with amplifiable genotype after viral rebound on lopinavir/ritonavir therapy. 14
Metabolic abnormalities, such as elevations of cholesterol, triglycerides and glucose, which have been observed with the use of PI-based combination therapy in adults, were uncommon in this study (two subjects developed Grade 3 elevations in cholesterol, whereas one additional subject developed Grade 3 elevations in both cholesterol and triglycerides). Few children had transaminase elevations, which are observed in adults receiving PI-based therapy. The most common laboratory alteration reported in this pediatric study was hyperamylasemia, observed in 6% of the subjects; however, elevated amylase concentrations were present at baseline in all of them, and one subsequently developed pancreatitis.
Results from this Phase I/II pediatric clinical trial demonstrate that a liquid coformulation of lopinavir/ritonavir was safe and well-tolerated by HIV-infected children, given that only two subjects discontinued treatment with lopinavir/ritonavir through 48 weeks. Administration of lopinavir/ritonavir was associated with substantial and persistent antiviral activity and immune function as manifested by the high percentage (79%) of subjects with plasma viral load <400 copies/ml and the increases in relative (percent) CD4 counts in most subjects at Week 48. Pharmacokinetic results indicate that a lopinavir/ritonavir dosing regimen of 300/75 mg/m2 BID in combination with an inducing NNRTI such as nevirapine provides lopinavir concentrations similar to those in adults at usual clinical doses, whereas a lopinavir/ritonavir dosing regimen of 230/57.5 mg/m2 BID provides lopinavir concentrations approximating adult exposure in the absence of an inducing NNRTI such as nevirapine.
The safety, tolerability and efficacy exhibited by lopinavir/ritonavir-containing regimens in the present study warrant their use in HIV-infected children. Although results from post hoc subgroup comparisons should be interpreted with caution, the virologic and immunologic response exhibited by ARV-naive subjects compared with ARV-experienced subjects suggests that the clinical benefit achieved with lopinavir/ritonavir might be compromised by the extent of prior ARV treatment experience.
We thank the children who participated in the trial, the children’s families/caregivers, the coinvestigators and the clinical coordinators at the study sites. T his work was supported by Abbott Laboratories, manufacturer of lopinavir/ritonavir (Kaletra).
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