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Early Virologic Rebound in a Pilot Trial of Ritonavir-Boosted Atazanavir as Maintenance Monotherapy
 
 
  Olle Karlstrom, MD,Filip Josephson, MD, and Anders Sonnerborg, MD, PhD Department of Infectious Diseases, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden; Department of Clinical Pharmacology, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden; and Department of Clinical Virology, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden. Funded by grants from the Swedish Research Council.
 
JAIDS early online publication Dec 7, 2006
 
"....In summary, these findings, including seemingly higher rates of viremia with monotherapy compared with triple therapy, reports of de novo development of PI mutations in treatment-naive patients, and suboptimal viral suppression in the CNS in a recently presented trial of boosted atazanavir monotherapy, lead us to question the relevance and ethics of conducting further large-scale monotherapy studies of boosted PIs, particularly because easily administered combination pills of NRTIs with a low toxicity profile are now available...." [see Discussion below]
 
ABSTRACT

Objective: To investigate the feasibility of ritonavir-boosted atazanavir monotherapy in HIV-1-infected patients with stable antiretroviral therapy (ART).
 
Design: Single-armed single-center pilot trial.
 
Methods: Adult HIV-1-infected patients, without protease inhibitor (PI) experience, were eligible if they had maintained a viral load <20 copies/mL for a minimum of 12 months on conventional ART. The trial regimen was atazanavir/ritonavir at a dose of 300/100 mg once daily. The atazanavir dose could be adjusted if plasma concentrations showed a low exposure. The study was intended to recruit 30 patients to be followed over 72 weeks. If 5 cases of virologic failure occurred during this period, the study was to be terminated.
 
Results: The study was terminated according to protocol when 15 of the planned 30 patients had been recruited, because 5 cases of virologic failure had occurred. In patients failing therapy, viral rebound was seen at weeks 12 through 16. Plasma atazanavir concentrations were not associated with the outcome. The median serum bilirubin concentration was significantly lower in the patients failing therapy, however. No PI resistance was found in samples from patients failing therapy.
 
Conclusions: Ritonavir-boosted atazanavir as maintenance monotherapy in HIV-1 infection might not be as potent as conventional ART. Serum bilirubin should be further studied as a biomarker of adequate atazanavir exposure.
 
Introduction
Presently, the recommended initial therapy for HIV-1 infection consists of 2 nucleoside reverse transcriptase inhibitors (NRTIs) combined with 1 nonnucleoside reverse transcriptase inhibitor (NNRTI) or 1 protease inhibitor (PI). Important longterm adverse effects include lipoatrophy, fat accumulation, and the metabolic syndrome, with component drugs often acting in synergy. Thus, it would be of great value if treatment could be simplified using as few drugs as possible, with these preferably being those with the least long-term toxicity.
 
The drug combinations are characterized by a high antiretroviral potency and a high genetic barrier to drug resistance. Both of these features are crucial for treatment success. It is notable that these qualities seem to be present in ritonavirboosted PIs in the absence of other antiretroviral drugs. For instance, the short-term potency of ritonavir-boosted lopinavir was not significantly different when combined with 2 NRTIs.1a Furthermore, loss of virologic efficacy requires several mutations in the HIV-1 genome. Indeed, no development of PI mutations has been found in clinical trials of previously untreated patients when using boosted PIs along with 2 NRTIs.1,2 Thus, successful long-term monotherapy with a boosted PI might be possible.
 
When PIs are used without ritonavir boosting, drug exposures are significantly lower. In this situation, the resistance barrier is confined to a single key mutation. This could be a reason why early trials exploring maintenance therapy using 1 PI or 1 PI plus 1 NRTI were disappointing, with a high frequency of virologic rebound.3-5 Several recently reported pilot trials exploring maintenance monotherapy with ritonavir-boosted indinavir,6 lopinavir,7-9 and, just recently, atazanavir10,11 have shown more promising results, with a lower frequency of viral rebound. Our pilot trial of maintenance monotherapy was conducted using boosted atazanavir because it has the most favorable metabolic profile12 among the approved boosted PIs and is easily administrated once daily. The trial included carefully selected patients previously naive to PI therapy, who had maintained virologic suppression for a minimum of 1 year.
 
METHODS
Study Design

Patients were recruited from the HIV clinic of the Karolinska University Hospital in Stockholm. The inclusion criteria were (1) HIV-1 infection, with no prior history of PI therapy; (2) ongoing triple-antiretroviral therapy (ART) with an HIV-1 RNA load <20 copies/mL for a minimum of 12 months before inclusion as determined by routine monitoring (ie, 2-3 times per year [latest HIV-1 RNA test ,30 days before baseline]); (3) age older than 18 years; and (4) in women, a negative pregnancy test result. Patients who reported taking concomitant medications known to interact with the study regimen and/or substance abuse were excluded.
 
Patients who fulfilled the inclusion criteria were switched from their prior regimen to the study medication at baseline. Follow-up visits were scheduled at weeks 4, 8, 12, 16, and 24 and every 3 months thereafter until week 72. At each visit, the following laboratory analyses were performed: CD4+ T-cell count; HIV-1 RNA load; complete blood cell count; and liver, kidney, and pancreatic function tests. Blood glucose, p-cholesterol (total, low density lipoprotein [LDL], and high density lipoprotein [HDL] cholesterol), and p-triglycerides were sampled while fasting; at the visits in study weeks 0, 4, and 12; and every 3 months thereafter. The plasma trough concentration (Ctrough) of atazanavir was determined at weeks 4 and 8 and at a presumed steady state after any dose adjustment.
 
The primary endpoint was the number of patients completing 72 weeks of therapy without experiencing virologic failure. The latter was defined as 2 consecutive plasma HIV-1 RNA samples above the limit of detection (>20 copies/mL). If the HIV-1 RNA level was above the limit of detection at any time, a new sample was drawn after 4 weeks. If the HIV-1 RNA level was still detectable at this time, the patient was thus defined as experiencing virologic treatment failure and the trial regimen was discontinued. If 5 such failures occurred among the 30 planned study participants, the study was to be terminated early. Secondary endpoints were the relation between atazanavir drug concentrations and outcome and changes in metabolic parameters.
 
Study Medication and Dosing
The drug regimen specified by the protocol was atazanavir/ritonavir at a dose of 300/100 mg once daily in a flat dose, to be taken with food. The first patient to fail (subject 3) exhibited low drug levels despite adequate compliance. The ritonavir booster dose was increased to 200 mg once daily, which did not affect the atazanavir concentration, however. Shortly thereafter, sufficient data were available at the Department of Clinical Pharmacology, Karolinska University Hospital, to distinguish adequately the range of atazanavir plasma Ctrough measurements reached in treatment with the ritonavir-boosted atazanavir regimen (presumed not to be inferior to lopinavir/ritonavir) and the range seen in treatment with the unboosted 400-mg once-daily regimen (which has failed to show a lack of inferiority with the same regimen). On this basis, the study protocol was changed, allowing a dose increase of atazanavir from 300 to 400 mg once daily (and possibly higher) in patients with a plasma concentration less than 500 ng/mL, if this was possible without inducing manifest jaundice. The ritonavir dose was specified always to remain at 100 mg once daily.
 
Adherence
The study drugs were dispensed to the participants at each study visit. Patients were instructed to return their used pill boxes. Adherence was assessed by pill counting and with patient interviews concerning missed doses since the last study visit.
 
Sample Size Considerations and Statistics
This was a 1-armed pilot study designed to examine the feasibility of treatment simplification with ritonavir-boosted atazanavir monotherapy in patients on stable triple ART. Historical data from our clinic indicated that the virologic treatment failure rate in compliant patients lacking background resistance who were treated with standard ART regimens would be ,3% over a 72-week period (equivalent to 1 in 30 patients). Five virologic failures in 30 patients (17%) over the study period would suggest an inferior efficacy to standard treatment. In this event, the trial was to be terminated early. The proportion of patients with jaundice using ritonavirboosted atazanavir has been approximately 10% in clinical trials (equivalent to 3 in 30 patients). We wanted to study the outcome of body fat composition after 72 weeks of treatment for 20 patients. On the basis of these considerations, a sample size of 30 participants was chosen. Between-group differences in total serum bilirubin and plasma atazanavir were compared by nonparametric analysis using the Mann-Whitney U test.
 
RESULTS
 
The first patient entered the study on October 14, 2004, and the last to be included (subject 16) was started on treatment on July 4, 2005. The study was terminated before complete recruitment at the end of November 2005 at the time of the fifth virologic failure (subject 15), as defined by the protocol. Ten patients switched to the study medication from an NNRTI-based regimen (4 failures), and 5 switched from abacavir/lamivudine/zidovudine (fixed-dose combination) (1 failure).
 
At the time of study termination, 32 patients had been asked to participate and 15 had been included from the T1 planned sample of 30 (Table 1).
 
The first patient with virologic treatment failure was repeatedly shown to have a low plasma concentration of atazanavir (250 ng/mL), despite an apparently high level of adherence and no interacting drugs. The dose of ritonavir was increased to 200 mg daily in this patient. This procedure did not increase the atazanavir concentration. The dose of atazanavir was increased in 3 patients (see section on study medication and dosing), 1 of whom had virologic treatment T2 failure (Table 2).
 
At study closure, 9 patients had had a plasma HIV-1 RNA load <20 copies/mL at all tested occasions, with a median observation time of 36 weeks (range: 16-48 weeks). One patient had stopped therapy because of jaundice (week 12). This patient had undetectable HIV RNA until exiting the trial. Five subjects had developed virologic treatment failure. Loss of virologic suppression was seen at week 12 in 3 participants and at week 16 in 2 subjects (see Table 2). There were no major differences between patients without virologic failure and those with failure regarding CD4+ T-cell nadir (median: 190 vs. 240 cells/mL), HIV-1 RNA level before the start of HIV therapy (median: 5.0 log vs 5.0 log copies/mL of plasma), or duration of undetectable HIV-1 RNA level before study entry (median: 25 vs. 20 months).
 

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Patients With Virologic Treatment Failure
Resistance

Genotypic resistance testing had been performed before the first HIV therapy in 1 of the 5 patients with virologic failure. No PI mutations were seen at that time. Using our standard genotypic assay, it was possible to sequence 1 sample from 1 of the failing subjects (subject 3: 3400 copies/mL). Using the more sensitive method, an additional 2 samples from each of the first 3 failing subjects (subjects 3, 8, and 10) were sequenced. Neither of these samples showed any primary resistance mutations, including I50L (data not shown).
 
Virologic Outcome in Relation to Atazanavir Trough Plasma Concentrations, Serum Bilirubin Levels, and Adherence
The median plasma Ctrough measurement did not differ between the 5 subjects with virologic treatment failure and the 7 patients without virologic treatment failure for whom Ctrough measurements were available (629 ng/mL; range: 250- 1529 ng/mL vs. 800 ng/mL; range: 171-1364 ng/mL; not significant [ns]). Neither did the average intraindividual coefficient of variation (CV) of repeat Ctrough measurements differ (0.39 vs. 0.47 [ns]). The median serum bilirubin concentration recorded during treatment was significantly higher in subjects on treatment not failing virologically than in those experiencing virologic failure (55.3 vs. 33.7 mmol/L; P = 0.014, Mann-Whitney U test). The distributions of average bilirubin concentrations in subjects failing and not failing virologically are shown in Figure 1. The median adherence rate (measured by F1 pill count) was 100% (range: 79%-100%). Adherence was 95% to 100% in all subjects except 2. One of these subjects (subject 10) had an adherence rate of 79%. This subject failed virologically. Her average Ctrough measurement, however, was the highest of all subjects (1529 ng/mL, CV = 0.44), and her average total serum bilirubin level was higher than the median of the subjects experiencing virologic failure. For the other subject (subject 11, treatment success, average Ctrough measurement = 986 ng/mL), adherence data were missing.
 
Interacting Drugs
In violation of the study protocol, acid-suppressing drugs known to interact with atazanavir were used by 2 subjects before viral failure; subject 8 was prescribed lansoprazole at a dose of 30 mg once daily to be used no more than occasionally and to be taken at least 3 hours after intake of atazanavir. The Ctrough measurement of atazanavir in this subject was greater than 714 ng/mL while using lansoprazole. Subject 10 was prescribed ranitidine at a dose of 300 mg once daily to be used only when needed, with the intake scheduled a few hours after atazanavir dosing. The atazanavir drug concentrations were closely monitored, with values ranging from 929 to 1643 ng/mL. None of the other patients (3 with failure, 9 without failure, and 1 stopping because of icterus) reported that they had taken any drugs known to interact with atazanavir or ritonavir during the course of the trial.
 

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Follow-Up
Four of the participants with virologic failure returned to their previous treatment regimens, whereas 1 kept to the study regimen, with the addition of 2 NRTIs. All 5 regained viral loads below the limit of detection after the change in therapy; the median follow-up was 7 months (range: 5-12 months).
 
Other Analyses
The rise in CD4+ T-cell counts on the study regimen seemed consistent with the increase on previous regimens. Because of the short-term follow-up for most patients, however, this was not further analyzed (data not shown). No significant changes were seen in levels of glucose, cholesterol (total, LDL, and HDL cholesterol) or triglycerides (data not shown).
 
FIGURE 1. The mean plasma Ctrough atazanavir and serum bilirubin measurements during the study in patients with virologic success (diamonds) or failure (squares). The median serum bilirubin concentration was significantly higher in patients classified as having virologic success (P = 0.014, Mann-Whitney U test), whereas the plasma Ctrough measurements did not differ significantly between groups.
 

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DISCUSSION
This pilot trial was conducted to study the feasibility of ritonavir-boosted atazanavir monotherapy as maintenance therapy of HIV-1 infection. The study was carefully performed at a single center, and only patients judged to be compliant were included. Subjects included were naive to PIs so as to minimize the risk of preexisting PI resistance. HIV-1 RNA had to have been below the limit of detection (<20 copies/mL) for a minimum of 1 year before study entry. Thereby, only patients with a low remaining viral burden with less risk for the development of resistance were included. Despite this rigorous patient selection, 5 patients failed virologically at a time when only 15 of planned 30 participants had been included. Therefore, the study was stopped according to protocol.
 
The first patient to fail in our study showed repeatedly low atazanavir concentrations, despite, apparently, being fully compliant with the study regimen and not using any interacting drugs. After this, the protocol was changed, based on local therapeutic drug monitoring (TDM) experiences (Josephson et al, manuscript submitted), to enable an increase in atazanavir dose until a Ctrough of >500 ng/mL was reached, if this was possible without inducing manifest jaundice. Despite this, another 4 patients failed. In the whole study population, no correlation was seen between atazanavir drug concentrations and virologic outcome.
 
At screening, we thoroughly discussed the interaction issue with each participant, with special regard to acid-suppressing drugs of all kinds. Two participants still used such agents, however. Both of these patients experienced virologic failure. In both subjects, the atazanavir concentration was closely monitored and was found to be well above the calculated minimum of 500 ng/mL. Increased indirect bilirubin, reflected as an increase of total serum bilirubin, is an exposure-dependent side effect of atazanavir caused by inhibition of UGT1A1, the enzyme that conjugates bilirubin. The magnitude of the serum bilirubin increase at a given atazanavir exposure is variable and depends to some extent on the genotype in the UGT1A1 promoter.13 Normal serum bilirubin values during atazanavir therapy have been associated with treatment failure; therefore, serum bilirubin has been suggested as a biomarker of adequate atazanavir exposure.14 In this study, the average atazanavir Ctrough measurements did not differ between subjects failing virologically and those who did not. Yet, the average serum bilirubin value was significantly lower in those who failed. One interpretation of our finding might be that the drug concentrations measured in the patients with virologic failure reflect improved drug adherence in the period immediately preceding a scheduled visit to the clinic and that serum bilirubin, to some extent, functions as a marker of long-term atazanavir exposure. Two sets of facts, however, are incongruent with this hypothesis. First, our adherence data do not support lower drug adherence in the failing patients. Second, published data on the kinetics of radioactively labeled unconjugated bilirubin imply a typical terminal half-life of approximately 10 hours,15 which is of the same magnitude as the half-life reported for ritonavir-boosted atazanavir.16 Thus, it is not evident that measurement of serum bilirubin would be a more powerful marker of cumulative atazanavir exposure than measurement of the actual drug concentration. Another possible interpretation of our finding, assuming that is it not attributable to pure chance, is that the degree of serum bilirubin increase at a given atazanavir exposure may not only be related to the UGT1A1 promoter genotype but to the intracellular availability of atazanavir. The site of drug action of PIs is intracellular, as is the site of action of atazanavir as an UGT1A1-inhibitor. In vivo intracellular drug concentration measurement is complicated and is seldom performed. Therefore, little is known about the relation between plasma and intracellular atazanavir concentrations. It is interesting to speculate that the low serum bilirubin values seen in patients experiencing virologic failure might correlate with low intracellular availability of atazanavir. This hypothesis should be further investigated.
 
Several pilot trials of boosted lopinavir and atazanavir monotherapy have recently been published or otherwise reported. In the study by Swindells et al,10 3 of 34 subjects treated with ritonavir/atazanavir maintenance monotherapy had failed virologically at week 24 (failure defined as plasma HIV RNA level >200 copies/mL on 2 consecutive occasions). Such a rate of viral rebound seems greater than would be expected in a highly compliant treatment population using a traditional triple-ART regimen. In a study by Arribas et al7 comparing lopinavir/ritonavir maintenance monotherapy with continued triple therapy, there were 3 virologic failures among 21 patients allocated to monotherapy as opposed to 1 failure among 21 patients with triple therapy. In these 2 trials, no PI resistance mutations were detected in patients with virologic failure. This was also the case in our study. A larger trial by Cameron et al8 included treatment-naive patients who were randomized to lopinavir/ritonavir monotherapy (preceded by 6 months of induction, including 2 NRTIs) or efavirenz-based triple therapy. Only 62% of the patients receiving lopinavir/ritonavir maintained an undetectable viral load throughout 72 weeks compared with 91% of the patients in the triple arm. Although most of the monotherapy patients who experienced viral rebound were resuppressed to <50 copies/mL without a change in the treatment regimen, 2 patients showed de novo PI mutations. In a third study by Norton et al9 comparing lopinavir/ritonavir monotherapy (from the start of therapy) versus lopinavir/ritonavir triple therapy, intermittent viremia was significantly more common in the monotherapy group. Also in this study, cases of de novo PI resistance were seen. Thus, although de novo resistance has not been reported in some monotherapy trials, including ours, it has been demonstrated to occur not only during monotherapy without prior induction but during maintenance monotherapy with a boosted PI. To place this in proper perspective, it should be noted that an analysis of 4 clinical trials enrolling 654 treatment-naive patients starting a lopinavir/ritonavir-based triple regimen, with a follow-up of 2 to 7 years, did not show any selection of PI resistance mutations in patients experiencing viremia.17-19
 
None of the previously mentioned monotherapy studies, including ours, have investigated HIV RNA levels in specialized physiologic compartments, such as the central nervous system (CNS), during PI monotherapy. This has been undertaken in a hitherto unpublished study of atazanavir/ritonavir maintenance monotherapy by Vernazza et al.11 After 24 weeks of therapy, 3 of 19 liquor samples showed detectable HIV RNA (2.2, 2.9, and 3.8 log10), despite undetectable levels of HIV RNA in plasma. Interestingly, all detectable cerebrospinal fluid (CSF) samples contained wild-type virus, which implies that in PI monotherapy, patients may not reach sufficient antiretroviral drug levels in the CNS. These worrisome results are in contrast with results obtained with 2 NRTIs plus 1 PI.20 The frequency of viral rebound seemed quite high in our study compared with some studies just mentioned. This can partly be explained by our stricter criteria for defining virologic failure, which were set to minimize the risk of resistance development. Furthermore, 2 of 4 patients with virologic failure intermittently used interacting acid-suppressing drugs (lansoprazole and ranitidine, respectively), which was specifically prohibited by our protocol. Excluding such use in large subpopulations may actually be difficult in routine clinical practice. The clinical consequences of cotreatment with acid-suppressing drugs in triple ART based on ritonavir-boosted atazanavir are not fully clarified. Our results imply the quite feasible notion that the deleterious effects of this interaction may be particularly acute in the setting of atazanavir monotherapy. It could be argued that residual CYP3A induction by the discontinued NNRTI, and consequent lower initial atazanavir exposures, might have affected the outcome of our study. Indeed 4 of 10 patients who switched from an NNRTI-based regimen failed, compared with 1 of 5 who switched from a triple NRTI-regimen (ie, zidovudine/lamivudine/abacavir fixed-dose combination). Loss of viral suppression was not seen until 3 to 4 months after switching therapy, and all cases of virologic failure showed wild-type virus. Furthermore, in the other mentioned studies, where NNRTIs were not part of the prior regimen, the timing of viremia was similar to that seen in our study (ie, at weeks 12-20). For these reasons, we find this hypothesis unlikely.
 
In summary, these findings, including seemingly higher rates of viremia with monotherapy compared with triple therapy, reports of de novo development of PI mutations in treatment-naive patients, and suboptimal viral suppression in the CNS in a recently presented trial of boosted atazanavir monotherapy, lead us to question the relevance and ethics of conducting further large-scale monotherapy studies of boosted PIs, particularly because easily administered combination pills of NRTIs with a low toxicity profile are now available. The finding that serum bilirubin was significantly correlated with virologic outcome in our material, although plasma concentrations of atazanavir were not, should be further explored. Serum bilirubin is less expensive than TDM, and might be better and more readily accessible as a marker of adequate atazanavir exposure.
 
 
 
 
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