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Interruption or deferral of antiretroviral therapy reduces markers of bone turnover compared with continuous therapy: The SMART Body Composition substudy
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Journal of Bone and Mineral Research Jan 8 2013
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.
Jennifer Hoy1, Birgit Grund2, Mollie Roediger2, Kristine E. Ensrud3, Indira Brar4, Robert Colebunders5, Nathalie De Castro6, Margaret Johnson7, Anjali Sharma8, Andrew Carr9, for the INSIGHT SMART Body Composition Substudy Group
1 The Alfred Hospital and Monash University, Melbourne, Australia; 2 University of Minnesota, Minneapolis, USA; 3 VA Medical Center and University of Minnesota, Minneapolis, USA; 4 Henry Ford Hospital, Detroit, USA; 5 Institute of Tropical Medicine, HIV AIDS Center, Antwerp, Belgium; 6 AP-HP, Groupe Hospitalier Lariboisiere Saint-Louis, F-75010, Paris, France; 7 Royal Free Hospital, London, UK; 8 SUNY Downstate Medical Center, New York, USA; 9 St. Vincent's Hospital and University of New South Wales, Sydney, Australia
Abstract
Bone mineral density (BMD) declines significantly in HIV patients on antiretroviral therapy (ART). We compared the effects of intermittent versus continuous ART on markers of bone turnover in the Body Composition substudy of the Strategies for Management of AntiRetroviral Therapy (SMART) trial and determined whether early changes in markers predicted subsequent change in BMD. For 202 participants (median age 44 years, 17% female, 74% on ART) randomised to continuous or intermittent ART, plasma markers of inflammation and bone turnover were evaluated at baseline, months 4 and 12; BMD at the spine (dual X-ray absorptiometry [DXA] and computed tomography) and hip (DXA) was evaluated annually. Compared to the continuous ART group, mean bone-specific alkaline phosphatase (bALP), osteocalcin, procollagen type 1 N-terminal propeptide (P1NP), N-terminal cross-linking telopeptide of type 1 collagen (NTX), and C-terminal cross-linking telopeptide of type 1 collagen (ßCTX) decreased significantly in the intermittent ART group, whereas RANKL and the RANKL:osteoprotegerin (OPG) ratio increased (all p<0.002 at month 4 and month 12). Increases in bALP, osteocalcin, P1NP, NTX, and ßCTX at month 4 predicted decrease in hip BMD at month 12, while increases in RANKL and the RANKL:OPG ratio at month 4 predicted increase in hip and spine BMD at month 12. This study has shown that compared with continuous ART, interruption of ART results in a reduction in markers of bone turnover and increase in BMD at hip and spine, and that early changes in markers of bone turnover predict BMD changes at 12 months.
Data was presented in part at the 13th International Workshop on Adverse Drug Reactions and Comorbidities in HIV, Rome, Italy, 14-16 July 2011
Introduction
Although adults infected with the human immunodeficiency virus (HIV) have increased fracture risk (1) and lower bone mineral density (BMD) compared with the general population (2-4), the relative contributions of HIV infection versus the use of antiretroviral therapy (ART) are unknown. The immune system also plays a key role in bone homeostasis, as both B and T lymphocytes regulate osteoclast activity (5), and higher circulating levels of interleukin 6 (IL-6) are independently associated with greater bone loss (6). In untreated HIV infection, osteocalcin is lower, and beta C-terminal cross-linking telopeptide of type 1 collagen (ßCTX) is higher, respectively, with more advanced HIV immunodeficiency (7-9). HIV may induce bone resorption by several mechanisms including increased receptor activator for nuclear factor-Κß ligand (RANKL) expression through chronic immune activation and increased synthesis of pro-inflammatory cytokines (10). Initiation of combination ART is associated with increases in markers of bone turnover, identified by increases in levels of both bone formation and resorption markers (7, 11-12). For ART-naïve patients initiating abacavir-lamivudine or tenofovir-emtricitabine with efavirenz, bone turnover markers increased to 24 weeks after ART initiation and remained relatively stable thereafter, and the increase in markers of bone turnover correlated with reduction in BMD, with r values ranging from -0.19 to -0.40 (11).
No prospective study has investigated the effects of cessation of ART on markers of turnover and inflammation, nor correlated changes in markers of bone turnover and inflammatory markers with subsequent changes in BMD on cessation of ART.
The INSIGHT Strategies for Management of AntiRetroviral Therapy (SMART) study was an international, randomized strategy treatment trial comparing intermittent, CD4+ T lymphocyte count-guided ART with continuous ART. BMD was measured at baseline and annually in a substudy (n=275) (13-14). In the intermittent ART group, participants stopped or deferred ART at baseline, and BMD increased or remained stable during the first year after study entry. In contrast, BMD decreased in the continuous ART group, by 0.8% per year at the hip and 0.4% per year at the spine by DXA, over 2.4 years mean follow-up (14). In order to investigate why stopping/deferring ART resulted in higher BMD compared with continuous ART, we evaluated markers of bone turnover and inflammation at baseline, month 4 and month 12. The aims of the analyses were: (1) To evaluate the effect of intermittent compared with continuous ART on bone turnover markers, regulators of bone turnover and inflammatory markers by comparing the randomized treatment groups in a subset of the SMART study; and (2) To determine whether early changes in biomarkers (baseline to month 4) predict change in BMD at the lumbar spine and hip at 12 months.
Overall Study Design
Participants in SMART (n=5472) were randomized to two groups: (i) intermittent, CD4+ T cell count-guided ART, where ART was stopped or deferred at study entry, and re-started when the CD4+ T cell count declined below 250 cells/μL, and stopped again at CD4+ T cell counts above 350 cells/ μL; or (ii) continuous ART. (13) ART drugs were not protocol-specified. The SMART study was approved by the institutional review board (IRB) or ethics committee at each clinical site. All participants provided written, informed consent. The study was registered at ClinicalTrials.gov: NCT00027352.
Discussion
This is the first randomized study in HIV patients to compare changes in markers of bone turnover and inflammation between continuous ART (continue or start ART) and intermittent ART (stop or defer ART, and resume ART at CD4 counts <250 cells/μL). Relative to continuous ART, we found significant reductions in markers of both bone resorption and bone formation in the intermittent ART group. Markers of bone formation and resorption both decreased from baseline to month 4, and remained stable thereafter to month 12 in the intermittent ART group. Conversely, these markers tended to increase in the continuous ART group.
The RANKL: OPG ratio increased in the intermittent ART group by Month 4, and remained elevated through Month 12. In some studies of postmenopausal osteoporosis, increased RANKL:OPG was associated with net bone loss, while no association with BMD was demonstrated in others (19). In our study the association was inverse; higher RANKL:OPG ratios were associated with increased BMD and reduction in other markers of bone resorption. The inverse association was also seen in a prospective study in 87 patients with HIV, which reported significant reductions in levels of RANKL and OPG as well as inflammatory markers,on initiation of ART, while markers of bone formation and resorption significantly increased. The OPG/sRANKL ratio remained unchanged over time and there was no association with markers of bone turnover (20).
RANKL, IL-6 and TNF-α significantly increased in the intermittent ART group and decreased in the continuous ART group. In contrast, osteoprotegerin levels and IL-1 remained relatively stable. Conditions associated with chronic immune activation and inflammation are well known to be associated with increased bone loss (e.g. rheumatoid arthritis and inflammatory bowel disease)(21-23). Inflammation results in increased RANKL expression and production by activated T cells and B cells, increased numbers of osteoclast precursors and reduction in production in osteoprotegerin, with resultant greater bone resorption and net bone loss (24-26). It is counterintuitive that the increases in RANKL and the RANKL:OPG ratio in the intermittent ART group occurred in association with reduction in markers of bone resorption and increased BMD. It is possible that ART increases bone turnover through a pathway that is not mediated by RANKL or osteoprotegerin, and that the increase in RANKL at cessation of ART occurred as a compensatory response to the reduction in markers of bone turnover. Alternatively, the increase in RANKL may be a reflection of increased immune activation and subsequent expression and production of RANKL by activated T cells associated with untreated HIV infection.
Early increases (from baseline to Month 4) in the markers of bone formation and bone resorption predicted decreases in BMD at the hip and spine at Month 12. The directions of associations between biomarkers and BMD were consistent across BMD at the hip (femoral neck and total hip) and spine (lumbar spine measured by DXA and qCT). However, only a few markers (ßCTX and the RANKL:OPG ratio for hip BMD, bALP and P1NP for spine BMD by qCT) remained independently associated with changes in BMD in multiple regression and the strength of the associations varied by BMD site and measurement modality. This reflects the complex interrelationships between the bone biomarkers, with none clearly dominating with respect to predicting change in BMD.
The randomized comparison provides evidence that the observed decline in markers of bone turnover in the continuous ART group versus the increase in the intermittent ART group are likely due to the differences in ART use. However, the difference between the two treatment groups may underestimate the effect of stopping ART, because in the intermittent ART arm, 36% of participants had recommenced ART by month 12, and 20% had not used ART at study entry. When the analysis was restricted to those participants in the intermittent ART arm who stopped ART and stayed off ART through month 12, the levels of markers of bone formation and resorption continued to decline through month 12, although most of the reduction was evident by month 4. Conversely, in the continuous ART group, when restricting the analysis to those who were using ART at study entry and continued to use ART through month 12, markers of bone turnover within this subset of participants tended to increase, but the magnitude of the increase was less than among those randomized to the continuous ART group who were not taking ART at baseline and started ART.
In the setting of ART, increased markers of bone turnover, as observed in the continuous ART group, have been consistently associated with loss of BMD, particularly over the first year of treatment (11-12). Elucidating the mechanism of ART-induced bone loss is confounded by changes in HIV-induced immune activation and inflammation. Is the significant early bone loss observed over 24-48 weeks after initiation of ART driven by immune reconstitution, as proposed by some (27-28), or by the direct effects of ART? In our study, increased CD4 T cell counts were associated with steeper decreases in spine BMD, pooled across treatment groups. It is not possible, however, to separate the effect of ART in our study from the effect of increased CD4 T cells. After adjustment for treatment group, there was no evidence for an association of CD4 T cell increases with bone loss.
The main strength of our study is the randomized comparison of continuous versus intermittent ART. There were several limitations to our study. About half of the participants randomized to intermittent ART had restarted ART by month 12. The ART regimens used in SMART were heterogeneous and may not be entirely representative of modern ART regimens; moreover, this study was not designed to evaluate the effects of specific drugs. The first follow-up time point was at month 4, so we could not determine which biomarker or group of biomarkers changed first. The analysis excluded 73 of the 275 randomized participants in the SMART Body Composition substudy, mostly because month 12 data were not available due to study closure. While the excluded participants were largely similar to those analyzed, they differed in some baseline characteristics. Finally, the proportion of women was higher and ART use at study entry was lower in one of the randomized treatment groups in the Body Composition substudy; however, after adjustment for sex and ART use at baseline in sensitivity analyses, results were similar to the unadjusted comparisons between treatment groups summarized in Figure 3.
In conclusion, this study has shown that interruption of ART results in a reduction in markers of bone turnover and increase in BMD at hip and spine relative to continuous ART, and that early changes in markers of bone turnover predict BMD changes at 12 months. The stabilization of bone loss after ART interruption, despite increased inflammation, suggests a deleterious effect of ART on bone, either directly or indirectly. The clinical implication of these findings is unknown given the imperative need for ART to prevent major HIV-related outcomes; once started, ART needs to be used continuously. The trend to initiate ART earlier in the course of HIV infection means that low BMD may become more prevalent as more patients use ART for longer, highlighting the need for prospective studies with longer follow-up.
Results
Baseline characteristics
Baseline characteristics of the study population are shown in Table 1. Median age was 44 years, 17% were female, 27% were black, and 56% were white. The treatment groups were well balanced, except that 62% of the intermittent ART group had HIV RNA <400 copies/mL at baseline, compared with 47% in the continuous ART group (p=0.03 for difference), more participants in the intermittent ART group had used ART at baseline (80% versus 68%; p=0.05), and more participants in the intermittent ART group had used protease inhibitors (p=0.04). Among those using ART at study entry, however, protease inhibitor use was balanced across treatment groups (p=0.15). The intermittent ART group also had a lower proportion of women (13% versus 23%); the difference was not statistically significant. Few participants had osteoporosis and none was receiving osteoporosis therapy. Baseline values for the bone turnover and regulatory markers and inflammatory cytokines are described in Table 2; differences between treatment groups were not significant.
Antiretroviral therapy
Of the participants assigned to continuous ART, 68% were using ART at baseline, and 32% started ART; 97% were on ART at month 4 and 91% at month 12. Of those assigned to intermittent ART, 80% were using ART at baseline and ceased ART, the others continued to defer ART according to the CD4-guided intermittent ART strategy. Through follow-up, 18% had recommenced ART at month 4 and 46% at month 12 due to CD4 cell decline or clinical events.
Changes in BMD and Markers of Bone Turnover and Inflammation by treatment group
Changes in BMD and estimated between-group differences are shown in Figure 2. By year 1, each BMD measure was significantly higher in the intermittent ART group (increase of 0.33% at the hip and 1.64% at the spine by DXA) compared with the continuous ART group (decrease of -1.04% at the hip, and -0.13% at the spine by DXA) at M12. Within the continuous ART group, mean BMD declined by all measures at hip and spine, although the decline was not statistically significant for spine BMD by DXA. In the intermittent ART group, BMD increased, although only the increase in spine BMD by DXA was significant.
Mean changes from baseline for (log transformed) markers of bone turnover, bone regulation and chronic inflammation at 4 and 12 months, and the between-group differences (change in the intermittent ART minus the continuous ART group) are presented in Figure 3. In the intermittent ART group, bALP, osteocalcin, P1NP, NTX, and ßCTX decreased, compared to an increase in the same markers in the continuous ART group. RANKL, the RANKL:OPG ratio and TNF-α increased in the intermittent ART group, and decreased in the continuous ART group. The changes were apparent by Month 4, and remained at about the same level at Month 12. Between-group differences in mean change were significant at both Months 4 and 12 for each of these biomarkers (all p-values <0.002). IL-6 followed a similar pattern to TNF-α, but the treatment difference was significant only at month 12 (p=0.05). There was no significant between-group difference in mean change in OPG or IL-1. Adjustment for sex, ART status at study entry, and baseline CD4 cell counts had minimal impact on the between-group comparison (data not shown).
Changes in Markers of Bone Turnover and Inflammation by first-year ART use
When restricting the analyses to participants who were on ART at baseline, and either remained on ART for 12 months (n=53), or stopped and remained off ART for 12 months (n=44) (Figure 1), differences between the ART groups at month 4 were similar to the intent-to-treat comparisons in all 202 participants. Differences tended to increase further by month 12, although most of the treatment difference was attained by month 4 (data not shown). There was no evidence for a difference between the treatment groups in OPG or IL-1.
Effect of Early Change in Markers (Baseline-Month 4) on Change in BMD (Baseline-Month 12)
Associations between early changes in bone biomarkers (Baseline to Month 4) and changes in BMD between baseline and Month 12 are summarized in Figure 4. For example, an increase of 1 log2 unit in bALP (2-fold increase from baseline to Month 4) was associated with a 2.3% per year steeper decrease in hip BMD. Pooled across treatment groups, increases in bone formation markers (bALP, osteocalcin) as well as increases in bone resorption markers (NTX and ßCTX) at Month 4 were significantly correlated with loss of total hip BMD, while increased RANKL and RANKL:OPG ratios were associated with increases of hip BMD. The association of increased P1NP with a decrease in hip BMD was borderline significant (p=0.06). The direction of the associations of these seven biomarkers with change in BMD were similar across all four BMD outcomes (total hip, femoral neck, spine by DXA and spine by qCT), although not all associations were significant. An increase in TNF-α by Month 4 was associated with increased spine BMD by qCT, but there was no evidence for an association of TNF-α, IL-1 or IL-6 with any of the other BMD outcomes. The analyses were adjusted for age, sex, race, BMI, HIV duration, ART use at study entry and baseline CD4 cell counts.
Changes in markers of bone turnover and inflammation were correlated (data not shown). When including all 11 bone biomarkers as predictors for change in BMD in multiple regression models, a backwards selection procedure retained early declines in ßCTX (p<0.001) and the RANKL:OPG ratio (p=0.03) as independent predictors for increase in total hip BMD. Results for the other BMD outcomes are summarized in Figure 4; superscripts to the p-values denote which bone markers were selected for the final model (s, *), and whether they were independently associated with the BMD outcome at year 1 (* for p < 0.05).
Changes in CD4+ T cell counts
In the intermittent ART group, mean CD4+ T cell counts declined by 198 and 232 cells/μL from baseline to months 4 and 12, respectively. In the continuous ART group, mean CD4+ T cell counts increased by 25 cells/μL to month 4, and 33 cells/μL from baseline to month 12. A Month 4 increase in CD4+ T cells by 100 cells/μL was associated with a decrease in spine DXA BMD by -0.40% (95% CI: -0.71 to -0.08%, p=0.01), and a decrease in spine qCT BMD by -0.89% (95% CI: - 1.44 to -0.35%, p=0.002) in univariate regression pooled across treatment groups. After adjustment for treatment group, these associations were not significant. There was no evidence for an association between changes in CD4+ T cell count at Month 4 and changes in hip BMD at Month 12.
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