MOAB02 Impacts of vitamin D and calcium supplementation on bone mineral density among perinatally HIV-infected adolescents: A 48-week randomized clinical trial Tavitiya Sudjaritruk, Linda Aurpibul, Torsak Bunupuradah, Suparat Kanjanavanit, Tawalchaya Chotecharoentanan, Sineenart Taejaroenkul, Pradthana Ounchanum, Piyarat Suntarattiwong, Thanyawee Puthanakit, on behalf of CAL-D Study Group NCT: 02426840

Background Adverse bone health is one of the common non-communicable conditions during the course of life-long HIV treatment1 Prevalence of low bone mineral density (BMD z-score <-2) among children and adolescents living with HIV ranged from 4-8% in high-income countries, up to 16-32% in middle income countries1 Several factors have been reported to be associated with bone demineralization1 Adverse bone health during adolescence may result in adult osteoporosis and bone fragility2 1Sudjaritruk T, et al. J Virus Erad. 2015;1:159-67. 2Young B, et al. Clin Infect Dis. 2011;52:1061–8.

Background Vitamin D is critical for calcium homeostasis and bone metabolism Adapted from Holick MF. Curr Opin Endocrinol Diabetes. 2002;9:87-98; Deluca HF. Am J Clin Nutr 2004;80:1689-96S.

Background Vitamin D is critical for calcium homeostasis and bone metabolism Clinical evidence supporting the benefit of vitamin D and calcium (VitD/Ca) supplementation on bone mineral status among HIV-infected youth is scarce

Objective To evaluate the impacts of VitD/Ca supplementation on BMD and bone metabolism among perinatally HIV-infected Thai adolescents Hypothesis: Dosage of vitamin D supplementation affects the changes of BMD and bone metabolism High-dose VitD/Ca supplement: A total of vitamin D 3,200 IU plus calcium 1.2 gm daily Normal-dose VitD/Ca supplement: A total of vitamin D 400 IU plus calcium 1.2 gm daily

Methods: Study design and population Study design: An ongoing, multicenter, 48-week, randomized, open-label study Study site: 5 pediatric HIV centers in Thailand Study population: Inclusion criteria: Exclusion criteria: Aged 10-20 years Perinatally acquired HIV infection Had a history of virologic suppression (plasma viral load <400 copies/ml) Received VitD/Ca supplement (VitD >400 IU and/or Ca >1 gm daily) Received pharmacologic treatment for low BMD Received medication that affects bone metabolism Had a history of bone fracture Pregnancy or breastfeeding

Methods: Randomization Randomization ratio 1:1 (computer-generated), open-labeled fashion, balanced within site FDC: fixed dose combination.

Methods: Outcome measurements BMD: Lumbar spine dual-energy X-ray absorptiometry (LS DXA) technique BMD z-score <-2 was defined as low BMD Bone biochemical markers: Bone metabolism-related hormone: 25-hydroxyvitamin D (25OHD), intact parathyroid hormone (iPTH) Bone turnover marker: alkaline phosphatase (ALP), C-terminal cross-linked telopeptide of type I collagen (CTX), procollagen type I amino-terminal propeptide (PINP)

Methods: Statistical analysis An interim analysis was performed Intention-to-treat analysis Stratified by baseline BMD z-score (low BMD vs. normal BMD) Wilcoxon rank sum test: between group comparison analysis Wilcoxon signed rank test: within group comparison analysis We conducted an interim analysis, using ITT The analysis were stratified by baseline BMD of participants

Results

Figure 1. Flow of study participants* Between April 2015 and October 2016: 166 HIV-infected adolescents were enrolled (Figure 1) 83 participants were randomly assigned to receive either high-dose or normal-dose VitD/Ca supplementation 67 participants (40%) had low baseline BMD 99 participants (60%) had normal baseline BMD

Table 1. Baseline characteristics of perinatally HIV-infected Thai adolescents Total (n = 166) High-dose group (n = 83) Normal-dose group P† Age, years 16.0 (14.4 - 17.7) 16.6 (14.6 - 18.1) 15.5 (14.0 - 17.2) 0.08 Male 79 (47.6) 40 (48.2) 39 (47.0) 0.88 WHO stage prior to ART Stage 1-2 Stage 3-4   74 (47.1) 79 (50.3) 35 (44.9) 40 (51.3) 39 (49.4) 0.51 CD4 T-cell percentage prior to ART, % 12 (3 - 20) 10 (3 - 20) 13 (3 - 20) 0.57 Current ART regimen NNRTI-based PI-based 105 (63.3) 61 (36.7) 52 (62.7) 31 (37.3) 53 (63.9) 30 (36.1) 0.62 Duration of ART, years 10.0 (7.0 - 12.3) 10.7 (7.1 - 12.5) 9.0 (7.0 - 12.1) 0.21 Current CD4 T-cell count, cells/mm3 711 (571 - 886) 710 (573 - 915) 711 (569 - 857) 0.69 Current plasma viral load, log10 copies/ml 1.6 (1.3 - 1.6) 0.46 Adherence to VitD/Ca supplementation, % 80 (70 - 99) 90 (70 - 100) 80 (70 - 90) 0.28 *Data presented as n (%) for categorical data and median (interquartile range) for continuous data. †The comparisons were performed using Pearson Chi-square for categorical data, and Wilcoxon rank sum tests for continuous data.

Table 2. Baseline bone mineral density and bone biochemical markers of perinatally HIV-infected Thai adolescents Laboratory parameters* Total (n = 166) High-dose group (n = 83) Normal-dose group P† Bone mineral density BMD z-score -1.5 (-2.3 to -0.3) -1.6 (-2.3 to -0.4) -1.5 (-2.3 to -0.2) 0.75 BMD z-score <-2 67 (40.4) 35 (42.2) 32 (38.6) 0.64 Bone biochemical markers Calcium, mg/dl 9.6 (9.3 - 9.9) 9.6 (9.3 - 9.8) 0.73 Phosphorus, mg/dl 4.4 (4.1 - 5.0) 4.4 (3.9 - 4.9) 4.5 (4.1 - 5.2) 0.20 25OHD, ng/ml 25.3 (20.7 - 33.2) 27.6 (21.0 - 35.7) 24.8 (19.5 - 29.9) 0.04 iPTH, pg/ml 41.7 (33.4 - 56.9) 41.4 (32.9 - 53.8) 42.5 (34.6 - 58.4) 0.30 Alkaline phosphatase, U/l 178 (125 - 272) 163 (109 - 263) 195 (137 - 283) 0.17 CTX, ng/l 1,350 (860 - 2,040) 1,270 (820 - 2,040) 1,390 (950 - 2,050) 0.26 PINP, μg/l 330 (165 - 609) 312 (145 - 592) 337 (177 - 625) 0.37 *Data presented as n (%) for categorical data and median (interquartile range) for continuous data. †The comparisons were performed using Pearson Chi-square tests for categorical data, and Wilcoxon rank sum tests for continuous data.

Change from baseline to week 48 Between group comparison P† Figure 2. Changes from baseline of bone mineral density and bone biochemical markers over the 48-week study follow-up (ITT analysis) Low BMD (n = 67) Normal BMD (n = 99) High-dose (n = 35) Normal-dose (n = 32) High-dose (n = 48) Normal-dose (n = 51) Parameters Change from baseline to week 48 Between group comparison P† BMD z-score 0.71 (0.29 to 1.11)‡ 0.49 (-0.40 to 1.19)‡ 0.10 (-0.25 to 0.66) -0.07 (-0.51 to 0.43) 0.07 25OHD, ng/ml 5 (-2 to 12)‡ 6 (3 to 12)‡ 5 (1 to 11)‡ 6 (2 to 10)‡ 0.41 iPTH, pg/ml -10 (-20 to 0)‡ -15 (-23 to -6)‡ -6 (-21 to 1)‡ -12 (-22 to -3)‡ 0.04 ALP, U/l -22 (-123 to 0)‡ -65 (-96 to -26)‡ -44 (-81 to -9)‡ -50 (-119 to -21)‡ 0.09 CTX, ng/l -170 (-450 to 0)‡ -420 (-720 to -130)‡ -330 (-740 to 6)‡ -320 (-620 to 30)‡ 0.39 PINP, µg/l -48 (-216 to 0)‡ -137 (-317 to -30)‡ -81 (-202 to 8)‡ -73 (-249 to 16)‡ 0.24 *Data is presented as median change from baseline (interquartile range). †P-value evaluates the overall difference in median change from baseline to week 48 between the two treatment groups (between group difference). ‡Indicates the median change from baseline to week 48 within the treatment groups (within group difference) is a statistical significant (P <0.05).

Discussions Parameter Our study Arpadi SM, et al3 (2012) Age 10-20 years 6-16 years Intervention VitD/Ca 3,200 IU/ 1.2 gm daily or VitD/Ca 400 IU/ 1.2 gm daily VitD/Ca 1,600 IU/ 1 gm daily or Placebo Duration 48 weeks 96 weeks BMD change LS BMD z-scores in adolescents with low BMD in both treatment groups No between-group differences in change of BMD z-scores from baseline TB BMD, TB BMC, LS BMD and LS BMC in both treatment groups No between-group differences in change of TB BMD and TB BMC from baseline Bone marker change iPTH, ALP, CTX, PINP in adolescents with low and normal baseline BMD in both treatment groups No between-group differences in change of bone markers, except for iPTH 25OHD in VitD/Ca supplemented group, but not in the placebo group Abbreviations: LS, Lumbar spine, TB, total body, TH, total hip. 3Arpadi SM, et al. Am J Clin Nutr. 2012;95:678-85.

Limitations Potential to differential measurement errors: open labeled fashion Total body DXA was not performed: a gold standard for evaluate BMD in adolescents Short duration of follow-up: too early to evaluate long-term outcomes Limited generalizability of results: focused on only perinatally acquired HIV infection, virologic suppressed, Asian population

Conclusions With the preliminary results: LS BMD z-scores were significantly increased in HIV-infected Thai adolescents with low baseline BMD who received VitD/Ca supplementation, regardless of dosage, over 48-week follow-up Bone biochemical markers were re-established in adolescents with low as well as normal baseline BMD in both treatment groups Supplementation of high-dose VitD/Ca did not show significant differences in the changes of BMD and bone metabolism outcomes compared to normal-dose VitD/Ca A prospective study with longer follow-up is warranted to confirm our findings

Acknowledgements Participants, study staffs, and laboratory staffs at Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand HIV-NAT, Thai Red Cross AIDS Research Centre, Bangkok, Thailand Nakornping Hospital, Chiang Mai, Thailand Chiang Rai Prachanukroh Hospital, Chiang Rai, Thailand Queen Sirikit National Institute of Child Health, Bangkok, Thailand Charoon Bhesaj Co., Ltd. for Oskept® (FDC tablet of vitamin D3 and calcium) supply throughout the study Funding support: The National Research University Project under Thailand's Office of the Higher Education Commission and the Chiang Mai University Fund