HIV and HCV Independently Lower BMD through Different Mechanisms N Maalouf, MD; S Zhang, PhD; H Drechsler, MD; J Cutrell, MD, I Farukhi, MD; R Castanon, MD; G Brown, MD; P Tebas, MD; and R Bedimo, MD

Role of HCV in Fracture Risk of HIV Patients HIV and HAART initiation increase fracture risk HCV co-infection is a significant risk factor for osteoporotic fractures in several cohorts of HIV-infected patients: –ANRS CO8 APROCO-COPILOTE cohort: HR: 3.6 (95% CI: 1.6–8.1) 1 –WIHS: HR: 1.86 ( ) 2 ; HOPS: HR: 1.99 (1.01–3.90) 3 However, the mechanisms of this increased risk (impact of HCV on BMD and bone turnover) is not clearly established. –It could be related to HCV-induced liver fibrosis –HCV is associated with higher levels of inflammatory markers (TNF- , IL-8). 4 These could in turn enhance osteoclastogenesis leading to excessive bone resorption and osteoporosis 3 1 Collin et al., AIDS May ; 23(8): 1021– Yin et al., AIDS 2010; 24:2679–2686; 3 Young et al., Clin Infect Dis 2011; 52:1061–1068; 4 Guerra. Dig Liver Dis 2007,39 Suppl 1:S76-82

Osteoporosis in HIV OSTEOPOROSIS and FRACTURE RISK Advancing age, Improved Survival Hypogonadism Glucocorticoids Low BMI, Malnutrition Tobacco, EtOH, Drugs Race/Ethnicity, Genetics HIV HAART (TDF) HCV The virus Immune reconstitution The virus (inflammation) Severity of liver disease? Traditional risk factors Disease specific factors

Impact of Severity of Liver Disease on Fracture Risk in HIV Patients US Veterans Cohort: 56,660 HIV patients (98.1% male; 31.2% HCV co-infected; mean age: 45.0 years) 1,2 HCV co-infection remained a strong independent predictor of osteoporotic fractures after controlling AST-to- platelet ratio (APRI; HR: 1.32; p= 0.001) or the presence of cirrhosis (HR: 1.30; CI: ; p=0.003). Johns Hopkins Cohort: 179 HIV/HCV patients 3 Severity of liver disease (METAVIR score) did not predict low BMD 1 Bedimo et al., AIDS 2012; 2 Maalouf et al., JBMR 2013; 3 El-Maouche et al. J Hepatol 2011

Goals of the study OSTEOPOROSIS and FRACTURE RISK Advancing age, Improved Survival Hypogonadism Glucocorticoids Low BMI, Malnutrition Tobacco, EtOH, Drugs Race/Ethnicity, Genetics HIV HAART (TDF) HCV The virus Immune reconstitution The virus (inflammation) Severity of liver disease? Traditional risk factors Disease specific factors Q1. What are the mechanisms?

Goals of the study OSTEOPOROSIS and FRACTURE RISK Advancing age, Improved Survival Hypogonadism Glucocorticoids Low BMI, Malnutrition Tobacco, EtOH, Drugs Race/Ethnicity, Genetics HIV HAART (TDF) HCV The virus Immune reconstitution The virus (inflammation) Severity of liver disease? Traditional risk factors Disease specific factors Q2. How much is HIV? How much HCV? Is there an interaction?

Study design  Prospective, cross-sectional study: 168 males with HIV, HCV, HIV/HCV co-infection and uninfected.  Included if age ≥ 40; eGFR ≥ 60; no known osteoporosis  All HIV patients were virologically suppressed on HAART;  All HCV patients were HCV treatment-naive  Study measurements:  bone mineral density (BMD) by DXA scan  Bone turnover markers: serum C-telopeptide (CTX), bone-specific alkaline phosphatase (BSAP) and osteocalcin (OC)  Regulatory cytokines: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG).  Statistics:  Groups means compared by ANOVA and by ANCOVA adjusting for age, race and BMI

Results: Patient Characteristics Variable HIV/HCV (N=28) HIV (N=62) HCV (N=45) Controls (N=33) P-Value (Unadjusted) Age* (years)55 (6)57 (8)55 (5)53 (8)0.048 Race / Ethnicity (% AA/C/H)71/25/432/55/1367/27/788/12/0< BMI* (Kg/m 2 )27.5 (6.0)27.5 (4.5)28.6 (5.3)29.9 (5.8)0.18 Smokers (%) Glucocorticoid Use (%) Alcohol Use (%) Tenofovir Use (%) Protease Inhibitors Use (%) *Data shown as mean (standard deviation)

Impact of HIV and HCV on T-Scores HIV and HCV independently lower T scores (smaller contribution for HCV). Effect most pronounced in femoral neck and total hip. No interaction between the two infections *Controlling for Age, BMI and Race Data shown as mean (standard deviation) BMD T-score Mean (SD) HIV/HCV (N=28) HIV (N=62) HCV (N=45) Controls (N=33) P- value Adj* HIV vs. non- HIV Adj* HCV vs. non- HCV Adj* HIV- HCV inter- action Fem Neck -1.6 (0.8)-1.4 (1.0)-1.2 (0.8)-0.6 (1.0) Total Hip -1.2 (0.7)-0.9 (0.9)-0.7 (0.7)-0.4 (0.8) 0.05< L-Spine -1.4 (1.4)-0.8 (1.7)-0.5 (1.5)-0.7 (1.6)

Impact of HIV and HCV on T-Scores

Impact of HIV and HCV on T-Scores Percent Osteoporosis at F Neck

HIV/HCV (N=28) HIV (N=62) HCV (N=45) Controls (N=33) P- value Adj* HIV vs. non- HIV Adj* HCV vs. non- HCV Adj* HIV- HCV inter- action Osteocalcin ng/mL 18.5 (6.3) 21.7 (8.9) 15.7 (6.5) 15.8 (5.1) < Bone Sp Alk Phos U/L 37.1 (12.1) 34.7 (11.4 )33.6 (10.1 )26.1 (8.7) < C-telopeptide (CTX) ng/mL 0.62 (0.24)0.58 (0.29) 0.45 (0.23) 0.41 (0.15) 0.01< Impact of HIV and HCV on Bone Markers *Controlling for Age, BMI and Race Data shown as mean (standard deviation) HIV groups had higher bone resorption and formation No increased resorption in HCV groups

Correlations of Bone Turnover Markers and Bone Mineral Density Total Hip BMD (g/cm 2 ) Serum Osteocalcin (ng/ml) r= -0.28, p<0.001 r= -0.21, p<0.001 Serum C-Telopeptide (ng/ml) Total Hip BMD (g/cm 2 )

Bone Turnover Coupling: The RANK/RANKL/OPG System RANK, RANKL, and OPG are members of TNF and TNF receptors superfamily. RANK and RANKL involved in formation and activation of osteoclast OPG is decoy receptor competing with RANK-RANKL cf. Ott, Endo Reviews, 2007 RANK: receptor activator of NFκB; RANKL: receptor activator of NFκB ligand OPG: osteoprotegerin

HIV/HCV (N=28) HIV (N=62) HCV (N=45) Controls (N=33) P- value Adj* HIV vs. non- HIV Adj* HCV vs. non- HCV Adj* HIV- HCV inter- action Osteocalcin ng/mL 18.5 (6.3) 21.7 (8.9) 15.7 (6.5) 15.8 (5.1) < Bone Sp Alk Phos U/L 37.1 (12.1) 34.7 (11.4 )33.6 (10.1 )26.1 (8.7) < C-telopeptide (CTX) ng/mL 0.62 (0.24)0.58 (0.29) 0.45 (0.23) 0.41 (0.15) 0.01< RANKL pmol/L 236 (306)131 (168) 190 (201) 179 (118) Osteoprotegerin (OPG) pmol/L 4.9 (2.5)4.3 (1.5) 4.9 (2.2) 3.6 (1.2) < < RANKL/OPG ratio 49 (53)34 (40) 40 (31) 52 (34) Impact of HIV and HCV on Bone Markers and Turnover Regulation *Controlling for Age, BMI and Race Data shown as mean (standard deviation) RANK: receptor activator of NFκB; RANKL: receptor activator of NFκB ligand

Conclusions  HIV and HCV independently lower BMD and T-scores (smaller contribution for HCV).  Effect most pronounced in femoral neck and total hip.  No interaction between the two infections.  HIV impact on BMD could be explained by increased turnover (resorption and formation markers).  See Cotter et al. 7 th IAS, MOPE077  Turnover doesn’t appear to be driven by RANK/RANKL/OPG system  HCV is not associated with increased bone resorption  Increased OPG and trends toward increased RANKL

Osteoporosis in HIV OSTEOPOROSIS and FRACTURE RISK Advancing age, Improved Survival Hypogonadism Glucocorticoids Low BMI, Malnutrition Tobacco, EtOH, Drugs Race/Ethnicity, Genetics HIV HAART (TDF) HCV Fibrosis Cirrhosis High Bone Turnover ? No  Turnover;  OPG? ?

Increased Bone Turnover in HIV: Potential Mechanisms HIV Infection Itself: –Dysregulation of Bone Metabolism: No changes in the RANKL and OPG Gonadal Hormones; PTH and Vitamin D –Increased Inflammation: TNF-alfa; IL-1, IL-6 HAART: –Immune reconstitution? VL control associated with lower BMD (El-Maouche. J Hepatol 2011) –Tenofovir: Renal insufficiency with secondary hyperparathyroidism Proximal tubular dysfunction? (Hamza, 7 th IAS; MOPE076)

Potential Mechanisms for Increased Bone Turnover in HIV: HAART Impact TDF/FTC/DRV/r vs RAL/DRV/r Significant increases in bone formation (P1NP) and resorption (CTx) following initiation of TDF/FTC + DRV/r vs. RAL + DRV/r. No difference in changes in inflammatory markers from baseline Bedimo et al., IAS 2013; Poster WEPE512

Acknowledgements Study funded by VA MERIT grant I01 CX A1 Thanks to Holly Wise and Joyce Ghormley, our study coordinators Thanks to IAS for giving us the opportunity to share our work Special thanks to all the study volunteers