Review of Studies on Bone Problems at ICAAC and Lipodystrophy Workshop

As you remember, several years ago when fat redistribution, or as it was called then "lipodystrophy", was initially uncovered in patients on HAART, initial reports (including from the Australian researchers) said that the use of protease inhibitors were the cause of lipodystrophy, body changes, or fat redistribution. Subsequent research findings found individuals who had only taken NRTIs were also suffering with fat redistribution or body composition changes (fat accumulation deep in the stomach--visceral adipose tissue--and/or fat depletion in the periphery --arms, legs, and face). The causes for this syndrome are not known but it's suggested that it may be related to a number of contributory factors including changes that occur due to HIV, an improvement or change in a person's immune system due to reduced virus load and improved immune status from HAART, and protease inhibitors, nucleosides. In fact, the prevailing current thinking is that several distinct or related syndromes may be going on at the same time leading to what may be different manifestations. Fat accumulation and fat depletion may be caused from different factors. Elevated lipids and glucose, and insulin resistance may result from different or overlapping causes. Interesting turns in research were reported from a number of studies in Toronto at the Lipodystrophy Conference. My first two reports discussed Human Growth Hormone and new data on lower every other day dosing and mitochondrial toxicity, and it's relationship to NRTIs and body composition changes.

In the recent year or so, there has been much discussion about bone problems people with HIV have been experiencing. Like what happened with lipodystrophy, initial reports including from the Washington University in St. Louis research group reported at the Feb 2000 Retrovirus Conference suggested protease inhibitors were associated with these bone problems. Several study findings from various reports presented at the Lipodystrophy Conference and at ICAAC suggested associations of bone problems with various potential causes including lower weight prior to starting antiretroviral therapy, elevated lactate levels associated with ddI and d4T (possibly associated with mitochondrial toxicity), protease inhibitors, duration of having HIV, and one study from Mallal and his Australian research group found correlation with reduction in subcutaneous fat or possibly with lipo-atrophy. I think we should be circumspect when reading all the various reports associating bone problems with particular causes. Opinions and study results may change and evolve like what happened with lipodystrophy. Hopefully, research into this problem will become more focused and developed, and a year or two from now study findings on the potential causes for bone problems may be different than they are now. Researchers are just starting to investigate the potential mechanisms of action for these problems.

Potential Causes or Mechanisms of Action Leading to Bone Problems

Pablo Tebas (Washington University in St. Louis) reported on evaluations of several indirect markers of bone formation and resorption and compared them to DEXA evidence of bone demineralization for 73 patients receiving protease inhibitor containing HAART.

Tebas reported increased excretions in urine and serum for markers of bone metabolism: 50% of the patients excreted >200 mg of urinary calcium per day; on average, patients had increased urine pyridinoline (41ｱ22 nmol/mmol creat), increased urinary deoxypridinoline (10ｱ6 nmol/mmol creat), increased serum bone alkaline phosphatae (188ｱ152 IU/L), and increased serum osteocalcin (20ｱ26 ug/l). Tebas also reported that serum alkaline phosphatase and urine n-teleopeptides were inversely correlated with DEXA t- and Z- scores for the lumbar spines. In other words, these serum markers went up as bone mineral density went down.

Protease Inhibitors May Inhibit In Vitro Conversion To Vitamin D

Tebas then reported in vitro findings suggesting that protease inhibitors (indinavir, ritonavir, and nelfinavir) may contribute to bone demineralization. A little scientific explanation they said in their report: two cytochrome P450 oxygenases help vitamin D activation to its most potent circulating metabolite: 1,25 (OH)₂ --vitamin D. Vitamin D-25-hydroxylase converts vitamin D to 25(OH)-vitamin D in the liver, which is activated to 1,25(OH)₂--vitamin D by 25(OH)-vitamin D-1a-hydroxylase in the kidney. The formation of bioactive 1,25(OH)₂ -vitamin D by 1a-hydroxylase activity is mandatory for vitamin D control of calcium homeostasis (balance).

Tebas examined whether indinavir, ritonavir, or nelfinavir inhibited 1a-hydroxylase, or the in vitro conversion leading to vitamin D. He reported data from these in vitro (test tube) studies demonstrating that high concentrations of ritonavir (11 ug/mL), indinavir (10 uM) and nelfinavir (6 ug/mL) impaired the bioactivation of vitamin D in the monocyte-macrophage cell line THP-1. All three protease inhibitors inhibited conversion of 25(OH)-vitamin D₃ to 1,25(OH)₂ -vitamin D in vitro in this cell line in the presence of ritonavir by 80%, indinavir 66%, and nelfinavir 31%. However, plasma levels were reportedly not reduced in the 73 patients raising the relevance of these findings.

Australian Study Reports on the Prevalence of Bone Mineral Density (BMD) Loss in 171 Australian patients and a Correlation of Change in Subcutaneous Fat With BMD Loss

David Nolan, from Royal Perth Hospital and Murdoch University in Australia, reported on his findings in determining the predictors of change in bone mineral density (BMD) over time in a large group of patients on HAART. His findings described below suggest an association between fat depletion or fat redistribution and bone mineral density loss.

Nolan conducted a cross-sectional analysis (one-time look) of 171 males in the Western Australia HIV cohort (group) using whole body DEXA scans, and lumbar spine z-scores in the most recent scan while patients were on therapy were compared. He reported relatively high rates of osteopenis (49%) and osteoporosis (17%), and relatively lower bone mineral density in patients receiving protease inhibitors (mean z-score -0.77ｱ1.16) compared to PI na夫e (-0.358ｱ1.18; p=0.055).

Nolan also conducted a longitudinal analysis in which rates of loss of BMD using serial DEXA scans from 64 patients on a stable regimen were compared in a multivariate analysis looking at multiple potentially predictive factors. From this analysis, he reported a correlation between changes in percentage of subcutaneous fat (mean change in % leg fat per year) and bone mineral density (mean increase z-score per year ｱ ; p=0.008), regardless of therapy. He also reported that patients receiving indinavir had greater increases in BMD than nelfinavir regimens, independent of changes in subcutaneous fat (p=0.0369, mulivariate analysis). He said this finding was in keeping with in vitro experimental data indicating that indinavir favors osteogenic differentiation of mesenchymal stem cells via retinoid signalling mechanisms. I don't think I buy this one.

A Second Australian Research Group Suggests Osteopenia May Be Associated With Elevated Lactate and Weight Prior to Starting HIV Therapy

Another Australian study reports similar findings. Dr. Andrew Carr and David Cooper (St Vincent's Hospital and the National Center in HIV Epidemiology and Clinical research, Australia) reported findings suggesting that osteopenia may be associated with elevated lactate levels, which in turn was associated with current ddI and current d4T therapy, and with lower body weight prior to antiretroviral therapy. I assume that patients had previously used other NRTIs.

Carr assessed bone density (t-score, z-score, and total mineral density) by DEXA in a group of 221 otherwise well HIV-infected men recruited to a lipodystrophy prevalence survey between November 1998 and February 1999 with the following data: demographics, smoking and exercise history, types and duration of all antiretroviral therapy, physician assessed lipodystrophy (overall and by region--evaluation by doctors and self-reports by patients may not be as accurate as objective assessments), CD4 counts, HIV viral load, fasting metabolic measures (lipid, glyclemia [suger], lactate, liver function, testosterone), and regional body fat and lean mass (DEXA and L4 abdominal CT).

32 patients were drug-na夫e, 42 were receiving NRTIs and 147 were receiving NRTI-PI therapy. Mean age was 43, 116 patients (52%) had lipodystrophy and 32 (14%) had lactic acidemia (>2.0 mmol/l). Osteoporosis (t-score <2.5 SD below normal) was found in 7 patients (3%) and osteopenia (t-score -1.0 to -2.5 SD) in 44 patients (22%). No patient had a fracture since being HIV-infected.

Carr and Cooper reported that the factors associated with osteopenia and osteoporosis were higher lactate (odds ratio 2.39 [95% CI; 1.39-4.11] per 1 mmol/l increase; p=0.002), and lower weight prior to commencing antiretroviral therapy (odds ratio 0.94 [95% CI: 0.90-0.98] per 1 kg increase; p=0.006). There was no other independent association found with any other parameter, including lipodystrophy at any site or NRTI, NNRTI, or PI type or duration of use. Lactic acidemia was in turn associated with current ddI use (odds ratio 6.10 [95% CI: 2.67-13.89]; p<0.0001), current d4T use (odds ratio 2.90 [95% CI: 1.25-6.71; p=0.013), and a greater rise in CD4 count on therapy (odds ratio 1.02 [95% CI: 1.01-1,04] per 10 cells increase; p=0.005).

No Improvement in Bone Mineral Density 48 Weeks After Switching to PI-Sparing Regimen

Another Australian research group assessed the effect on bone mineral density 48 weeks after switching from a protease inhibitor to a PI-sparing regimen. 80 HIV-infected patients with lipodystrophy enrolled in the PILR study. Determination of bone mineral density and t-scores by DEXA scans were performed at screening, weeks 12, 24 and 48 after randomization to either continue the PI regimen or switch to a non-PI regimen. Baseline levels of testosterone, osteocalcin and insulin-like growth factor-1 (IGF-1) were correlated with bone mineral density. J Hoy reported that 21 of 74 (28.4%) had evidence of osteopenia at baseline, and a further 7 (9.5%) had osteoporosis. There was no change in the proportion of individuals with osteopenia over the 48 weeks after switching to PI sparing regimen. I think that this could mean the PI had nothing to do with osteopenia or the damage was not reversible yet. There was no association between baseline insulin level (IGF-1), testosterone levels, duration of antiretroviral therapy, duration of PI use, CD4 count, and baseline t-scores. Age, total body weight, total body fat, lean body mass and osteocalcin levels were correlated with total bone mineral density. Slight decreases were observed in the leg and total bone mineral density at week 48, but there were no statistically significant differences between the randomized treatment groups.

Osteopenia May Be Due to HIV Rather Than HIV Antiretroviral Drugs

Dr. Graeme Moyle, from Chelsea & Westminster Hospital in London, UK, reported data from his study using DEXA scans suggesting that osteopenia in persons with HIV-infection may be due to HIV rather than antiretroviral therapies.

DEXA scans were performed on 51 males and 1 female HIV+ never treated patients (control group), 22 male PI-treated patients, and 10 NRTI-only treated male patients. All treated patients had physical signs suggestive of fat redistribution. T-scores based on whole body bone mineral density were calculated and included in a multivariate analysis. Insufficent data were available to investigate the relationship of metabolic parameters with bone mineral density.

Median viral load was 116,850 copies/ml in never-treated patients, 101 copies/ml in the PI-treated, and 2563 copies/ml in the NRTI-only treated patients (p<0.001), and CD4 count was 159 in never-treated patients, 390 in PI-treated and 452 in NRTI-only group (p<0.001). Median duration of therapy was 570 and 396 days for PI and NRTI-only group. Total bone mineral density was similar (1.21, 1.17, and 1.17 in the 3 groups, respectively). T-scores were non-significantly lower in the never-treated group (-0.12) than the PI (-0.6) and NRTI-only group (-0.69; p=0.39). Moyle reported that based on time from HIV test to diagnosis of osteopenia, use of therapy appeared to have a significant protective effect on bone mineral density (p=0.022 log rank). This was confirmed by a Cox proportional hazards model in which both PI and NRTI-only were associated with reduced hazard ratio of osteopenia (0.17 [95% CI 0.06-0.56] and 0.32 [95% CI 0.13-0.82], respectively). Presence of a low (< median value 62,247 copies/ml) viral load also approached significance (0.52 [95% CI 0.26-1,01]). Moyle suggests virologic control with therapy may diminish the risk of osteopenia.

Two Additional Reports of Factors Possibly Associated With Bone Problems

There were two reports presented on small numbers of patients and suggesting associations of potential factors contributing to bone problems. Dr. Marshall Glesby, from Cornell University Medical College in NYC, reported in an oral presentation findings of a small case-control study (two matched groups) of avascular necrosis (AVN) of the femoral head in 14 HIV-infected individuals diagnosed in 1992-2000. All but two were after 1996 and 8 (57%) had bilateral AVN. Median CD4 and mean HIV viral load at the time of AVN were 164 cells and 3.8 log (6,300 copies/ml). Cases and controls did not differ significantly by year of HIV diagnosis, race, gender, HIV risk factor, HCV or HBV status, history of alcoholism, smoking, cancer, diabetes or pancreatitis, CD4, nadir CD4, or HIV viral load, random glucose & tryglicerides. The duration of prior antiretroviral therapy prior to ANV was longer in the cases (median 32 verses 14 months; p=0.06). 71% of cases used protease inhibitors prior to, and 57% at the time of, AVN diagnosis versus 57% and 32%, respectively, of controls at comparable time points (p=0.50 and 0.18, respectively). In matched univariate analysis, prior PCP (odds ratio 6.3; 95% CI 1.3-31; p=0.02), CD4 rise >50 cells from nadir (odds ratio 4.3; CI 0.85-22; p=.08), or known prior corticosteroid use (odds ratio 6.6; CI 0.72-61; p=0.10), PI use at time of AVN (odds ratio 4.0; CI 0.77-21; p=0.10), and d4T use at time of AVN were associated with AVN. In multivariate analysis, only PCP remained independently associated with AVN. It was suggested by observers that the association with PCP might be due to prior steroid therapy or more advanced prior HIV disease.

Laurent Roudiere, from Hopital Necker in Paris, France, reported on 7 patients out of a cohort of 508 HIV-infected patients including 280 on triple therapy, who were diagnosed with osteonecrosis of the two femoral heads or one knee, between 1998 and May 2000. No case had been diagnosed before 1998. Symptoms were pain and limitation of the joint(s), and diagnosis was confirmed by bone scintiscan and/or magnetic resonance imaging. Four of six patients with hip osteonecrosis had to undergo hip replacement surgery. Patients had low CD4 nadirs (median 18 cells), long exposure to NRTIs before HAART (median 34.5 months), long exposure to HAART including a PI (median 32 months), significant responses to HAART (median CD4 and viral load at time of osteonecrosis, 280 cells and 19 copies/ml) and 5/7 had fat redistribution. While on therapy including a PI, all had elevated plasma tryglicerides and/or cholesterol (median maximum plasma levels 4.55 and 6.20 umol/l, respectively) and 3 had developed diabetes. Only one patient had chronically received corticosteroids (18 months of prednisone, 20 mg/day, stopped 1 year before osteonecrosis) and none had excessive alcohol intake or a history of trauma, radiotherapy, sickle-cell anemia or systemic lupus erythematosus. Roudiere concluded that about 2% of treated patients and 10% of patients with lipodystrophy in his cohort involved osteonecrosis. He suggested the osteonecrosis be considered in a case definition for lipodystrophy.

Spanish Study in 45 HIV-infected Males Reports Finds Osteopenia in NRTI-Only Treated Patients

In a poster at ICAAC MJ Galindo, from Hospital Clinico in Valencia, Spain reported on a study of the factors related to the alterations on bone mineral density present in HIV. They analyzed lumbar spine bone mineral density using DEXA, nutritional status and calcium-phosphorous metabolism parameters (serum calcium, phosphorus and PTH levels, calcium and AMPc urinary excretion during 24-hour) in 45 infected male patients between June and December 1996. Thirty-two patients (71.1%) were on treatment with dual therapy without PI and 13 (28.9%) were na夫e. The patients were 35 years old, 34 (75.4%) had been intravenous drug users all of them were asymptomatic, none had AIDS criteria, and the average CD4 was 212. Osteopenia was defined as Z-score between -1 and -2.5, and osteoporosis as Z score less than -2.5 according to WHO criteria. They reported that 28 of 45 patients (62%) had evidence of osteopenia, but none of them had osteoporosis criteria. No differences in nutritional status, mineral metabolism or in treatment regimens were observed between patients with and without osteopenia.  

French Research Group Suggest Duration of HIV is a Factor in Developing Osteopenia

In a second poster at ICAAC C Allavena and a research group from Nantes, France (Hotel Dieu) reported on a study exploring the rols of HIV therapy in osteopenia developing. Eighty-five HIV-infected patients (70 males, 15 females, mean age: 39.4) were consecutively enrolled in a cross-sectional study. Dual energy x-ray absorptiometry was performed to measure fat mass, lumbar spine and proximal femur bone mineral densities (BMD), with T and Z-scores. Osteopenia was defined as a lumbar T-score < -1.5 SD in male and < -1 SD in female, and osteoporosis as a lumbar T-score < -3.2 SD in male and < -2.5 SD in female. Osteopenia was present in 3/15 (20%) treatment-na夫e patients and 30/70 (43%) patients on treatment (p=0.09). Among the patients on treatment, osteopenia was present in 40.4% of patients on PI, 45.4% of patients not receiving PI. Osteoporosis was absent in treatment-na夫e group and present in 6.6% of patients receiving treatment (p=0.22). Mean Z-score (L1-L4) was -1.13 ｱ 0.13 SD in males, i.e. 83% of normal for age-matched population. Osteopenia was related to the duration of HIV infection and there was no relation with ART experience, duration of PI use nor presence of lipodystrophy. The authors concluded osteopenia and osteoporosis are more frequent in patients with prolonged duration of HIV infection with no clear association with use of potent ART.