Lithocholic Acid, a Metabolite of the Microbiome, Increases Oxidative Stress in Breast Cancer.

In breast cancer patients, the diversity of the microbiome decreases, coinciding with decreased production of cytostatic bacterial metabolites like lithocholic acid (LCA). We hypothesized that LCA can modulate oxidative stress to exert cytostatic effects in breast cancer cells. Treatment of breast cancer cells with LCA decreased nuclear factor-2 (NRF2) expression and increased Kelch-like ECH associating protein 1 (KEAP1) expression via activation of Takeda G-protein coupled receptor (TGR5) and constitutive androstane receptor (CAR). Altered NRF2 and KEAP1 expression subsequently led to decreased expression of glutathione peroxidase 3 (GPX3), an antioxidant enzyme, and increased expression of inducible nitric oxide synthase (iNOS). The imbalance between the pro- and antioxidant enzymes increased cytostatic effects via increased levels of lipid and protein oxidation. These effects were reversed by the pharmacological induction of NRF2 with RA839, tBHQ, or by thiol antioxidants. The expression of key components of the LCA-elicited cytostatic pathway (iNOS and 4HNE) gradually decreased as the breast cancer stage advanced. The level of lipid peroxidation in tumors negatively correlated with the mitotic index. The overexpression of iNOS, nNOS, CAR, KEAP1, NOX4, and TGR5 or the downregulation of NRF2 correlated with better survival in breast cancer patients, except for triple negative cases. Taken together, LCA, a metabolite of the gut microbiome, elicits oxidative stress that slows down the proliferation of breast cancer cells. The LCA-oxidative stress protective pathway is lost as breast cancer progresses, and the loss correlates with poor prognosis.

The Effect of Discontinuing Treatment With Blosozumab: Follow-up Results of a Phase 2 Randomized Clinical Trial in Postmenopausal Women With Low Bone Mineral Density.

Administration of blosozumab, a humanized monoclonal antibody that binds sclerostin, increases bone formation and bone mineral density (BMD) in postmenopausal women with low BMD. To evaluate the effect of discontinuing blosozumab, we studied women enrolled in a 1-year randomized, placebo-controlled phase 2 trial for an additional year after they completed treatment. Of the 120 women initially enrolled in the study, 106 women completed treatment and continued into follow-up; 88 women completed 1 year of follow-up. At the beginning of follow-up, groups remained balanced for age, race, and body mass index, but lumbar spine and total hip BMD were increased in prior blosozumab groups, reflecting an anabolic treatment effect. At the end of follow-up, 1 year after discontinuing treatment, lumbar spine BMD remained significantly greater than placebo in women initially treated with blosozumab 270 mg every 2 weeks (Q2W) and blosozumab 180 mg Q2W (6.9% and 3.6% above baseline, respectively). Total hip BMD also declined after discontinuation of treatment but at 1 year after treatment remained significantly greater than placebo in women initially treated with blosozumab 270 mg Q2W and blosozumab 180 mg Q2W (3.9% and 2.6% above baseline, respectively). During follow-up, median serum P1NP was not consistently different between the prior blosozumab groups and placebo. A similar pattern was apparent for median serum C-terminal telopeptide of type 1 collagen (CTx) levels, with more variability. Mean serum total sclerostin concentration increased with blosozumab, indicating target engagement, and declined to baseline after discontinuation. There were no adverse events considered related to prior treatment with blosozumab. Anti-drug antibodies generally declined in patients who had detectable levels during prior treatment. These findings support the continued study of blosozumab as an anabolic therapy for treatment of osteoporosis.

Gynecologic effects of arzoxifene in postmenopausal women with osteoporosis or low bone mass.

OBJECTIVE: The aim of this study was to report the gynecologic safety findings from the Generations trial, a phase 3 study of the selective estrogen receptor modulator (SERM), arzoxifene. METHODS: A predefined objective of the trial was to evaluate the effects of arzoxifene on the genital tract. Gynecologic examinations were performed yearly, and further gynecologic assessment, including endometrial biopsy, was performed in a predefined subset of women and in those who developed vaginal bleeding. RESULTS: Overall, 9,354 women were randomized (4,678 to placebo, 4,676 to arzoxifene 20 mg/d). There were 13 adjudicated cases of endometrial cancer (placebo, 4 cases; arzoxifene, 9 cases: P = 0.165) and 6 cases of endometrial hyperplasia (placebo, 2 cases; arzoxifene, 4 cases). Endometrial thickness, assessed at 24- and 36-month transvaginal ultrasounds in a subset of women, increased slightly in women assigned to arzoxifene compared with baseline and women in placebo. At the last measurement, 3 (1.7%) women assigned to placebo and 21 (10.2%) assigned to arzoxifene had an endometrial thickness greater than 5 mm (P < 0.001 for difference between treatment groups). Endometrial polyps were more common in women treated with arzoxifene (n = 37) than in women treated with placebo (n = 18; P < 0.05). Vulvular and vaginal inflammation, including mycotic infections, and vaginal discharge were reported more frequently in women treated with arzoxifene than in women treated with placebo, as were urinary tract infections. CONCLUSIONS: Gynecologic events were generally more common in women treated with arzoxifene than in women treated with placebo. The gynecologic effects of arzoxifene seem to differ from those of raloxifene, although both SERMs have a benzothiophene structure. Although all SERMs influence cells through the estrogen receptor, they need to be evaluated independently in terms of their effects on various tissues, including the genital tract.

Teriparatide reduces bone microdamage accumulation in postmenopausal women previously treated with alendronate.

Suppression of bone turnover by bisphosphonates is associated with increased bone microdamage accumulation in animal models. Our objective was to study the effects of teriparatide treatment on changes in microdamage accumulation at the iliac crest in previously treatment-naïve patients or in those switched from alendronate to teriparatide. Sixty-six postmenopausal women with osteoporosis (mean age, 68.0 yr; and mean BMD T-score of -2.8 at lumbar spine and -1.7 at total hip; 62% with prevalent fractures) entered this prospective, nonrandomized study and started with 24-mo 20 microg/d subcutaneous teriparatide treatment in monotherapy: 38 patients stopped previous alendronate treatment (10 mg/d or 70 mg/wk for a mean duration of 63.6 mo) and switched to teriparatide, whereas 28 were previously treatment naïve. Thirty-one paired biopsies with two intact cortices were collected and analyzed for microstructure and microdamage accumulation at baseline and after 24 mo of teriparatide administration. After 24 mo of teriparatide treatment, crack density (Cr.Dn), crack surface density (Cr.S.Dn), and crack length (Cr.Le) were decreased in previously alendronate-treated patients, whereas only Cr.Le was reduced in former treatment-naïve patients. Patients with lower initial femoral neck BMD also showed a higher reduction of microdamage accumulation. Better bone microarchitecture correlated positively, whereas bone turnover markers and age did not correlate with reduced microdamage accumulation on teriparatide. In conclusion, teriparatide reduces microdamage accumulation in the iliac crest of patients previously treated with alendronate. There is insufficient evidence to suggest that age or bone turnover would be associated with this change.

Low bone mineral density is associated with bone microdamage accumulation in postmenopausal women with osteoporosis.

Marked suppression of bone turnover by bisphosphonates is associated with increased bone microdamage accumulation in animal models. The purpose of this study was to test the hypothesis that long-term treatment with alendronate (ALN) results in accumulation of microdamage in bone in women after menopause. Sixty-six postmenopausal women with osteoporosis (mean age of 68.0 years and mean BMD T-score of -1.7 at total hip and -2.8 at lumbar spine; 62% with prevalent fractures) were evaluated in this cross-sectional analysis. Thirty-eight had been treated previously with ALN (10 mg/day or 70 mg/week for a mean duration of 63.6 months) while twenty-eight were treatment naive (TN). Without adjustments, crack surface density (Cr.S.Dn) and crack density (Cr.Dn) were not different between ALN and TN patients. After adjustment for potential confounders (age, prevalent fractures, femoral neck BMD, activation frequency and center), Cr.Dn was elevated in ALN patients (P=0.028 and P=0.069 for Cr.S.Dn). In ALN patients only, lower femoral neck BMD (Cr.S.Dn, r=-0.58, P=0.003; Cr.Dn, r=-0.54, P=0.005) and increased age (Cr.S.Dn, r=0.43, P=0.03; Cr.Dn, r=0.43, P=0.03) were associated with microdamage accumulation. Among potential confounders, femoral neck BMD was the only independent predictor for these correlations (P=0.04 for Cr.Dn and P=0.03 for Cr.S.Dn). We conclude that increased microdamage accumulation may occur in low BMD patients treated with alendronate.

Increases in BMD correlate with improvements in bone microarchitecture with teriparatide treatment in postmenopausal women with osteoporosis.

UNLABELLED: Increases in BMD are correlated with improvements in 2D and 3D trabecular microarchitecture indices with teriparatide treatment. Therefore, improvements in trabecular bone microarchitecture may be one of the mechanisms to explain how BMD increases improve bone strength during teriparatide treatment. INTRODUCTION: Bone strength is determined by BMD and other elements of bone quality, including bone microarchitecture. Teriparatide treatment increases BMD and improves both cortical and trabecular bone microarchitecture. Increases in lumbar spine (LS) BMD account for approximately 30-41% of the vertebral fracture risk reduction with teriparatide treatment. The relationship between increases in BMD and improvements in cortical and trabecular microarchitecture has not yet been studied. MATERIALS AND METHODS: The relationship between increases in BMD and improvements in cortical and trabecular microarchitecture after teriparatide treatment was assessed using data from a subset of patients who had areal BMD measurements and structural parameters from transiliac bone biopsies in the Fracture Prevention Trial. 2D histomorphometric and 3D microCT parameters were measured at baseline and 12 (n = 21) or 22 (n = 36) mo. LS BMD was assessed at baseline and 12 and 18 mo, and femoral neck (FN) BMD was measured at baseline and 12 mo. Pearson correlation was performed to assess the relationship between actual changes in BMD and actual changes in microarchitectural parameters. RESULTS: Changes in LS BMD at 12 mo were significantly correlated with improvements in trabecular bone structure at 22 mo: 2D bone volume (r = 0.45, p = 0.02), 2D mean wall thickness (r = 0.41, p = 0.03), 3D bone volume (r = 0.48, p = 0.006), 3D trabecular thickness (r = 0.44, p = 0.01), 3D trabecular separation (r = -0.37, p = 0.04), 3D structural model index (r = -0.54, p = 0.001), and 3D connectivity density (r = 0.41, p = 0.02). Changes in LS BMD at 18 mo had similar correlations with improvements in bone structure at 22 mo. Changes in FN BMD at 12 mo were significantly correlated with changes in 2D mean wall thickness (r = 0.56, p = 0.002), 3D bone volume (r = 0.51, p = 0.004), 3D trabecular thickness (r = 0.44, p = 0.01), 3D trabecular separation (r = -0.46, p = 0.01), and 3D structural model index (r = -0.55, p = 0.001). CONCLUSIONS: Increases in BMD are correlated with improvements in trabecular microarchitecture in iliac crest of patients with teriparatide treatment. Therefore, improvements in trabecular bone microarchitecture may be one of the mechanisms to explain how BMD increases improve bone strength during teriparatide treatment.

Sustained nonvertebral fragility fracture risk reduction after discontinuation of teriparatide treatment.

UNLABELLED: A follow-up in 1262 women was conducted after the discontinuation of teriparatide. The hazard ratio for combined teriparatide group (20 and 40 microg) for the 50-month period after baseline was 0.57 (p = 0.002), suggesting a sustained effect in reducing the risk of nonvertebral fragility fracture. INTRODUCTION: Treatment with teriparatide [rhPTH(1-34)] 20 and 40 microg once-daily subcutaneous dosing significantly reduced the risk of nonvertebral fragility fractures over a median exposure of 19 months. MATERIALS AND METHODS: All participants in the Fracture Prevention Trial were invited to participate in a follow-up study. Prior treatment assignments were revealed, and patients were able to receive osteoporosis treatments without restriction. RESULTS: Approximately 60% of the 1262 patients received an osteoporosis treatment at some time during follow-up, with greater use in the former placebo group than in the combined former teriparatide group (p < 0.05). The hazard ratios for nonvertebral fragility fractures in each teriparatide group relative to placebo were statistically significant for the 50-month period including treatment and follow-up (p < 0.03). In the follow-up period, the hazard ratio was significantly different between the 40 mug and combined groups versus placebo but not for the 20 microg group versus placebo. However, the 20 and 40 microg groups were not different from each other. Kaplan-Meier analysis of time to fracture showed that the fracture incidence in the former placebo and teriparatide groups diverged during the 50-month period including teriparatide treatment and follow-up (p = 0.009). Total hip and femoral neck BMD decreased in teriparatide-treated patients who had no follow-up treatment; BMD remained stable or further increased in patients who received a bisphosphonate after teriparatide treatment. CONCLUSIONS: While the study design is observational, the results support a sustained effect of teriparatide in reducing the risk of nonvertebral fragility fractures up to 30 months after discontinuation of treatment.

Early changes in biochemical markers of bone formation predict BMD response to teriparatide in postmenopausal women with osteoporosis.

UNLABELLED: The relationship between early changes in biochemical markers of bone turnover and the subsequent BMD response to daily teriparatide therapy in women with postmenopausal osteoporosis was studied. Changes in five biochemical markers, obtained from a subset of women enrolled in the Fracture Prevention Trial, were examined. Early increases in the PICP and the PINP were the best predictors of BMD response to teriparatide in this analysis. INTRODUCTION: Early reductions in biochemical markers of bone turnover with antiresorptive therapy negatively correlate with subsequent increases in BMD. We undertook this analysis to determine if early changes in biochemical markers with teriparatide therapy predict subsequent increases in BMD. MATERIALS AND METHODS: In the Fracture Prevention Trial, 1637 postmenopausal women with osteoporosis were randomized to receive daily, self-administered, subcutaneous injections of placebo, teriparatide 20 microg/day, or teriparatide 40 microg/day. Serum concentrations of two bone formation markers (bone-specific alkaline phosphatase [bone ALP] and the carboxy-terminal extension peptide of procollagen type 1 [PICP]) and urinary concentrations of two bone resorption markers (free deoxypyridinoline [DPD] and N-terminal telopeptide [NTX]) were assessed in a trial population subset (n = 520) at baseline and at 1, 3, 6, and 12 months. We also assessed serum concentrations of another bone formation marker, the amino-terminal extension peptide of procollagen type 1 (PINP), in a subset of 771 women at baseline and 3 months. Lumbar spine (LS) BMD was measured by DXA at baseline and 18 months. Femoral neck BMD was measured at baseline and 12 months. RESULTS AND CONCLUSION: Baseline bone turnover status correlated positively and significantly with BMD response. The highest correlations occurred for the LS BMD response to teriparatide 20 microg/day. Among all studied biochemical markers, increases in PICP at 1 month and PINP at 3 months correlated best with increases in LS BMD at 18 months (0.65 and 0.61, respectively; p < 0.05). The relationships between these two biochemical markers and the LS BMD response were stronger than the corresponding relationships for the femoral neck BMD response. Using receiver operator curve analysis, we determined that the increases in PICP at 1 month and PINP at 3 months were the most sensitive and accurate predictors of the LS BMD response.

Early changes in biochemical markers of bone formation correlate with improvements in bone structure during teriparatide therapy.

CONTEXT: Biochemical markers of bone turnover may reflect bone structure during anabolic treatment. OBJECTIVE: The objective was to evaluate associations between changes in biochemical markers and structural and dynamic bone parameters during teriparatide treatment. DESIGN: This study was a randomized, multicenter, double-blind, placebo-controlled fracture prevention trial, with 20-month median treatment duration for biopsy subset. SETTING: The trial was conducted at 11 clinical study sites. PATIENTS: Sixty-one postmenopausal women with osteoporosis who had paired transiliac biopsy specimens participated in the study. INTERVENTIONS: Once-daily sc injections of either placebo or teriparatide (20 or 40 microg) were administered. MAIN OUTCOME MEASURES: The study measured: 1) serum and urinary biochemical markers of bone formation [bone alkaline phosphatase and procollagen I C-terminal propeptide (PICP)] and resorption (N-telopeptide and deoxypyridinoline); and 2) structural and dynamic analyses of bone biopsies, including two-dimensional (2D) histomorphometry and three-dimensional (3D) micro-computed tomography evaluations measured at baseline (n = 57) and 12 (n = 21) or 22 (n = 36) months. RESULTS: U-N-telopeptide/creatinine and serum-PICP correlated with bone structure and dynamic indices at baseline, respectively. Changes in bone alkaline phosphatase at 1 month correlated with changes at 22 months in 2D wall thickness (r = 0.73; P = 0.001), trabecular bone volume (trabecular bone volume per total volume, BV/TV) (r = 0.58; P < 0.05), marrow star volume (r = -0.51; P = 0.05); 3D trabecular thickness (r = 0.49; P < 0.05), and BV/TV (r = 0.54; P < 0.05). Changes in PICP at 1 month correlated with changes in wall thickness (r = 0.60; P = 0.01), and 2D BV/TV (r = 0.51; P < 0.05) at 22 months. Changes in markers at 6 or 12 months were not associated with changes in structural or dynamic parameters. CONCLUSIONS: Early (1-month) changes in biochemical markers of bone formation, but not resorption, correlated with improvements in bone structure after 22 months of teriparatide therapy.