Three-year denosumab treatment in postmenopausal Japanese women and men with osteoporosis: results from a 1-year open-label extension of the Denosumab Fracture Intervention Randomized Placebo Controlled Trial (DIRECT).

SUMMARY: A 12-month extension phase of DIRECT in Japanese subjects with osteoporosis showed that total 3 years of denosumab treatment in Japanese postmenopausal women and men with osteoporosis was associated with low fracture rates, persistent bone turnover marker (BTM) reductions, continuous bone mineral density (BMD) increases, and a favorable overall benefit/risk profile. INTRODUCTION: The DIRECT trial demonstrated that 2 years of treatment with denosumab 60 mg subcutaneously every 6 months significantly reduced the incidence of vertebral fracture compared to placebo in Japanese postmenopausal women and men with osteoporosis. The purpose of this study is to evaluate the efficacy and safety of denosumab treatment for up to 3 years. METHODS: This study includes a 2-year randomized, double-blind, placebo-controlled phase and a 1-year open-label extension phase in which all subjects received denosumab. The data correspond to 3 years of denosumab treatment in subjects who received denosumab (long-term group) and 1 year of denosumab treatment in subjects who received placebo (cross-over group) in the double-blind phase. RESULTS: Eight hundred and ten subjects who completed the double-blind phase enrolled into the extension phase, and 775 subjects completed the study. All subjects received denosumab with daily supplements of calcium and vitamin D. The cumulative 36-month incidences of new or worsening vertebral fractures and new vertebral fractures were 3.8 and 2.5 %, respectively, in the long-term group. In this group, the BMD continued to increase, and the reduction in BTMs was maintained. In the cross-over group, comparable BMD increases and BTMs reductions to those of in their first year of the long-term group were confirmed. Adverse events did not show a notable increase with long-term denosumab administration. One event of osteonecrosis of the jaw occurred in the cross-over group. CONCLUSIONS: Three-year denosumab treatment in Japanese subjects with osteoporosis showed a favorable benefit/risk profile.

Age- and gender-related changes in ligament components.

The age- and gender-related changes in extracellular matrix components (elastin, elastin cross-links, fibrillin, collagen and glycoprotein) and mineral components (calcium, Ca; phosphorus, P) in human lumbar yellow ligaments were investigated using samples obtained from surgical specimens. The mineral (Ca and P) contents increased with ageing (r = 0.703 and r = 0.772, respectively), whereas the contents of matrix components tended to decrease with ageing (elastin r = -0.261, elastin cross-links r = -0.213, fibrillin r = 0.494; collagen r = -0.322 and glycoprotein r = -0.143). Comparison of the male and female groups revealed that the ligament elastin content and elastin cross-links decreased in the male group, whereas the ligament collagen content decreased in the female group significantly in an age-dependent manner (r = -0.788, r = -0.753 and r = -0.721, respectively). These findings demonstrate age- and gender-related changes in mineral and matrix components (especially elastin and collagen) in the lumbar yellow ligaments in the Japanese population. It is suggested that elastin and collagen metabolism in ligaments changes both with age and according to gender.

[Quantitative changes of elastin, fibrillin and collagen in abdominal aortic aneurysms].

To examine quantitative changes of elastin, fibrillin and collagen in abdominal aortic aneurysms, including ruptured abdominal aortic aneurysms (RAAA), inflammatory abdominal aortic aneurysms (IAAA) and abdominal aortic aneurysms (AAA) were measured. Items measured included the desmosine content of the aorta (desmosine 1) or of the elastin fraction (desmosine 2), fibrillin content in the aorta, hydroxyproline in the aorta, collagen percent and elastin percent, and were compared with control samples from the nonaneurysmal aortic segments. The elastin contents (desmosine 2) in RAAA, IAAA and AAA were significantly lower than those of controls. The content of the desmosine 2 from IAAA and AAA did not show a negative association with Ca. The fibrillin contents of the aorta from RAAA, IAAA and AAA were significantly higher than those of controls. The collagen content in the RAAA aorta was significantly higher than that of controls. There was a correlation of the ratio of fibrillin to elastin components (fibrillin/desmosine 1 or fibrillin/desmosine 2 or fibrillin/elastin%) and the ratio of collagen to elastin components (collagen/desmosine 1 or collagen/desmosine 2 or collagen/elastin%). These results indicated that increasing fibrillin and collagen might be a complementary result of decreasing elastin crosslinks in the aorta. This phenomenon was markedly in RAAA.

[Qualitative change in the elastin from the calcified portion of human artery].

To examine the qualitative changes of elastin and the aorta related to calcification of human arteries, biochemical properties were measured, including calcium (Ca), phosphorus (P) and magnesium (Mg) contents in the aorta or in the elastin fraction in calcification, cholesterol content in atherosclerosis, desmosine content of cross-link, free thiol contents (free SH/total SH) and hydrophobic properties in the elastin fraction from the calcified portion, adjacent sites and another normal artery. The results from different sites of the calcified abdominal artery are as follows: The contents of Ca, P and Mg in aorta and the elastin fraction from the calcification site were higher than those at other sites. Moreover, Ca in the aorta and elastin fraction correlated positively with P and Mg. The content of cholesterol in the calcification site was the same as at other sites and did not correlate with Ca, P or Mg. The content of desmosine in the calcification site was significantly lower than that in different sites. In addition, its content was negatively associated with Ca and P in the elastin fraction and with the aortic Mg. The content of free thiol in the calcification site was similar to the other sites and correlated negatively with Ca and P in the aorta. The hydrophobicity in the calcification was similar to that at other sites, and was negatively associated with Ca and Mg in the elastin fraction.

Long-term n-3 fatty acid deficiency induces no substantial change in the rate of protein synthesis in rat brain and liver.

The influence of long-term n-3 fatty acid deficiency on the rate of protein synthesis in rat brain and liver was investigated in relation to learning behavior or a presumed survival time-shortening factor (SSF) in rapeseed oil, using a large-dose [3H]phenylalanine (Phe) injection method. When Wistar rats were made n-3 fatty acid-deficient by feeding a safflower oil (alpha-linolenate-deficient) diet for 2 generations, conditions under which the safflower oil group had been shown to exhibit altered learning behaviors, compared with the perilla oil group, no significant changes in the rate of protein synthesis were observed compared with the perilla oil (alpha-linolenate-sufficient) or rapeseed oil (alpha-linolenate-sufficient but SSF-containing) groups. However, the rapeseed oil group had a reduced specific radioactivity of free Phe in the cerebral cortex, compared with the safflower oil group. In contrast to the reported observation of very long-term n-3 fatty acid deficiency inducing an almost 2-fold increase in the rate of protein synthesis in the brain, our results indicate that altered learning behavior resulting from n-3 fatty acid deficiency in rats is not associated with any substantial changes in the rate of protein synthesis in the brain.

Characteristic change of urinary elastin peptides and desmosine in the aortic aneurysm.

To estimate elastin metabolism in aneurysm, urinary levels of desmosine and elastin peptide in patients (n=23, range 54 to 85 years old) with aneurysm were measured by ELISA and compared between two control groups divided by age (<10 years old and >20 years old). The amounts of urinary desmosine and elastin peptide in the aneurysm group were significantly increased compared with those in the older control group (>20 years old). There was a correlation between urinary desmosine and elastin peptide in the young group. On the other hand, no such correlation was observed in the aneurysm group and the older control group. The distribution of the ratio (desmosine/elastin peptide) in the aneurysm group was different from that of the young control group. We conclude that assay of elastin peptide and desmosine in urine are useful in characterizing elastin degradation in a patient with aneurysm.

Analysis of elastin metabolism in patients with late-onset focal dermal elastosis.

Late-onset focal dermal elastosis has recently been described as new clinical entity characterized by pseudoxanthoma elasticum-like eruptions and an accumulation of normal-appearing elastic fibres in the dermis. Elastin and collagen contents of the skin of 2 patients were 2- and 1.4-fold higher than in the skin of controls, respectively. A focal accumulation of elastin but not of fibrillin-1 was observed by immunohistochemical staining. The levels of type I and III collagen and elastin mRNAs isolated from cultured patient fibroblasts were elevated 2-3-fold compared with control fibroblasts. There was no significant change in the excretion of elastin peptides in the urine of patients and controls. These results suggest that the focal accumulation of elastic fibres in the patient skin may be related to overexpression of elastin rather than to altered degradation of elastin.

Comparison of elastolytic activity between experimental aneurysm and experimental diabetes mellitus.

In order to clarify the degradation of elastin under abnormal conditions, we examined the aortic elastolytic activity in rat experimental diabetes mellitus induced by treatment with streptozotocin and in rat experimental aneurysm induced by treatment with an inhibitor of lysyloxidase (beta-aminopropionitrile: BAPN). Measurement of the aortic elastolytic activity used 14C-labeled elastin as the substrate, and the determined value was compared with the aortic lysosomal enzyme (acid phosphatase) activity. In the case of experimental diabetes, the aortic elastolytic activity was not changed, but the aortic acid phosphatase activity was significantly increased compared with the control. In the case of the experimental aneurysm, the aortic elastolytic activity measured after 2 and 3 weeks was increased compared with each control. There was a negative correlation (r=-0.435, n=36) between the elastolytic activity and the cross-linking (desmosine) content in the aorta. The ratio of elastolytic activity to desmosine content was significantly increased compared with the control. Therefore, the degradation of aortic elastin in the experimental aneurysm was caused by elastase, not by lysosomal enzymes. We concluded that an elastase-like enzyme mainly contributed to the degradation of elastin in the experimental aneurysm since the inhibitory pattern of the elastolytic activity in the experimental aneurysm was similar to that of pancreatic elastase.

p53 and p21(WAF1) expression in lymphocytic thyroiditis and thyroid tumors.

To clarify the roles of increased apoptosis and cell proliferation in chronic autoimmune lymphocytic thyroiditis and thyroid tumorigenesis, expression of p53 and p21(WAF1) proteins was immunohistochemically investigated in a series of 158 cases. Positive epithelial cells were quantified to give numbers per unit square and to score for distribution. They were found scattered in nontumorous thyroid tissue, their numbers increasing with the severity of thyroiditis and the correlation between expression of the two proteins, regardless of the presence or absence of thyroid neoplasms. Simultaneous expression of both proteins was occasionally found in the same cells by analysis of serial histologic sections. In thyroid tumors, increased expression was found to be diffuse, focal, or scattered for the distribution of p53- or p21(WAF1)-immunopositive cells in accordance with tumor cell dedifferentiation, showing significant correlation between expression of the two proteins. Correlated with these findings, enhanced apoptosis along with decreased Bcl-2 expression and increased Ki-67 labeling in lymphocytic thyroiditis and thyroid tumors was also confirmed in the same series, using in situ DNA nick-end labeling and immunohistochemical methods. Increased expression of p53 and/or p21(WAF1) proteins was thus suggestive of possible DNA damage and increased apoptosis in autoimmune thyroiditis. In addition, a significant correlation between protein overexpression and dedifferentiation of thyroid tumor cells was apparent.

Significant correlation of telomerase activity in thyroid papillary carcinomas with cell differentiation, proliferation and extrathyroidal extension.

Telomerase activity was examined by telomeric repeat amplification protocol assay in thyroid disease states, including adenomas and carcinomas, and correlated with clinicopathological features. Of a total of 26 papillary carcinomas, 16 cases (61.5%) were positive, with the poorly differentiated subtype being predominant (P < 0.05). A significantly more shortened terminal restriction fragment length (P < 0.05), higher incidence of extrathyroidal extension (P < 0.001), and more elevated Ki-67 labeling indices (P < 0.002) were also found in telomerase-positive than in telomerase-negative papillary carcinomas. Of four follicular carcinomas, 3 cases (75.0%) were positive. Positive telomerase activity in follicular adenomas (9/23 cases, 39.1%) and lymphocytic thyroiditis (12/22 cases, 54.5%) appeared to be mainly caused by infiltrating lymphocytes. However, three cases of atypical adenoma with relatively increased Ki-67 labeling indices were positive, suggesting a possibility of malignant potential. The good correlations with extrathyroidal invasiveness, Ki-67 labeling indices and poor differentiation of papillary carcinomas, established by multivariate analysis, suggest that this parameter might have potential application in the estimation of tumor progression and prognosis, and in clinical management.

Comparison of ELISA and HPLC for the determination of desmosine or isodesmosine in aortic tissue elastin.

We developed a rapid and simple method for estimating tissue elastin content by measuring desmosine (D) in tissue hydrolysates by competitive ELISA. We compared the ELISA previously reported HPLC methods. When D or isodesmosine (ID) in hydrolysate of the same elastin preparation were measured by the two different methods, a good linear relationship was obtained (r = 0.854 for human aorta or r = 0.938 for rabbit aorta, respectively). The ELISA method can detect as little as 6 pmol/ml and it may be useful in monitoring elastin metabolism in patients with various connective tissue diseases.

15-Deoxyspergualin "rescue therapy" for methylprednisolone-resistant rejection of renal transplants as compared with anti-T cell monoclonal antibody (OKT3).

A randomized trial with OKT3, an anti-T cell monoclonal antibody or with 15-deoxyspergualin against methylprednisolone-resistant rejection crisis was performed in 25 posttransplant patients immunosuppressed with prednisolone and cyclosporine. At least temporary reversal of rejection was observed in 58.3% of patients treated with 15-deoxyspergualin. This reversal rate may be quite comparable to 61.5% seen in patients treated with OKT3. Adverse effects with 15-deoxyspergualin were related to bone marrow suppression, while those with OKT3 were pyrexia, gastrointestinal symptoms, and herpes infection. In contrast to OKT3, which may act by modulating T cell surface antigen, 15-deoxyspergualin may be effective somewhere in the later stages of the rejection cascade.