Angelina V. Ciaccia

Safety and adverse effects associated with raloxifene: multiple outcomes of raloxifene evaluation.

OBJECTIVE: To examine the effect of raloxifene on major adverse events that occur with postmenopausal estrogen therapy or tamoxifen. METHODS: The Multiple Outcomes of Raloxifene Evaluation, a multicenter, randomized, double-blind trial, enrolled 7,705 postmenopausal women with osteoporosis. Women were randomly assigned to raloxifene 60 mg/d or 120 mg/d or placebo. Outcomes included venous thromboembolism, cataracts, gallbladder disease, and endometrial hyperplasia or cancer. RESULTS: During a mean follow-up of 3.3 years, raloxifene was associated with an increased risk for venous thromboembolism (relative risk [RR] 2.1; 95% confidence interval [CI] 1.2–3.8). The excess event rate was 1.8 per 1,000 woman-years (95% CI −0.5–4.1), and the number needed to treat to cause 1 event was 170 (95% CI 100–582) over 3.3 years. Risk in the raloxifene group was higher than in the placebo group for the first 2 years, but decreased to about the same rate as in the placebo group thereafter. Raloxifene did not increase risk for cataracts (RR 0.9; 95% CI 0.8–1.1), gallbladder disease (RR 1.0; 95% CI 0.7–1.3), endometrial hyperplasia (RR 1.3; 95% CI 0.4–5.1), or endometrial cancer (RR 0.9; 95% CI 0.3–2.7). CONCLUSION: Raloxifene was associated with an increased risk for venous thromboembolism, but there was no increased risk for cataracts, gallbladder disease, endometrial hyperplasia, or endometrial cancer. LEVEL OF EVIDENCE: I

Raloxifene effect on frequency of surgery for pelvic floor relaxation.

OBJECTIVE To assess the effects of raloxifene therapy on the frequency of surgery for pelvic floor relaxation in postmenopausal women. METHODS This analysis used safety data through 3 years of treatment from three double‐masked, placebo‐controlled, randomized trials of raloxifene, which included 6926 postmenopausal women with uteri at entry. Studies 1 and 2 enrolled 969 nonosteoporotic, postmenopausal women who were assigned to 30, 60, or 150 mg per day raloxifene or placebo. Study 3 enrolled 5957 osteoporotic, postmenopausal women randomized to raloxifene 60 or 120 mg per day or placebo. Indications for any reported pelvic operations were identified, including procedures performed for pelvic organ prolapse or urinary incontinence. RESULTS A total of 34 (1.51%) women in the placebo group and 35 (0.75%) raloxifene‐treated women underwent surgical procedures for pelvic floor relaxation. The odds ratio (and 95% confidence interval) for pelvic floor repair in women assigned to raloxifene was 0.50 (0.31, 0.81). Thus, raloxifene therapy was associated with a significantly reduced risk for pelvic floor surgery (P < .005). CONCLUSION Raloxifene does not increase pelvic floor relaxation. An apparent protective effect on pelvic floor function warrants further investigation.

Selective estrogen receptor modulators: tissue selectivity and differential uterine effects

Selective estrogen receptor modulators (SERMs) are compounds that bind to estrogen receptors and produce estrogen-like (agonist) effects in some tissues and estrogen-blocking (antagonist) effects in other tissues. One of the goals of SERM research has been to develop compounds that provide the potential benefits of estrogen in the skeleton and cardiovascular system, but avoid the negative effects of estrogen in other tissues. Estrogen therapy has been consistently associated with endometrial stimulation, including glandular proliferation, hyperplasia and cancer. In contrast, the presence or degree of endometrial stimulation observed with SERMs varies by compound. The purpose of this review is to differentiate the endometrial effects of compounds that display a SERM-like profile. Molecular mechanisms involving SERM binding to estrogen receptors, preclinical uterine effects in both tissue culture and animal models, and endometrial findings in clinical experience are discussed. There are several SERMs commercially available or in development. The favorable safety profile of raloxifene in the uterus differentiates it from the others. Future SERM development will continue to focus on finding compounds that exhibit minimal endometrial stimulation.

Transition from estrogen-progestin to raloxifene in postmenopausal women: effect on vasomotor symptoms.

OBJECTIVE: To compare vasomotor symptoms after transition from estrogen–progestin therapy to raloxifene 60 mg/d with and without a placebo washout. METHODS: Postmenopausal women currently taking continuous combined estrogen–progestin therapy (conjugated equine estrogen, 0.625 mg/medroxyprogesterone acetate, 2.5 or 5 mg) daily for 5 or more months were enrolled. Women were randomized to 1 of 4 blinded regimens: 1) 12 weeks estrogen–progestin; 2) 12 weeks placebo; 3) 4 weeks placebo, then 8 weeks raloxifene; or 4) 12 weeks raloxifene. For the final 36 weeks, all subjects received raloxifene. Vasomotor symptoms were assessed by patient diaries. RESULTS: A total of 266 women (mean age 57.5) were enrolled. Mean hot flush frequency at baseline was approximately 1 per week in the entire population, with 16% of women reporting hot flushes. Mean frequency and severity of hot flushes during the first 12 weeks of the study were statistically greater in the 3 groups transitioned off estrogen–progestin (range of hot flushes per week: 4 weeks, 11–12; 8 weeks, 18–24; 12 weeks, 13–16), as compared with those continuing estrogen–progestin, with no difference between these 3 groups (P ≥ .1). Approximately 50–70% of these women reported hot flushes, generally rated as mild to moderate by participants, after estrogen–progestin discontinuation. CONCLUSION: A large proportion of women discontinuing estrogen–progestin experience hot flushes. Raloxifene does not appear to increase the frequency or severity of vasomotor symptoms in women discontinuing estrogen–progestin more than that observed with placebo treatment after estrogen–progestin discontinuation. Transition from estrogen–progestin to raloxifene with no washout period therefore may be acceptable. LEVEL OF EVIDENCE: I

Antiresorptive treatment of postmenopausal osteoporosis: review of randomized clinical studies and rationale for the Evista alendronate comparison (EVA) trial.

SUMMARY Standard pharmacological antiresorptive therapy for the prevention and/or treatment of postmenopausal osteoporosis now consists of four categories of drugs: estrogens, a selective estrogen receptor modulator (SERM), bisphosponates, and calcitonin. All of these drugs have been studied in randomized controlled trials, but meaningful comparisons of the efficacy of drugs have been difficult due to differences in baseline risks for fracture and differences in study design, including calcium and vitamin D supplementation, definition of fracture, and discontinuation rates. The current paper reviews results from pivotal studies of antiresorptive therapies with fracture as a primary endpoint, as well as head-to-head trials comparing these therapies using surrogate markers of fracture risk, and introduces the first head-to-head trial with fracture as a primary endpoint. The Evista* Alendronate Comparison (EVA) trial, a multi-center, double-blind, double-dummy, randomized trial with two active treatment arms is currently underway to compare directly the osteoporotic fracture risk reduction efficacy of raloxifene and alendronate in postmenopausal women with osteoporosis as defined by bone mineral density. The results from this trial will permit more informed judgment by practitioners and provider groups concerning the relative clinical utility of these two drugs. * Evista is a registered trade name of Eli Lilly and Company, Indianapolis, IN, USA

Heparin Promotes Proteolytic Inactivation by Thrombin of a Reactive Site Mutant (L444R) of Recombinant Heparin Cofactor II

A heparin cofactor II (HCII) mutant with an Arg substituted for Leu444 at the P1 position (L444R-rHCII) was previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). The present study characterizes the effect of glycosaminoglycans on the substrate versus inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry of inhibition of L444R-rHCII with α-thrombin (compared with minus glycosaminoglycan) but decreased it with R93A,R97A,R101A-thrombin, a mutant thrombin that does not bind glycosaminoglycans. Dermatan sulfate decreased the stoichiometry of inhibition of L444R-rHCII with both proteinases. SDS-polyacrylamide gel electrophoresis showed no proteolysis of L444R-rHCII when incubated with R93A,R97A,R101A-thrombin in the absence or the presence of glycosaminoglycan or with α-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with α-thrombin/heparin. A time course of α-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the α-thrombin activity being regained after 6 h of incubation. In contrast, all other combinations of inhibitor, proteinase, and glycosaminoglycan resulted in complete and sustained inhibition of the proteinase. Heparin fragments of 8-20 polysaccharides in length rapidly accelerated L444R-rHCII inhibition of both α-thrombin and R93A,R97A,R101A-thrombin. After extended incubations, R93A,R97A,R101A-thrombin was completely inhibited by L444R-rHCII with all the heparin fragments, but approximately 30-50% of α-thrombin activity remained with fragments long enough to bridge HCII-thrombin. These results collectively indicate that ternary complex formation, mediated by heparin, increases L444R-rHCII inactivation by α-thrombin.

The Effect of Retinoic Acid on Thymidine Incorporation and Morphology of Corneal Stromal Fibroblasts

The effect of retinoic acid on DNA synthesis and cell morphology was studied using corneal stromal fibroblasts in culture. All-trans retinoic acid induces an increase in DNA synthesis after 24 hours of exposure. Autoradiographic studies of 3H-thymidine incorporation into corneal stromal cells exposed to 10(-6) M retinoic acid for 24 hours showed an increase in labeling which ranged from 19.2% to 67.6% over control cultures. Scintillation analysis of labeled cultures also showed an increase in incorporation of 3H-thymidine into cells treated with 10(-6) M retinoic acid, with increases ranging from 21.8% to 114.7% above control cultures. Exposure of cultured corneal stromal cells to 10(-6) M retinoic acid resulted in a dramatic change in cell morphology such that they changed from spindle-shaped to round, flattened cells which were epithelioid in appearance. These data demonstrate that biological activity of retinoic acid in stromal fibroblasts and imply a role for vitamin A in maintenance of stroma structure and function.

17β-Estradiol, But Not Raloxifene, Decreases Thrombomodulin in the Antithrombotic Protein C Pathway

Raloxifene is a nonsteroidal selective estrogen receptor modulator (SERM) that mimics the effects of estrogen on some plasma lipids and may have direct effects on the vascular wall. The objective of this study was to determine the effects of 17β-estradiol, raloxifene, and LY139,478 (a related benzothiophene SERM) on the anticoagulant protein C pathway. In human vascular endothelial cells activated with interleukin-1 (IL-1), we demonstrated decreased thrombomodulin-dependent protein C activation. 17β-estradiol reduced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells by decreasing thrombomodulin expression. In contrast, raloxifene and LY139,478 enhanced the anticoagulant properties of both unstimulated and IL-1-activated endothelial cells through upregulation of thrombomodulin. Regulation of the protein C pathway via thrombomodulin on vascular endothelium may be a novel mechanism by which SERMs could potentially confer cardioprotective effects and reduce the thrombotic ...

Arginine 200 of Heparin Cofactor II Promotes Intramolecular Interactions of the Acidic Domain IMPLICATION FOR THROMBIN INHIBITION

Heparin cofactor II (HCII) is presumed to be a physiological inhibitor of the serine proteinase thrombin. The reaction between HCII and thrombin is quite unique, because it involves an unusual HCII-reactive site loop sequence of Leu444-Ser445, requires the presence of glycosaminoglycans for optimal activity and involves a protein-protein interaction besides the reactive site loop-active site interaction characteristic of serine proteinase inhibitor-serine proteinase pairs. Two mutations at a unique HCII residue, Arg200 → Ala or Glu, were generated by site-directed mutagenesis. The mutations did not alter either HCII binding to heparin-Sepharose or HCII inhibition of thrombin in the presence of heparin or dermatan sulfate, suggesting that Arg200 is not part of the glycosaminoglycan binding site of HCII. In the absence of glycosaminoglycan, there was a significant increase in α-thrombin inhibition by the Arg200 mutants as compared with wild type recombinant HCII (wt-rHCII), whereas inhibition rates with chymotrypsin were identical. Inhibition of γT-thrombin, which lacks anion-binding exosite 1 ((ABE-1), the region of α-thrombin that interacts with the acidic domain of HCII), was significantly reduced compared with α-thrombin, but the reduction was more dramatic for the Arg200-rHCII mutants. Hirugen, which binds to ABE-1 of α-thrombin, also diminished inhibition of α-thrombin by the Arg200-rHCII mutants to nearly wt-rHCII levels. Both Arg200-rHCII mutants had significantly increasedk a values as compared with wt-rHCII, whereas thek d rates were unchanged. Collectively, these results suggest that the improved inhibitory activity of the Arg200-rHCII mutants is mediated by enhanced interactions between the acidic domain and ABE-1, resulting in an increased HCII-thrombin association rate.