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.

17beta-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 17beta-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. 17beta-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 risk associated with HRT in compromised patients.

A pharmacological review of selective oestrogen receptor modulators.

Selective oestrogen receptor modulators (SERMs) are structurally diverse non-steroidal compounds that bind to oestrogen receptors and produce oestrogen agonist effects in some tissues and oestrogen antagonist effects in others. SERMs are being evaluated for a number of oestrogen-related diseases, including post-menopausal osteoporosis, hormone-dependent cancers, and cardiovascular disease. Several compounds that exhibit a SERM profile are currently available for clinical use, including clomiphene, tamoxifen, and toremifene (which are triphenylethylenes) and raloxifene (a benzothiophene). Clomiphene is used for the induction of ovulation in sub-fertile women attempting pregnancy. Tamoxifen and toremifene are both used to treat breast cancer. Tamoxifen may have beneficial effects on bone mineral density and serum lipids. The effects of toremifene on serum lipids are similar to that of tamoxifen. Both compounds have stimulatory effects on the endometrium. Raloxifene, indicated for the treatment and prevention of post-menopausal osteoporosis, has beneficial effects on bone mineral density and serum lipids, but does not increase the risk of endometrial hyperplasia or endometrial cancer. Recently, raloxifene was shown to reduce the incidence of vertebral fractures in otherwise healthy women with osteoporosis; in the same study, a reduced incidence of breast cancer was also observed. Similar to oestrogens, SERMs increase the incidence of venous thromboembolism. Several newer compounds that exhibit a SERM profile are also in clinical development, including other triphenylethylenes (droloxifene, idoxifene) and benzothiophenes (LY353381.HCl), benzopyrans (EM-800), and naphthalenes (CP-336,156).

Raloxifene: a new choice for treating and preventing osteoporosis.

Selective estrogen receptor modulators (SERMs) are a new class of drugs that provide a new option for addressing the health challenges of postmenopausal women. This review discusses the proposed mechanism of action of SERMs and describes clinical findings on raloxifene, a SERM now available for treating and preventing osteoporosis.

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.

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 alpha-thrombin inhibition by the Arg200 mutants as compared with wild type recombinant HCII (wt-rHCII), whereas inhibition rates with chymotrypsin were identical. Inhibition of gammaT-thrombin, which lacks anion-binding exosite 1 ((ABE-1), the region of alpha-thrombin that interacts with the acidic domain of HCII), was significantly reduced compared with alpha-thrombin, but the reduction was more dramatic for the Arg200-rHCII mutants. Hirugen, which binds to ABE-1 of alpha-thrombin, also diminished inhibition of alpha-thrombin by the Arg200-rHCII mutants to nearly wt-rHCII levels. Both Arg200-rHCII mutants had significantly increased ka values as compared with wt-rHCII, whereas the kd 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.

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 alpha-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 alpha-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with alpha-thrombin/heparin. A time course of alpha-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the alpha-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 alpha-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 alpha-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 alpha-thrombin.

Characterization of recombinant heparin cofactor II expressed in insect cells.

Recombinant human heparin cofactor II (rHCII) was expressed as a fully active protein in the High-Five insect cell line. A maximal protein concentration of 6 micrograms/10(6) cells was achieved 2 days postinfection. Approximately 40 micrograms of partially purified rHCII was routinely recovered from 50 ml of media after sequential heparin and Q-Sepharose affinity adsorption. rHCII had a slightly lower apparent molecular weight than blood plasma HCII (pHCII) due to differences in N-glycosylation. Like pHCII, rHCII formed a stable bimolecular complex with thrombin when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The thrombin and chymotrypsin inhibitory properties of rHCII and pHCII were quite similar. In the absence of glycosaminoglycan, the thrombin inhibition rate (k2 x 10(-4) M-1 min-1) was 2.29 +/- 0.36 for rHCII and 3.38 +/- 0.34 for pHCII. Chymotrypsin inhibition rates (k2 x 10(-5) M-1 min-1) were 6.2 +/- 2.0 for rHCII and 8.0 +/- 2.6 for pHCII. In the presence of glycosaminoglycans, the maximal thrombin inhibition rate (k2 x 10(-3) M-1 min-1) for rHCII was 10.4 +/- 2.5 at 100 micrograms/ml heparin and 16.0 +/- 4.3 at 1000 micrograms/ml dermatan sulfate compared to 9.0 +/- 0.7 at 200 micrograms/ml heparin and 18.5 +/- 5.3 at 1000 micrograms/ml dermatan sulfate for pHCII. HCII inhibition of thrombin was blocked by a synthetic sulfated hirudin peptide in both the presence and the absence of glycosaminoglycan. The present report describes for the first time the expression and characterization of HCII in a baculovirus system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rHCII for future structure-function studies.