Persistence at 12 months with denosumab in postmenopausal women with osteoporosis: interim results from a prospective observational study.

UNLABELLED: To determine persistence with subcutaneous denosumab every 6 months in women being treated for osteoporosis, we conducted a single-arm prospective, observational study in the United States and Canada. Among 935 patients enrolled, 12-month persistence was 82%, with 66 patients (7%) reporting serious adverse events and 19 patients (2%) reporting fractures. INTRODUCTION: Increased persistence with osteoporosis therapy is associated with reduced fracture risk. Denosumab reduced fracture risk in clinical trials; persistence in community settings is undetermined. This study evaluates persistence with denosumab in community practice in the United States (US) and Canada. METHODS: In a 24-month multicenter, prospective, single-arm, observational study, women being treated for osteoporosis were enrolled ≤4 weeks after the first subcutaneous injection of denosumab. For this 12-month prespecified interim analysis, endpoints include persistence (one injection at study entry and another within 6 months + 8 weeks), attributes associated with persistence (univariate analysis), and serious adverse events (SAEs). RESULTS: Among 935 patients (mean age 71 years), mean baseline T-scores were -2.18 (femoral neck) and -2.00 (lumbar spine); 50% of patients had experienced osteoporotic fracture(s). At 12 months, 82 % of patients were persistent with denosumab. Baseline factors significantly (p < 0.05) associated with higher persistence included use of osteoporosis medications >5 years previously, lumbar spine T-score > -2.5, and treatment by female physicians (US). Lower persistence was associated (p < 0.05) with psychiatric diagnoses including depression, southern US residence, being divorced, separated, or widowed (US), and prior hip fracture (Canada). SAEs were reported in 66 patients (7%); no SAEs of osteonecrosis of the jaw, atypical femoral fracture, fracture healing complications, hypocalcemia, eczema, or hypersensitivity were reported. Nineteen patients (2%) reported osteoporotic fractures. CONCLUSIONS: The 12-month persistence observed in this single-arm open-label study of US and Canadian community practice extends the evidence regarding denosumab's potential role in reducing fracture risk in postmenopausal women with osteoporosis.

Final results of the DAPS (Denosumab Adherence Preference Satisfaction) study: a 24-month, randomized, crossover comparison with alendronate in postmenopausal women.

UNLABELLED: The final analysis of this 2-year, randomized, crossover study showed that postmenopausal women with osteoporosis were more adherent, compliant, and persistent with subcutaneous denosumab injections every 6 months than with once-weekly alendronate tablets. After receiving both treatments, women reported greater satisfaction with injectable denosumab and preferred it over oral alendronate. INTRODUCTION: Osteoporosis patients who are non-compliant or non-persistent with therapy may have suboptimal clinical outcomes. This 2-year, randomized, open-label, crossover study compared treatment adherence between subcutaneous denosumab, 60 mg every 6 months, and oral alendronate, 70 mg once weekly. METHODS: Postmenopausal women at 25 centers in the USA and Canada with bone mineral density T-scores -4.0 to -2.0 and no prior bisphosphonate use received alendronate then denosumab, or denosumab then alendronate, over successive 12-month periods. Adherence required both compliance (denosumab injections 6 months apart or ≥ 80% of alendronate tablets) and persistence (both denosumab injections or ≥ 2 alendronate doses in the last month and completion of the treatment period). RESULTS: Of the 250 women enrolled (124 alendronate, 126 denosumab), 221 entered the second year (106 denosumab, 115 alendronate). Denosumab was associated with less non-adherence than alendronate (first year, 11.9% vs 23.4%; second year, 7.5% vs 36.5%). Risk ratios for non-adherence, non-compliance, and non-persistence favored denosumab in both years (p < 0.05). Of 198 subjects expressing treatment preference, 183 (92.4%) preferred the injections over the oral therapy. BMD improved further when subjects received denosumab after alendronate and remained stable when they received alendronate after denosumab. CONCLUSION: Based on the final results of this crossover study after women had received each treatment for up to 1 year, postmenopausal women with osteoporosis were more adherent, compliant, and persistent with subcutaneous denosumab injections every 6 months than with once-weekly alendronate tablets and reported increased treatment preference and satisfaction with injectable denosumab over oral alendronate.

Adherence, preference, and satisfaction of postmenopausal women taking denosumab or alendronate.

UNLABELLED: In this study, 250 women with osteoporosis were randomized to 12 months with subcutaneous denosumab 60 mg every 6 months or oral alendronate 70 mg once weekly, then crossed over to the other treatment. The primary endpoint, treatment adherence at 12 months, was 76.6% for alendronate and 87.3% for denosumab. INTRODUCTION: The purpose of this study is to evaluate treatment adherence with subcutaneous denosumab 60 mg every 6 months or oral alendronate 70 mg once weekly. METHODS: In this multicenter, randomized, open-label, 2-year, crossover study, 250 postmenopausal women with low bone mineral density received denosumab or alendronate for 12 months, then the other treatment for 12 months. The alendronate bottle had a medication event monitoring system cap to monitor administration dates. Definitions were as follows: compliance, receiving both denosumab doses 6 (± 1) months apart or 80-100% of alendronate doses; persistence, receiving both denosumab doses and completing the month 12 visit within the visit window or ≥ 2 alendronate doses in the final month; adherence, achieving both compliance and persistence. This report includes data from the first 12 months. RESULTS: The primary study endpoint, adherence in the first 12 months, was 76.6% (95/124) for alendronate and 87.3% (110/126) for denosumab. Risk ratios for denosumab compared with alendronate at 12 months were 0.58 (p = 0.043) for non-adherence, 0.48 (p = 0.014) for non-compliance, and 0.54 (p = 0.049) for non-persistence. Subject ratings for treatment necessity, preference, and satisfaction were significantly greater for denosumab and ratings for treatment bother were significantly greater for alendronate. Adverse events were reported by 64.1% of alendronate-treated subjects and 72.0% of denosumab-treated subjects (p = 0.403). The most common adverse events were arthralgia, back pain, pain in extremity, cough, and headache (each in <10% of subjects in each group). CONCLUSIONS: Significantly greater treatment adherence was observed for subcutaneous administration of denosumab every 6 months than for oral alendronate once weekly.

Preference and satisfaction with a 6-month subcutaneous injection versus a weekly tablet for treatment of low bone mass.

UNLABELLED: The Preference and Satisfaction Questionnaire (PSQ) compares patient preference and satisfaction between a 6-month subcutaneous injection and weekly oral tablet for treatment of bone loss. Patients preferred and were more satisfied with a treatment that was administered less frequently, suggesting the acceptability of the 6-month injection for treatment of bone loss. INTRODUCTION: The PSQ compares patient preference and satisfaction between a 6-month subcutaneous injection and a weekly oral tablet for treatment of bone loss. METHODS: Postmenopausal women with low bone mass who enrolled in two separate randomized phase 3 double-blind, double-dummy studies received a 6-month subcutaneous denosumab injection (60 mg) plus a weekly oral placebo or a weekly alendronate tablet (70 mg) plus a 6-month subcutaneous placebo injection. After 12 months, patients completed the PSQ to rate their preference, satisfaction, and degree of bother with each regimen. RESULTS: Most enrolled patients (1,583 out of 1,693; 93.5%) answered >or=1 item of the PSQ. Significantly more patients preferred and were more satisfied with the 6-month injection versus the weekly tablet (P < 0.001). More patients reported no bother with the 6-month injection (90%) than the weekly tablet (62%). CONCLUSION: Patients preferred, were more satisfied, and less bothered with a 6-month injection regimen for osteoporosis.

Differential regulation of receptor-stimulated cyclic adenosine monophosphate production by polyvalent cations in MC3T3-E1 osteoblasts.

Extracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3-E1 osteoblasts if polyvalent cations differentially modulate the agonist-stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1 (PGE)-stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)-stimulated cAMP production. In contrast, these cations had no effect on forskolin- or cholera toxin-induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12-myristate 13-acetated (PMA) mimicked the effects of cations on PGE1- and PTH-stimulated cAMP accumulation in MC3T3-E1 cells, respectively, diminishing and augmenting the responses. Moreover, down-regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1- and enhancing PTH-stimulated cAMP production, indicating involvement of PKC-dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd(3+)-mediated suppression of PGE1-stimulated cAMP, suggesting actions independent of Gm. Thus, polyvalent cations may either stimulate or inhibit hormone-mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in vivo under different hormonal conditions.

A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor.

The presence of a cation-sensing mechanism in osteoblasts is suggested by the ability of specific cations to stimulate osteoblastic proliferation in culture and to induce de novo bone formation in some experimental models. Our study examines whether extracellular cations stimulate osteoblasts through the recently identified G protein-coupled calcium receptor (CaR). We found that CaR agonists, calcium (Ca2+), gadolinium (Gd3+), aluminum (Al3+), and neomycin, stimulated DNA synthesis in murine-derived MC3T3-E1 preosteoblasts, whereas magnesium (Mg2+), nickel (Ni2+), cadmium (Cd2+), and zinc (Zn2+) had no effect. With the exception of Mg2+, the cation specificities and apparent affinities were similar to that reported for CaR. CaR agonists also stimulated DNA synthesis in C3HT10(1/2) fibroblasts, but not in mesangial PVG, CHO, hepatic HTC, COS-7 cells, or malignant transformed ROS17/2.8 and UMR-106 osteoblasts. In addition, similar to other growth factors, CaR agonists activated transcription of a serum response element luciferase reporter construct (SRE-Luc) stably transfected into MC3T3-E1 osteoblasts, but had no effect on SRE-Luc transfected into CHO and COS-7 cells. We were unable to detect CaR expression by Northern analysis using a mouse CaR-specific probe or to amplify CaR mRNA by reverse transcribed polymerase chain reaction in MC3T3-E1 osteoblasts. These findings suggest that an extra-cellular cation-sensing mechanism is present in murine-derived osteoblasts that is functionally similar to but molecularly distinct from CaR.

Forskolin inhibits protein kinase C-induced mitogen activated protein kinase activity in MC3T3-E1 osteoblasts.

We recently demonstrated that stimulation of DNA synthesis in MC3T3-E1 osteoblasts involves cross-talk between protein kinase C (PKC)-dependent pathways and activation of possible nonreceptor tyrosine kinases. In the current investigation we examined whether the Raf-1/MAP kinase kinase (MKK)/mitogen-activated protein kinase (MAPK) cascade integrates cross-talk between G protein-coupled second messengers and protein tyrosine phosphorylation in osteoblasts. We investigated the effects on DNA synthesis, protein tyrosine phosphorylation, and Raf-1, MKK, and MAPK activities of PKC activation by phorbol 12-myristate 13-acetate (PMA) and of cAMP elevation by forskolin (FSK) in MC3T3-E1 osteoblasts. We found that PMA-stimulated DNA synthesis was associated with increments in tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) and activation of Raf-1, MKK, and MAPK in these cells. FSK treatment of osteoblasts, which raised intracellular cAMP levels and inhibited DNA synthesis, blocked PKC-stimulated tyrosine phosphorylation of p44mapk (ERK1) and p42mapk (ERK2) as well as inhibited PKC-stimulated MAPK and Raf-1 activities. Despite this, PMA activated the intermediate MKK step of the Raf-1/MKK/MAPK cascade in the presence of FSK. The differential inhibition of PMA-stimulated Raf-1 and MKK activities by FSK suggests that PKC activates both Raf-1-dependent and -independent pathways in MC3T3-E1 osteoblasts. Moreover, the noncoordinate effects of FSK on PMA-stimulated MKK and MAPK activities indicates the presence of a additional distal cAMP-dependent inhibitory mechanisms.

Okadaic acid-sensitive protein phosphatases dephosphorylate MARCKS, a major protein kinase C substrate.

The myristoylated alanine-rich C kinase substrate (MARCKS) undergoes a rapid and, in certain circumstances, transient increase in phosphorylation in response to stimuli that activate protein kinase C. We have investigated the protein-serine/threonine phosphatase activity responsible for reversing the phosphorylation of MARCKS. In cell-free assays, protein phosphatases 1, 2A and 2C (PP1, PP2A and PP2C) all dephosphorylate recombinant MARCKS or a synthetic peptide based on its phosphorylation site domain. In intact Swiss 3T3 cells, okadaic acid, a specific inhibitor of PP1 and PP2A, had little effect on MARCKS phosphorylation on its own, but largely prevented the dephosphorylation of MARCKS that occurred following activation of protein kinase C by bombesin with subsequent receptor blockade. These results indicate that although the dephosphorylation of MARCKS can be mediated by PP2C in vitro, this protein is dephosphorylated by okadaic acid-sensitive phosphatases in the intact cell.

Differentiation of MC3T3-E1 osteoblasts is associated with temporal changes in the expression of IGF-I and IGFBPs.

We examined the relationship between osteoblast maturation and temporal changes in the secretion of IGF-I and the IGF-binding proteins (IGFBPs) in the MC3T3-E1 model of osteoblast development. IGF-I was present at low levels in conditioned media in proliferating preosteoblasts (3.7 +/- 1.7 ng/micrograms DNA and 3.9 +/- 0.6 at culture (days 3 and 9) and increased progressively in postmitotic differentiating osteoblasts, reaching a maximal concentration of 13.1 +/- 1.5 ng/micrograms DNA by day 25 of culture. We also observed an increase in IGF-I mRNA expression. Using Western ligand blot and immunoblot techniques, we found that IGFBP-2, -4, and -5 also displayed temporal differences in expression during MC3T3-E1 development. We observed a sustained increase in IGFBP-2, -4, and -5 mRNA expression between days 10-14, coincident with the onset of differentiation. IGFBP-2 and IGFBP-4 protein concentrations increased in parallel with IGFBP mRNA expression, but IGFBP-5 levels peaked between days 8-14 of culture, and declined thereafter in spite of persistent IGFBP-5 mRNA levels. These findings suggest complex transcriptional and post-transcriptional regulation of IGFBP metabolism during osteoblast development. Thus, IGF-I and IGFBP production are regulated during osteoblast development. In turn, time-dependent changes in IGF-I and modulation of IGF-I bioavailability by IGFBPs may regulate the osteoblastic developmental sequence.

The effect of selective estrogen receptor modulators on parameters of the hypothalamic-pituitary-gonadal axis.

The SERMs currently in clinical practice or in late stages of clinical development have been studied primarily for their effects on the breast, cardiovascular, bone, and reproductive systems. The effect of SERMs on the hypothalamic-pituitary-gonadal (HPG) axis has not been the primary focus of the studies conducted thus far. However the effect of SERMs on the HPG axis and the associated regulation of endocrine parameters may play an important role in their overall clinical profile. In this review the effects of selected SERMs on the HPG axis in premenopausal women, postmenopausal women, and men are summarized.

Denosumab compared with risedronate in postmenopausal women suboptimally adherent to alendronate therapy: efficacy and safety results from a randomized open-label study.

Denosumab has been shown to reduce new vertebral, nonvertebral, and hip fractures in postmenopausal women with osteoporosis. In subjects who were treatment-naïve or previously treated with alendronate, denosumab was associated with greater gains in bone mineral density (BMD) and decreases in bone turnover markers when compared with alendronate-treated subjects. This trial was designed to compare the efficacy and safety of denosumab with risedronate over 12 months in postmenopausal women who transitioned from daily or weekly alendronate treatment and were considered to be suboptimally adherent to therapy. In this randomized, open-label study, postmenopausal women aged ≥55 years received denosumab 60 mg subcutaneously every 6 months or risedronate 150 mg orally every month for 12 months. Endpoints included percentage change from baseline in total hip BMD (primary endpoint), femoral neck, and lumbar spine BMD at month 12, and percentage change from baseline in sCTX-1 at months 1 and 6. Safety was also assessed. A total of 870 subjects were randomized (435, risedronate; 435, denosumab) who had a mean (SD) age of 67.7 (6.9) years, mean (SD) BMD T-scores of -1.6 (0.9), -1.9 (0.7), and -2.2 (1.2) at the total hip, femoral neck, and lumbar spine, respectively, and median sCTX-1 of 0.3 ng/mL at baseline. At month 12, denosumab significantly increased BMD compared with risedronate at the total hip (2.0% vs 0.5%), femoral neck (1.4% vs 0%), and lumbar spine (3.4% vs 1.1%; p<0.0001 at all sites). Denosumab significantly decreased sCTX-1 compared with risedronate at month 1 (median change from baseline of -78% vs -17%; p<0.0001) and month 6 (-61% vs -23%; p<0.0001). Overall and serious adverse events were similar between groups. In postmenopausal women who were suboptimally adherent to alendronate therapy, transitioning to denosumab was well tolerated and more effective than risedronate in increasing BMD and reducing bone turnover.

Application of QSARs and VFARs to the rapid risk assessment process at US EPA.

With continued development of new chemicals and genetically engineered microbes as potential agents for terrorism and industrial development, there is a great need for the continued development and application of quantitative structure activity relationships (QSARs) and virulence factor activity relationships (VFARs). Development and application of QSARs and VFARs will facilitate efficient and streamlined use of dwindling resources and assessment of risks associated with exposures to chemical and biological agents. To facilitate the continued development of QSARs and VFARs at US Environmental Protection Agency, a two day workshop was organized June 20-21, 2006, in Cincinnati, OH, USA. This article summarizes the workshop report by highlighting the importance of continued QSAR research, the current state of VFAR science, and the guidance provided to the National Homeland Security Research Center and National Risk Management Research Laboratory by an expert panel for the continued use and development of computational approaches.

Molecular to pharmacologic control of osteoblast proliferation and differentiation.

Control of osteoblast growth and development can be characterized from receptor mediated events to nuclear messengers controlling gene transcription. From this analysis it is possible to formulate a model to explain the reciprocal relationship between growth and differentiation as well as differential cytokine modulation of osteoblast function. Central to this model are putative tissue specific transcriptional switches (possibly of the bHLH gene superfamily) that may repress proliferation and permit the regulation of mature osteoblast phenotypic characteristics. This model proposes that in post-mitotic differentiated osteoblasts, tissue specific transcription factors determine the capacity to express osteoblastic characteristic, whereas receptor activated signalling cascades, namely, cAMP/protein kinase A, receptor serine/threonine kinase, and vitamin D receptor-dependent pathways, regulate mature osteoblast-specific gene expression. Activated differentiation switches also may feedback to transcriptionally repress proliferation. Conversely, in preosteoblasts, in which differentiation switches are turned off, distinct signalling cascades involving tyrosine kinases, PKC, and calcium/calmodulin regulate proliferation. Proliferating preosteoblasts also exhibit negative modulation of maturation either through inactivation of putative tissue-specific transcription factors and/or through AP-1 dependent phenotype suppression of genes expressed in mature osteoblast. Thus, the final outcome of transcriptional regulation of osteoblast function results from complex interactions between signalling pathways and permissive differentiating transcription factors. Though many aspects of this model remain speculative and require confirmation, it serves as a useful conceptual framework to further investigate the differential control of osteoblast proliferation and differentiation that may lead to improved pharmacologic ways to manipulate bone formation in vivo.

Influence of dietary fat on factors in serum that regulate thyroid cell metabolism.

The type of dietary fat affected the production of cAMP by cultured thyroid cells incubated with mouse and rat sera. Greater amounts of cAMP were produced with serum from mice fed 30% rac-1(3)-palmitoyl glycerol and 4% safflower oil (PG + SO) than with serum from mice fed 30% rac-1(3)-palmitoyl glycerol (PG). The serum from mice fed PG + SO gave a response similar to that with calf serum. Sera were separated into lipoprotein and aqueous fractions by centrifugation. A combination of both the lipoprotein and aqueous fractions of serum from mice fed PG + SO was required for the increased response. Proteolysis of the aqueous fraction of the serum from either mice fed PG or rats fed 30% hydrogenated coconut oil (HCO) reduced the amount of cAMP produced by the thyroid cells. However, the same effect was not seen with aqueous fractions of calf serum or serum from mice fed PG + SO or rats fed HCO plus 5% corn oil. These findings suggest that there are at least three factors in serum capable of regulating thyroid cell metabolism that are controlled by the type of fat fed the animal.

Developmental regulation of osteocalcin expression in MC3T3-E1 osteoblasts: minimal role of the proximal E-box cis-acting promoter elements.

Osteoblasts undergo a temporal sequence of development characterized by transcriptional upregulation of osteoblast-specific genes. Basic helix-loop-helix (bHLH) transcription factors may control this developmental process through binding to E-box cis-acting elements in developmentally regulated genes. To investigate the role of bHLH proteins in MC3T3-E1 osteoblasts, which undergo a developmental sequence in vitro, we analyzed the transcriptional control of osteocalcin gene expression by stable transfection of an osteocalcin promoter-luciferase chimeric gene (p637OC-luc) and assessed the role of E-box cis-acting elements in osteocalcin promoter by DNA binding assays. We compared our findings in MC3T3-E1 osteoblasts with transient DNA transfections and DNA binding experiments in Ros 17/2.8 osteoblasts. We found that the activity of 637-OC luciferase promoter was low in undifferentiated 5-day-old cultures but increased in parallel with endogenous osteocalcin message expression in mature MC3T3-E1 osteoblasts, consistent with developmental stage-specific transcriptional upregulation of the osteocalcin gene. We identified two putative E-box elements in the proximal osteocalcin promoter, E-box 1 (CACATG) at -102 and E-box 2 (CAGCTG) at position -149. In gel mobility shift assays, factors present in nuclear extracts derived from differentiated osteoblast bound to oligonucleotide probes containing the E-box 1 and E-box 2 elements. Binding to the E-box 2 probe was not specific for the core CAGCTG element, whereas the CACATG site in E-box 1 oligonucleotide was required for specific binding of these nuclear factors. Stable transfection of p637OC-luc containing a mutant E1 site (p637OC-luc E1m), however, did not alter the developmental upregulation of osteocalcin promoter activity in MC3T3-E1 osteoblasts. Moreover, the E-box 1 mutation had no effect on either basal or vitamin D-stimulated activity of the osteocalcin promoter in Ros 17/2.8 osteoblasts in transient transfection experiments. These data suggest that osteoblasts contain underfined factors that bind to the E-box 1 CACATG site in the proximal osteocalcin promoter; however, this E-box element does not play a significant role in the developmental stage-specific regulation of the osteocalcin gene in MC3T3-E1 osteoblasts.

Utilization of quantitative structure-activity relationships (QSARs) in risk assessment: alkylphenols.

Alkylphenols are a class of environmentally pervasive compounds, found both in natural (e.g., crude oils) and in anthropogenic (e.g., wood tar, coal gasification waste) materials. Despite the frequent environmental occurrence of these chemicals, there is a limited toxicity database on alkylphenols. We have therefore developed a "toxicity equivalence approach" for alkylphenols which is based on their ability to inhibit, in a specific manner, the enzyme cyclooxygenase. Enzyme-inhibiting ability for individual alkylphenols can be estimated based on the quantitative structure-activity relationship developed by Dewhirst (1980) and is a function of the free hydroxyl group, electron-donating ring substituents, and hydrophobic aromatic ring substituents. We evaluated the toxicological significance of cyclooxygenase inhibition by comparison of the inhibitory capacity of alkylphenols with the inhibitory capacity of acetylsalicylic acid, or aspirin, a compound whose low-level effects are due to cyclooxygenase inhibition. Since nearly complete absorption for alkylphenols and aspirin is predicted, based on estimates of hydrophobicity and fraction of charged molecules at gastrointestinal pHs, risks from alkylphenols can be expressed directly in terms of "milligram aspirin equivalence," without correction for absorption differences. We recommend this method for assessing risks of mixtures of alkylphenols, especially for those compounds with no chronic toxicity data.

Effect of raloxifene on sexual function in postmenopausal women.

OBJECTIVE: To determine the effects of raloxifene on sexual function in postmenopausal women with pre-existing vaginal atrophy treated with vaginal estrogen cream. METHODS: A total of 187 naturally postmenopausal women, 42-80 years of age, with signs of genitourinary atrophy were enrolled in this 6-month, multicenter, parallel-group study. Subjects were randomized to oral raloxifene HCl 60 mg daily or matching placebo; the same subjects were also randomized to receive one application of either vaginal conjugated estrogen cream 0.5 g twice weekly for 6 months or non-hormonal vaginal moisturizer twice weekly for 3 months, followed by conjugated estrogen cream for 3 months. Both investigators and subjects were masked to the identity of the oral medication. The vaginal preparations were administered in an open-label fashion. The Sexual Activity Questionnaire (SAQ) was administered at baseline and at 3 and 6 months. Safety was assessed throughout the study. RESULTS: A total of 102 women were sexually active at baseline and, of these, 82 were also sexually active at the 6-month end-point. At 6 months, raloxifene and placebo, in the presence of vaginal conjugated estrogen cream, were both associated with improvement from baseline in vaginal dryness and reduced discomfort during sexual activity. There were no significant differences between raloxifene and placebo groups in any SAQ item. Enjoyment of sexual activity significantly increased from baseline with raloxifene but not with placebo. No difference in adverse events was observed between groups. CONCLUSION: Raloxifene had no negative effects on sexual function in postmenopausal women with vaginal atrophy who were treated concomitantly with vaginal estrogen cream.

Nonmyristoylated MARCKS complements some but not all of the developmental defects associated with MARCKS deficiency in mice.

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, is a widely expressed, prominent substrate for protein kinase C. Although the exact function of MARCKS has not been elucidated, targeted disruption of the MARCKS gene (Macs) in mice has shown that MARCKS plays a crucial role in the development of the central nervous system. Mice deficient in MARCKS exhibited universal perinatal death with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (Stumpo et al., Proc. Natl. Acad. Sci. USA 92, 944-948, 1995). In the present studies, a transgene consisting of approximately 3.4 kb of promoter from the human MARCKS gene (MACS), with an epitope tag sequence inserted at the carboxyl terminus of the MARCKS coding region, was able to complement completely MARCKS deficiency in mice. Thus, the human transgene contained all of the elements necessary for normal developmental expression of MARCKS. To test the importance of MARCKS myristoylation to its developmental role, an otherwise identical transgene was constructed in which the glycine at the amino terminus of MARCKS was mutated to an alanine. This mutation, which resulted in the expression of nonmyristoylated MARCKS, was successful in partially rescuing the Macs null phenotype. Specifically, about 25% of these mice survived the perinatal period; these survivors appeared to develop normally except for slightly decreased body size. In both the survivors and the nonsurvivors, all of the known anatomical defects associated with MARCKS deficiency were corrected by expression of the nonmyristoylated human protein. These results indicate that myristoylation of MARCKS is not required for the protein to correct many of the developmental abnormalities characteristic of its deficiency.

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).