Economic Evaluation of Senshio® (Ospemifene) for the Treatment of Vulvovaginal Atrophy in Scotland.

BACKGROUND: Local oestrogens, the current treatment for vulvar and vaginal atrophy (VVA), are not suitable for all women. Standard of care (SoC) consists of over-the-counter lubricants and moisturisers. Senshio® (ospemifene) provides a treatment option for postmenopausal women who are not candidates for local vaginal oestrogen therapy who would otherwise have an unmet clinical need. OBJECTIVES: The aim of this study was to estimate the cost-effectiveness of ospemifene, a selective oestrogen receptor modulator, for the treatment of moderate to severe symptomatic VVA in postmenopausal women who are not candidates for local vaginal oestrogen therapy. METHODS: The Scottish Medicines Consortium (SMC) recently evaluated the clinical and cost-effectiveness evidence of ospemifene plus SoC compared with SoC alone. A cost-effectiveness study, from a National Health Service (NHS) Scotland perspective over a lifetime time horizon, was submitted to the SMC. The cohort-based Markov model used robust clinical evidence from two large pivotal phase III randomised controlled studies and included four health states classified by dyspareunia symptom severity: none, mild, moderate and severe. The movement of women between health states was dependent on the effectiveness of treatment in reducing dyspareunia. Extensive sensitivity analyses were undertaken to assess the level of confidence associated with the base-case results. RESULTS: Treatment with ospemifene was associated with an additional cost of £847 per patient and an increase in quality-adjusted life-years (QALY) of 0.06 per patient. Ospemifene had an incremental cost-effectiveness ratio of £14,138 per QALY. In the probabilistic sensitivity analysis, there was a probability of 89% that ospemifene was cost-effective at a threshold of £20,000 per QALY gained. Ospemifene remained cost-effective under all scenario analyses. The SMC reviewed the clinical and economic evidence and judged that the evidence demonstrated a robust case to support prescribing ospemifene in NHS Scotland. CONCLUSION: Ospemifene is a cost-effective intervention that has recently been accepted by the SMC for the treatment of postmenopausal women with moderate to severe VVA who are not candidates for local oestrogen.

Antimicrobial, Antitumor and Side Effects Assessment of a Newly Synthesized Tamoxifen Analog.

BACKGROUND: Tamoxifen citrate is a very prevalent drug marketed under several trade names like Apo-Tamox, Nolvadex, Tamec, Tamizam, and Tamoplex. This molecule is approved by the FDA for breast cancer treatment. Some studies have shown that tamoxifen has anti-tuberculosis and antiparasitic activities. Like any drug, tamoxifen possesses side effects, more or less dangerous. AIMS: Basically, this work is a comparative study that aims to: primarily compare the antimicrobial and antitumor activities of tamoxifen and a newly synthesized tamoxifen analog; and to determine the molecule with lesser side effects. METHODS: Three groups of mice were injected with tamoxifen citrate and compound 2(1,1-bis[4-(3- dimethylaminopropoxy)phenyl]-2-phenyl-but-1-ene dihydrochloride) at doses corresponding to C1 (1/10), C2 (1/50), and C3 (1/100) to compound 2 lethal dose (LD50 = 75 mg/kg) administered to adult mice. A group of noninjected mice served as a study control. RESULTS: Experimental results suggest that compound 2 has better antitumor and antimicrobial activity than tamoxifen citrate besides its lower toxicity effects. CONCLUSION: The results obtained from the present study confirmed the antitumor and antimicrobial effect of tamoxifen citrate and its hematological side effects. Compound 2 seems to be more effective than tamoxifen citrate for antitumor and antimicrobial treatment while having less hematological side effects and less disruption of the blood biochemical parameters. These findings encourage us to perform further studies on compound 2 and test it for other therapeutic uses for which tamoxifen was found effective.

A cost-effectiveness analysis of vaginal carbon dioxide laser therapy compared with standard medical therapies for genitourinary syndrome of menopause-associated dyspareunia.

BACKGROUND: Topical vaginal estrogen therapy is considered the gold standard treatment for genitourinary syndrome of menopause-associated dyspareunia, but early investigations of energy-based devices show promise for patients with contraindications or those who are refractory to vaginal estrogen cream therapy. Although evaluating safety, efficacy, and long-term outcomes for novel technologies is critically important when new technologies become available to treat unmet healthcare needs, evaluation of the costs of these new technologies compared with existing therapies is also critically important but often understudied. OBJECTIVE: We sought to perform a cost-effectiveness analysis of 3 therapies for genitourinary syndrome of menopause, including vaginal estrogen therapy, oral ospemifene therapy, and vaginal CO2 laser therapy and determine if vaginal laser therapy is a cost-effective treatment strategy for dyspareunia associated with genitourinary syndrome of menopause. STUDY DESIGN: An institutional review board-exempt cost-effectiveness analysis was performed by constructing a decision tree using decision analysis software (TreeAge Pro; TreeAge Software, Inc, Williamstown, MA) using integrated empirical data from the published literature. Tornado plots and 1-way and 2-way sensitivity analyses were performed to assess how changes in the model's input parameters altered the overall outcome of the cost-effectiveness analysis model. RESULTS: All 3 treatment methods were found to be cost-effective below the willingness-to-pay threshold of $50,000.00 per quality-adjusted life year for moderate dyspareunia. The incremental cost-effectiveness ratio for vaginal CO2 laser therapy was $16,372.01 and the incremental cost-effectiveness ratio for ospemifene therapy was $5711.14. Although all 3 treatment strategies were on the efficient frontier, vaginal CO2 laser therapy was the optimal treatment strategy with the highest effectiveness. In a 1-way sensitivity analysis of treatment adherence, vaginal CO2 laser therapy was no longer cost-effective when the adherence fell below 38.8%. Vaginal estrogen cream and ospemifene therapies remained cost-effective treatment strategies at all ranges of adherence. When varying the adherence to 100% for all strategies, oral ospemifene therapy was "dominated" by both vaginal CO2 laser therapy and vaginal estrogen cream therapy. In a 2-way sensitivity analysis of vaginal CO2 laser therapy adherence and vaginal CO2 laser therapy cost, vaginal CO2 laser therapy still remained the optimal treatment strategy at 200% of its current cost ($5554.00) when the adherence was >55%. When the cost fell to 20% of its current cost ($555.40), it was the optimal treatment strategy at all adherence values above 29%. CONCLUSION: This study showed that vaginal fractional CO2 laser therapy is a cost-effective treatment strategy for dyspareunia associated with GSM, as are both vaginal estrogen and oral ospemifene therapies. In our model, vaginal CO2 laser therapy is the optimal cost-effective treatment strategy, and insurance coverage should be considered for this treatment option if it is proven to be safe and effective in FDA trials.

Incidence of venous thromboembolism among postmenopausal women prescribed ospemifene, selective estrogen receptor modulators for noncancer indications, or untreated vulvar and vaginal atrophy.

OBJECTIVE: Ospemifene is a nonsteroidal selective estrogen receptor modulator (SERM) for the treatment of moderate symptomatic vulvar and vaginal atrophy (VVA) due to menopause. A postauthorization safety study is currently examining the incidence of venous thromboembolism (VTE) among postmenopausal women receiving ospemifene or other SERM (raloxifene, bazedoxifene, or tamoxifen, for noncancer indications), or with untreated VVA. METHODS: This interim analysis used the US MarketScan Commercial and Medicare Supplemental claims database from 2013 to 2017 to identify incident VTE. The incidence rate and 95% confidence interval of VTE during the first continuous course of treatment (or continuous untreated time for the untreated cohort) were calculated for each cohort overall and by age group, with sensitivity analyses examining incidence in the short term (up to 90 days) and long term (all available follow-up, regardless of treatment changes). RESULTS: Analyses included 8,188 ospemifene users, 11,777 other SERM users, and 220,242 women with untreated VVA. The incidence per 1,000 person-years and 95% confidence interval of VTE were 3.7 (1.7-7.1) for ospemifene, 11.5 (8.9-14.6) for other SERM, and 11.3 (10.8-11.7) for untreated VVA. Stratification by age and altering the time frame for analysis produced results with similar patterns to the primary analysis. CONCLUSIONS: This interim analysis of an ongoing study suggests a favorable safety profile for ospemifene with respect to VTE. Comparative analyses with covariate adjustment will be performed when data accrual is complete.

Photodegradation of (E)- and (Z)-Endoxifen in water by ultraviolet light: Efficiency, kinetics, by-products, and toxicity assessment.

Endoxifen is an effective metabolite of a common chemotherapy agent, tamoxifen. Endoxifen, which is toxic to aquatic animals, has been detected in wastewater treatment plant (WWTP) effluent. This research investigates ultraviolet (UV) radiation (253.7 nm) application to degrade (E)- and (Z)-endoxifen in water and wastewater and phototransformation by-products (PBPs) and their toxicity. The effects of light intensity, pH and initial concentrations of (E)- and (Z)-endoxifen on the photodegradation rate were examined. Endoxifen in water was eliminated ≥99.1% after 35 s of irradiation (light dose of 598.5 mJ cm-2). Light intensity and initial concentrations of (E)- and (Z)-endoxifen exhibited positive trends with the photodegradation rates while pH had no effect. Photodegradation of (E)- and (Z)-endoxifen in water resulted in three PBPs. Toxicity assessments through modeling of the identified PBPs suggest higher toxicity than the parent compounds. Photodegradation of (E)- and (Z)-endoxifen in wastewater at light doses used for disinfection in WWTPs (16, 30 and 97 mJ cm-2) resulted in reductions of (E)- and (Z)-endoxifen from 30 to 71%. Two of the three PBPs observed in the experiments with water were detected in the wastewater experiments. Therefore, toxic compounds are potentially generated at WWTPs by UV disinfection if (E)- and (Z)-endoxifen are present in treated wastewater.

Post-marketing assessment of generic tamoxifen in Brazilian breast cancer patients.

Generic formulations of tamoxifen are commonly prescribed to oestrogen receptor-positive breast cancer patients at the Brazilian National Cancer Institute (INCA). We carried out a post-marketing surveillance of the generic tamoxifen formulation in current use at INCA, by comparing plasma concentrations of the parent drug and metabolites obtained with the generic vs the reference formulation. Thirty patients participated in an open-label, bracketed protocol, comprising 3 successive phases of 30-32 days each: the generic formulation was used in phases 1 and 3 and the reference formulation in phase 2. Two blood samples were collected in the last 4 days of each phase, for LC-MS/MS quantification of tamoxifen and metabolites in plasma. The median plasma concentrations (ng/mL) for the reference formulation were as follows: tamoxifen, 135.0 (CI 95% 114.2-155.8); endoxifen, 35.3 (30.0-40.8); and 4-hydroxytamoxifen, 4.8 (4.2-5.4). The endoxifen/tamoxifen plasma concentration ratio was 0.27 (0.21-0.25). ANOVA detected no statistically significant difference in plasma concentrations of tamoxifen, metabolites or the endoxifen/tamoxifen ratio among the three phases. The genetic component (rGC) of the CYP2D6-mediated conversion of tamoxifen into endoxifen, estimated using the repeated drug administration procedure across the three phases, was 0.87, pointing to an important component of genetic variability. In conclusion, this first post-marketing surveillance trial of oncologic generic drugs carried out in Brazilian patients verified the switchability between the reference and the generic tamoxifen formulation currently used at our institution. The adopted bracketed protocol adds confidence to this conclusion and may serve as a frame for future trials of post-marketing assessment of other generic drug products.

Ospemifene versus local estrogen: adherence and costs in postmenopausal dyspareunia.

Aim: Objective was to compare adherence and persistence, as well as direct healthcare costs and utilization, of ospemifene to available local estrogen therapies (LETs). Patients & methods: This retrospective database study used integrated medical and pharmacy claims data from the IQVIA Real-World Data Adjudicated Claims - US Database. Results: Ospemifene patients had significantly greater adherence and persistence compared with the other nonring LETs. Ospemifene had the lowest mean outpatient costs of any of the LET cohorts, including the estradiol vaginal ring. Total all-cause healthcare costs were also significantly less for ospemifene patients compared with all other LETs.

Transformation of tamoxifen and its major metabolites during water chlorination: Identification and in silico toxicity assessment of their disinfection byproducts.

The selective estrogen receptor modulator tamoxifen is the most commonly used drug for the treatment and prevention of breast cancer. Tamoxifen is considered as a pro-drug since it is known to exert its pharmacological effect through its major active metabolites, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen, which are mainly excreted in the urine in the days following administration. In the present work, the reactivity of tamoxifen and its major active metabolites in free chlorine-containing water was investigated for the first time. Under the studied chlorination conditions, tamoxifen was fairly stable whereas its metabolites were quickly degraded. A total of thirteen chlorinated byproducts were tentatively identified by ultra-high performance liquid chromatography coupled to high-resolution hybrid quadrupole-Orbitrap tandem mass spectrometry. Time-course profiles of the identified byproducts were followed in real wastewater samples under conditions that simulate wastewater disinfection. A preliminary assessment of their acute aquatic toxicity at two trophic levels by means of quantitative structure-activity relationship models showed that the identified byproducts were up to 110-fold more toxic than the parent compounds.

Assessment of ospemifene or lubricants on clinical signs of VVA.

INTRODUCTION: Ospemifene is a new oral estrogen receptor agonist/antagonist with tissue-selective effects approved for the treatment of moderate to severe dyspareunia, a symptom of postmenopausal vulvar and vaginal atrophy (VVA). AIM: The aim of the study is to assess ospemifene or lubricant use on the clinical signs of VVA. METHODS: Subjects in three double-blind, placebo-controlled clinical trials were randomized to ospemifene or placebo. In two of the trials, women were provided nonhormonal lubricants for use as needed, and a preplanned evaluation of the frequency of lubricant use was performed. Additionally, a post hoc placebo group analysis for impact of lubricant use or nonuse on physiologic effects of the percentage of superficial and parabasal cells (maturation index) and vaginal pH was conducted. A secondary preplanned end point included visual examination of the vagina (clinical signs of vaginal dryness, petechiae, pallor, friability, and redness of the mucosa) comparing change from baseline to end of treatment for the ospemifene 60-mg/day group and vs. placebo. MAIN OUTCOME MEASURES: The primary end points in the phase 3 clinical trials included the percentage of superficial cells, parabasal cells, vaginal pH, and most bothersome symptoms compared with placebo. RESULTS: There was no significant difference in physiologic effects between placebo lubricant users vs. nonusers in either 12-week study. Compared with baseline, substantially more subjects receiving ospemifene 60 mg/day than placebo showed complete resolution of clinical signs of VVA after 12 and 52 weeks of treatment. CONCLUSIONS: Ospemifene substantially improved clinical signs of VVA. Within the placebo group, there was no difference in physiologic effects in lubricant users vs. nonusers. Based on gynecologic evaluation of the vagina, benefits were apparent at 12 weeks and sustained for 52 weeks in the ospemifene-treated subjects with significant improvement over placebo. In these three clinical trials, in contrast to ospemifene-treated women, placebo subjects who utilized lubricants had no improvement in their underlying vaginal physiology.

Hands-off linear interaction energy approach to binding mode and affinity estimation of estrogens.

With this work we target the development of a predictictive model for the identification of small molecules which bind to the estrogen receptor alpha and, thus, may act as endocrine disruptors. We propose a combined thermodynamic approach for the estimation of preferential binding modes along with corresponding free energy differences using a linear interaction energy (LIE) ansatz. The LIE model is extended by a Monte Carlo approach for the computation of conformational entropies as recently developed by our group. Incorporating the entropy contribution substantially increased the correlation with experimental affinity values. Both squared coefficients for the fitted data as well as the more meaningful leave-one-out cross-validation of predicted energies were elevated up to r(Fit)² = 0.87 and q(LOO)² = 0.82, respectively. All calculations have been performed on a set of 31 highly diverse ligands regarding their structural properties and affinities to the estrogen receptor alpha. Comparison of predicted ligand orientations with crystallographic data retrieved from the Protein database pdb.org revealed remarkable binding mode predictions.

Ospemifene: first global approval.

Ospemifene (Osphena™) is an oral selective estrogen receptor modulator (SERM), with tissue-specific estrogenic agonist/antagonist effects. QuatRx Pharmaceuticals conducted the global development of the agent before licensing it to Shionogi for regulatory filing and commercialization worldwide. Ospemifene is the first non-estrogen treatment approved for moderate to severe dyspareunia in women with menopause-related vulvar and vaginal atrophy. The drug is approved in the USA, and application for EU regulatory approval is underway. This article summarizes the milestones in the development of ospemifene leading to this first approval for moderate to severe dyspareunia, a symptom of postmenopausal vulvar and vaginal atrophy.

Assessment of the impact of CYP3A polymorphisms on the formation of α-hydroxytamoxifen and N-desmethyltamoxifen in human liver microsomes.

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CL(int))] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min⁻¹ · pmol cytochrome P450⁻¹). There was no significant difference in CL(int) values between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CL(int) with recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CL(int) for N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6ß-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.

Assessment of vascular effects of tamoxifen and its metabolites on the rat perfused hindquarter vascular bed.

Tamoxifen has been suggested to produce beneficial cardiovascular effects, although the mechanisms for these effects are not fully known. Moreover, although tamoxifen metabolites may exhibit 30-100 times higher potency than the parent drug, no previous study has compared the effects produced by tamoxifen and its metabolites on vascular function. Here, we assessed the vascular responses to acetylcholine and sodium nitroprusside on perfused hindquarter vascular bed of rats treated with tamoxifen or its main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen, and endoxifen) for 2 weeks. Plasma and whole-blood thiobarbituric acid reactive substances (TBARS) concentrations were determined using a fluorometric method. Plasma nitrite and NOx (nitrite + nitrate) concentrations were determined using an ozone-based chemiluminescence assay and Griess reaction, respectively. Treatment with tamoxifen reduced the responses to acetylcholine (pD(2) = 2.2 +/- 0.06 and 1.9 +/- 0.05 after vehicle and tamoxifen, respectively; P < 0.05), while its metabolites improved these responses (pD(2) = 2.5 +/- 0.04 after N-desmethyl-tamoxifen, 2.5 +/- 0.03 after 4-hydroxy-tamoxifen, and 2.6 +/- 0.08 after endoxifen; P < 0.01). Tamoxifen and its metabolites showed no effect on endothelial-independent responses to sodium nitroprusside (P > 0.05). While tamoxifen treatment resulted in significantly higher plasma and whole blood lipid peroxide levels (37% and 62%, respectively; both P < 0.05), its metabolites significantly decreased lipid peroxide levels (by approximately 50%; P < 0.05). While treatment with tamoxifen decreased the concentrations of markers of nitric oxide formation by approximately 50% (P < 0.05), tamoxifen metabolites had no effect on these parameters (P > 0.05). These results suggest that while tamoxifen produces detrimental effects, its metabolites produce counteracting beneficial effects on the vascular system and on nitric oxide/reactive oxygen species formation.

Organ specificity of DNA adduct formation by tamoxifen and alpha-hydroxytamoxifen in the rat: implications for understanding the mechanism(s) of tamoxifen carcinogenicity and for human risk assessment.

Tamoxifen is an anti-oestrogen widely used in the adjuvant therapy of breast cancer and is also used as a prophylactic to prevent the disease in high-risk women. An increased risk of endometrial cancer has been observed in both settings. In rats, tamoxifen potently induces liver carcinomas and also induces uterine tumours when given neonatally. It forms DNA adducts in rat liver via the formation of alpha-hydroxytamoxifen, the ultimately reactive form being generated by sulfotransferase. In order to investigate the formation of tamoxifen-derived DNA adducts in other rat tissues, female Fischer F344 or Sprague-Dawley rats were treated with tamoxifen or alpha-hydroxytamoxifen by gavage or by intraperitoneal injection, daily for 1, 4 or 7 days, and DNA adducts were detected by (32)P-postlabelling analysis. Tamoxifen formed DNA adducts in the liver but not in other tissues (uterus, stomach, kidney, spleen and colon). alpha-Hydroxytamoxifen also formed adducts at high levels in liver, but with the exception of single animals (1/8) in which a low level of adducts was detected in the stomach in one case, and in the kidney in the other; it also did not give rise to adducts in other tissues. The results suggest that tamoxifen is a genotoxic carcinogen in rat liver, but a non-genotoxic carcinogen in rat uterus, making it, uniquely, a carcinogen with more than one mechanism of action. Mutagenicity experiments conducted in Salmonella typhimurium strains expressing bacterial or human N,O-acetyltransferase did not provide evidence that either alpha-hydroxytamoxifen or alpha-hydroxy-N-desmethyltamoxifen undergoes metabolic activation by acetylation. The confinement of ST2A2, the isozyme of hydroxysteroid sulfotransferase that can activate the compounds, mainly to rat liver is the possible reason for the formation of ducts in the liver but not in other organs of the rat.

An assessment of the value of ultrasonographic screening for endometrial disease in postmenopausal women without symptoms.

OBJECTIVE: Our purpose was to evaluate the use of transvaginal ultrasonography for the detection of endometrial disease in a population of postmenopausal women who were without symptoms. STUDY DESIGN: Postmenopausal women were screened for potential inclusion in 2 multicenter, double-blind, placebo-controlled studies of 2 years' duration to evaluate the safety and efficacy of idoxifene in the prevention of osteoporosis. Baseline endometrial evaluation was performed by transvaginal ultrasonography and aspiration biopsy of the endometrium. RESULTS: A total of 1926 women were screened by transvaginal ultrasonography, and 1833 of them had endometrial thickness < or =6 mm. Five cases of endometrial abnormality (adenocarcinoma [n = 1] and atypical hyperplasia [n = 4]) were detected in the 1750 women from this cohort who underwent biopsy. The negative predictive value was >99%. One case of adenocarcinoma was detected in the 42 women who had endometrial thickness >6 mm and underwent biopsy. However, the sampling rate (45%) of women with endometrial thickness >6 mm was too low for confidence in the positive predictive value of 2%. CONCLUSIONS: Despite a high negative predictive value, transvaginal ultrasonography may not be an effective screening procedure for detection of endometrial abnormality in untreated postmenopausal women who are without symptoms.

Sonographic assessment of the endometrium in osteopenic postmenopausal women treated with idoxifene.

Idoxifene is a novel selective estrogen receptor modulator that has shown beneficial effects on bone turnover and lipid metabolism in clinical studies. Preclinical studies have demonstrated that idoxifene has estrogen antagonist activities on the endometrium. This paper describes the results of a double-blind, placebo-controlled, and dose ranging study involving 331 osteopenic postmenopausal women who were treated with either placebo or idoxifene (2.5, 5, or 10 mg/day) for 12 weeks. In these women, endometrial assessment was carried out by transvaginal sonography and endometrial biopsy on selected patients at baseline and on all women at the end of treatment. Women with an endometrial thickness greater than 10 mm were excluded from the study. Aspiration endometrial biopsy was performed on women with an endometrial thickness between 6 and 10 mm at baseline and on all women after treatment. Of the 298 biopsies performed in the subjects at the end of treatment, 99% of the women were reported to have either a benign or atrophic endometrium (85%) or insufficient tissue for diagnosis (14%). Proliferative histologic features were reported in two cases (1%) (2.5 mg idoxifene) and atypical hyperplasia in one placebo patient. Even though idoxifene use was associated with a dose related increase in endometrial thickness as evaluated by transvaginal sonography, no relationship was established between endometrial histologic features and change in endometrial thickness. On histologic analysis, the increase in endometrial thickness seen on transvaginal sonography was not associated with proliferative or hyperplastic change in the epithelial (glandular) endometrial tissue. In 48 patients (16% of total) transvaginal sonography showed endometrial thickening of 5 mm or more over the study period. The endometrial histologic features were benign in all these patients. Nineteen percent of women developed intraluminal fluid, even though endometrial thickness was normal and unchanged and histologic features were normal. Our data show that after 3 months of treatment, no significant pathologic changes of the endometrium were observed. Our data indicate that measurements of endometrial thickness by transvaginal sonography may falsely suggest the presence of endometrial pathologic changes in some postmenopausal women treated with idoxifene. Additional testing using saline infusion sonohysterography is an important part of the transvaginal sonography protocol in equivocal or abnormal cases to exclude focal lesions such as polyps. In addition, our data indicate that pathologic changes of the endometrium are extremely rare in the treated group, indicative of its short term safety. Continued investigation such as this will be needed to establish long term safety.

Assessment of cell proliferation by AgNOR scores and Ki-67 labeling indices and a comparison with potential doubling times.

The potential doubling time (T[pot]) has been proposed as a pretreatment estimator of intratreatment tumor proliferative capacity. The assay has several limitations, however, including the inability to discriminate between host and tumor cells in diploid tumors and the need for prospective labeling of patients with a halogenated pyrimidine. Often a separate biopsy is needed as well. In this study, Ki-67 immunostaining and AgNOR silver staining were examined as possible alternative methods by comparing them with the T[pot] in an in vitro system using two different cell lines under varying growth conditions. Using CaSki cells, nutritional status was varied to alter proliferation rates, and a strong correlation was found between Ki-67 labeling index and AgNOR scores (r = 0.927, P = 0.0003). Both Ki-67 labeling index and AgNOR scores had statistically significant inverse correlations with cell doubling time (T[d]), length of S phase (T[s]), and T(pot), as well as a positive correlation with iododeoxyuridine (IdUrd) labeling index. As an alternative method of inhibiting proliferation, 4-hydroxytamoxifen (4OH-TAM), the potent anti-estrogenic metabolite of tamoxifen, was used to inhibit the proliferation of estrogen-dependent MCF-7 cells. Treatment with the anti-estrogen resulted in decreased Ki-67 labeling index and AgNOR scores of MCF-7 cells, correlating with a decrease in IdUrd labeling index and inversely correlating with T(pot). The significant correlations among Ki-67 labeling index, AgNOR scores, and these other measures of proliferation suggest their potential value as indicators of proliferative activity. Retrospective markers, such as Ki-67 labeling index or AgNOR score, may be particularly attractive clinically in that prospective labeling of patients with halogenated pyrimidines could be avoided; however, extensive clinical testing will be required before the clinical usefulness of these markers can be established.

An assessment of the role of domain F and PEST sequences in estrogen receptor half-life and bioactivity.

The estrogen receptor (ER) is a rapidly turning over protein, with a half-life of ca. 3-4 h in estrogen target cells. Sequence analysis of the human ER reveals a putative PEST sequence, sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T), in the carboxy-terminal F domain of the protein. Since PEST sequences have been implicated in the rapid turnover of some proteins, we have used site-directed mutagenesis to investigate the role of the F region containing PEST residues in the stability and bioactivity of the receptor. A truncated form of ER lacking the last 41 amino acids of the protein and encompassing the PEST sequences (amino acids 555 to 567) was made by mutagenesis of the ER cDNA. Pulse-chase experiments, involving immunoprecipitation of [35S]methionine/[35S]cysteine labeled receptors or of receptors covalently labeled with tamoxifen aziridine followed by gel electrophoresis, were used to determine the half-life of the wild-type and truncated ERs. These experiments showed that the turnover rate of the receptors expressed in Chinese hamster ovary and monkey kidney (COS-1) cells was 3 to 5 h and that elimination of the PEST residues did not have a significant effect on the degradation rate of the protein. Moreover, deletion of the last 41 amino acids (F domain) of the ER did not affect transactivation ability, ligand binding affinity, or the phosphorylation pattern of the receptor. Therefore, the role of domain F in ER function remains unclear, but it is not a determinant of the relatively rapid rate of ER turnover in cells.