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Five studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The general hypothesis was that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function for the purposes of fixed-time artificial insemination and embryo production. The specific objectives were to determine the effect of route and vehicle, type of aromatase inhibitor, and duration of aromatase inhibitor treatment (short vs prolonged) on ovarian follicles in cattle, and to test the efficacy of an aromatase inhibitor-based protocol to synchronize ovulation in cattle. In the first experiment, heifers were treated with letrozole intravenously (n=10) or intramuscularly (n=10) or allocated in iv and im control groups (n=5/group). During the second experiment, heifers were divided randomly into two groups (n=15/group) and an intravaginal device containing 1 g of letrozole or a blank device (control) was inserted. The third experiment was designed with the goal of formulating and testing an intravaginal device that provides biologically active circulating concentrations of an aromatase inhibitor for a minimum of 4 days. The biological significance of the pharmacokinetic differences between the letrozole intravaginal devices resulting from the third study was evaluated during the fourth study. A final study was designed to determine the effect of stage of the estrous cycle on the proportion of animals that ovulated and the synchrony of ovulation of heifers treated with an aromatase inhibitor-based ovulation-synchronization protocol and to determine subsequent pregnancy outcomes. In all the studies, the effects of aromatase inhibitor on ovarian function were assessed by transrectal ultrasound examination of the ovaries, and blood samples were collected for hormone concentration determination. Results demonstrated that route of administration, or more precisely, the nature of iii the vehicle used for the administration of letrozole (intravenous, intramuscular depot, short release intravaginal or prolonged release intravaginal) has an impact on the effects of letrozole on hormonal profiles and ovarian dynamics. The intramuscular route appeared to provide a prolonged release of letrozole from the injection site which had a marked effect on estradiol production, dominant follicle lifespan, and CL form and function. Letrozole treatment during the ovulatory follicle wave by means of a gel-based intravaginal releasing device during the second study resulted in more rapidly growing dominant follicles and larger ovulatory follicles, delayed ovulation (by 24 h) of a single follicle and formation of a CL that secreted higher levels of progesterone. A wax-based vehicle allowed for a steady and continuous delivery of the active compound over the treatment period. During the third study, the addition of a letrozole-containing gel coating increased the rate of initial absorption and hastened the increase on plasma concentrations of the active ingredient, while the letrozole-containing wax-based vehicle prolonged drug-delivery from the intravaginal device. When tested in vivo during the fourth study, we confirmed that letrozole-impregnated intravaginal devices formulated with a wax base plus a gel coat vehicle was most suitable for the application of a letrozole-based protocol for the synchronization of ovulation in cattle, since it effectively delivered elevated concentrations of letrozole, and reduced estradiol production resulting in increased follicular growth and lifespan, without adversely affecting progesterone production. The application of a letrozole-impregnated intravaginal device for 4 days, combined with PGF treatment at device removal and GnRH 24 h post-device removal increased the percentage of ovulations and synchrony of ovulation in cattle, regardless the stage of the estrous cycle at initiation of treatment. As observed in previous studies, the effects observed could be associated with an increase in circulating LH iv concentrations. However, the effects of treatment on gonadotropin concentrations are inconclusive, possibly due to inadequate sampling frequency. The impact of letrozole treatment of oocyte fertility remains unknown. The results of the five experiments support our general hypothesis that non-steroidal aromatase inhibitors have potential as a steroid-free option for the control of ovarian function in cattle. However, further research is needed in order to elucidate the effects of letrozole treatment during the proestrous on oocyte competence and fertility of the resulting ovulations in cattle.

Pastaraisiais metais vyksta labai daug elgsenos endokrininės kontrolės tyrimų. Mūsų darbe mes tyrėme galimą estrogenų vaidmenį vištų patelių lytinės elgsenos difirenciacijai. Eksperimentiniai gyvūnai 8 embrioninio vystymosi dieną buvo aveikti aromatazės inhibitoriumi, fadrozoliu, tokiu būdu blokuojant estradiolio gamybą. Kontroliniams gyvūnams buvo suleistas fizioliginis tirpalas. Suaugusių vištų elgsena buvo stebima dvejuose skirtinguose lytinės elgsenos testuose: su suaugusiu intaktiniu (nepaveiktu) gaidžiu ir su intaktine (kontroline) višta. Testuojant su gaidžiu fadrozoliu paveiktos vištos rodė mažesnį pasiruošimą kopuliuoti su gaidžiu, turėjo polinkį dominuoti ir vengti gaidį. Testuojant su kontroline višta, eksperimentiniai paukščiai rodė vyrišką elgseną - sparno rėžimą, bandymus kopuliuoti ir giedojimą. Kelios fadrozoliu paveiktos vištos rodė pilną kopuliacinės elgsenos seką, tame tarpe kloakų kontakto judesius. Mūsų tyrimai palaiko hipotezę, kad estrogenai yra pagrindinis faktorius, lemiantis naminių vištų lytinės elgsenos diferenciaciją. / In recent years much scientific reseach is devoted to the endocrine control of behaviour. In our work we studied the possible role of estrogens in differentiation of sexual behaviour in female chickens. Eksperimental animals were treated on day 8 of embrionic development with an aromataze inhibitor, fadrozole, thus blocking the oestradiol production. Control animals received injections of vehicle (saline). In adulthood behaviour of hens was observed in two different sexual behaviour tests: with mature non-treated (intact) cocks and with non-treated (control) hens. When tested with a cock, fadrozole-treated hens showed reduced readiness to copulate with a cock, tended to dominate and to avoid a cock. When tested with a control hen, experimental birds displayed male-type behaviour – waltzing, mount attempts and crowing,. Some of fadrozole-treated hens showed a full sequence of copulatory behaviour, including cloacal contact movement. Our study supports the hypothesis that oestrogens play a major role in differentiation of sexual behaviour in the domestic chickens.

Foram estudadas 20 pacientes na menacme portadoras de leiomioma uterino, sintomáticas, que utilizaram anastrozol na dose 1 mg/dia por três meses consecutivos. Durante o tratamento acompanhou-se o volume do conjunto úteroleiomiomas com ultra-sonografia, no momento inicial e após um mês e três meses do início do uso da medicação. Além disso, foi observada a evolução da sintomatologia relacionada ao leiomioma (hipermenorréia, dismenorréia e metrorragia), presença de sintomas ligados ao hipoestrogenismo e dosagens séricas de FSH, estradiol, colesterol total e frações, triglicérides e glicose. Obtivemos redução média do volume do conjunto útero-leiomiomas de 9,32% até 31%. Houve diminuição significante dos sintomas relacionados ao leiomioma do útero. Não ocorreram sintomas ligados ao hipoestrogenismo. Não se comprovou modificação significante dos demais parâmetros estudados. Concluiu-se que o inibidor da aromatase, o anastrozol, na dosagem utilizada, foi efetivo na redução volumétrica do conjunto útero-leiomiomas, além de proporcionar significativa melhora dos sintomas relacionados a essa doença, sem alterar, no entanto, os valores de FSH, estradiol, colesterol total e frações, triglicérides e glicose. / We studied 20 patients in menacme with leiomyoma in the uterus, referring symptoms, using anastrozol 1 mg per day for three months. During this treatment we monitored the uterus-leiomyoma volume using ultrasound, at the beginning, one month after and three months after the beginning of the use of the medication. We also observed the leiomyoma symptoms evolution, presence of usual symptoms of hipoestrogenism and seric values of FSH, estradiol, cholesterol and its fractions triglycerides and glicemia. We obtained reduction in uterus-leiomyoma volume of 9.32% in average, in maximum 31%. There was significative reduction in symptoms related with leiomyoma. There were not symptoms of hipoestrogenism. We not observed significative modifications in the other studied parameters. This study concluded that the aromatase inhibitor, anastrozol, in this dosage, is effective in uterus-leiomyomas reduction, cause significative reduction of the symptoms related with this illness, without change the seric dosage of FSH, estradiol, total cholesterol and fractions, triglycerides and glicemia.

Foram estudadas 20 pacientes na menacme portadoras de leiomioma uterino, sintomáticas, que utilizaram anastrozol na dose 1 mg/dia por três meses consecutivos. Durante o tratamento acompanhou-se o volume do conjunto úteroleiomiomas com ultra-sonografia, no momento inicial e após um mês e três meses do início do uso da medicação. Além disso, foi observada a evolução da sintomatologia relacionada ao leiomioma (hipermenorréia, dismenorréia e metrorragia), presença de sintomas ligados ao hipoestrogenismo e dosagens séricas de FSH, estradiol, colesterol total e frações, triglicérides e glicose. Obtivemos redução média do volume do conjunto útero-leiomiomas de 9,32% até 31%. Houve diminuição significante dos sintomas relacionados ao leiomioma do útero. Não ocorreram sintomas ligados ao hipoestrogenismo. Não se comprovou modificação significante dos demais parâmetros estudados. Concluiu-se que o inibidor da aromatase, o anastrozol, na dosagem utilizada, foi efetivo na redução volumétrica do conjunto útero-leiomiomas, além de proporcionar significativa melhora dos sintomas relacionados a essa doença, sem alterar, no entanto, os valores de FSH, estradiol, colesterol total e frações, triglicérides e glicose. / We studied 20 patients in menacme with leiomyoma in the uterus, referring symptoms, using anastrozol 1 mg per day for three months. During this treatment we monitored the uterus-leiomyoma volume using ultrasound, at the beginning, one month after and three months after the beginning of the use of the medication. We also observed the leiomyoma symptoms evolution, presence of usual symptoms of hipoestrogenism and seric values of FSH, estradiol, cholesterol and its fractions triglycerides and glicemia. We obtained reduction in uterus-leiomyoma volume of 9.32% in average, in maximum 31%. There was significative reduction in symptoms related with leiomyoma. There were not symptoms of hipoestrogenism. We not observed significative modifications in the other studied parameters. This study concluded that the aromatase inhibitor, anastrozol, in this dosage, is effective in uterus-leiomyomas reduction, cause significative reduction of the symptoms related with this illness, without change the seric dosage of FSH, estradiol, total cholesterol and fractions, triglycerides and glicemia.

BACKGROUND:In breast cancer endocrine therapy, post-therapy Ki-67 assay of biopsy material predicts recurrence-free survival but is invasive and prone to sampling error. 18F]Fluorodeoxyglucose (FDG) positron emission tomography (PET) has shown an early agonist or 'flare' response to tamoxifen and estradiol, but has not been tested in response to estrogen-lowering aromatase inhibitors (AIs). We hypothesized that decreased agonistic response to AIs would result in early FDG uptake decline. We also measured early response to trastuzumab (T), another targeted agent for breast cancer with differing mechanisms of action. Our study was designed to test for an early decline in FDG uptake in response to AI or T and to examine association with Ki-67 measures of early response.METHODS:Patients with any stage of newly diagnosed or recurrent breast cancer were eligible and enrolled prior to initiation (or resumption) of AI or T therapy. FDG PET and tissue biopsy were planned before and after 2 weeks of AI or T therapy, with pretreatment archival tissue permitted. Cutoffs of greater than or equal to]20% decline in standardized uptake value (SUV) as FDG PET early response and less than or equal to]5% post-treatment expression as Ki-67 early response were defined prior to analysis.RESULTS:Forty-two patients enrolled, and 40 (28 AI, 12 T) completed serial FDG-PET imaging. Twenty-two patients (17 AI, 5 T) had newly diagnosed disease, and 23 (14 AI, 9 T) had metastatic disease (5 newly diagnosed). Post-treatment biopsy was performed in 25 patients (63%) and was either refused or not feasible in 15. Post-treatment biopsy yielded tumor in only 17/25 cases (14 AI, 3 T). Eleven of 14 AI patients with post-therapy tissue showed FDG PET early response, and there was 100% concordance of PET and post-therapy Ki-67 early response. For the T group, 6/12 showed an FDG PET early response, including 2/3 patients with post-therapy biopsy, all with Ki-67 >5%.CONCLUSIONS:Substantial changes in FDG PET SUV occurred over 2 weeks of AI therapy and were associated with low post-therapy proliferation. SUV decline was seen in response to T, but few tissue samples were available to test association with Ki-67. Our results support further investigation of FDG PET as a biomarker for early response to AI therapy.

Indiana University-Purdue University Indianapolis (IUPUI) / Oral endocrine therapy (OET) is standard therapy for millions of estrogen receptor-positive breast cancer survivors (ER+BCS). OET reduces recurrence, mortality, and metastasis. ER+BCS often do not take their OET as recommended due to adverse side effects. The purpose of this dissertation was to develop an explanatory framework of decision making by women with ER+ breast cancer who report experiencing side effects from OET. This project was comprised of two components. The first component was a systematic review with three main findings: (1) side effects negatively impact OET non-adherence, (2) there is an absence of decisional supports provided to or available for ER+BCS who are experiencing OET side effects,, and (3) ER+BCS likely have unmet decisional needs related to OET. The second component was a grounded theory study that included 31 ER+BCS reporting OET side effects. During a single semi-structured interview, participants described the experience of OET over time. This study produced two qualitatively derived projects. First, a theoretical framework was developed that depicted four stages through which the experience of OET decision making unfolded. The stages were (1) being told what I need to do to live, (2) doing what I need to do to live, (3) enduring what I need to do to live, and (4) deciding how I want to live. Second, a typology was developed that depicted six sources of external decisional supports (healthcare providers, husbands, other breast cancer survivors, friends and family, the internet and other media sources, and God) that met four types of decisional needs (information about OET and its side effects, in-depth discussions about side effects, help in managing side effects, and emotional support). Findings can be used to develop interventions, such as decision aids, to promote quality decision making in women experiencing OET side effects.

Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase is the cytochrome P450 enzyme that converts androgens to estrogens. Aromatase is the target of the aromatase inhibitor class of drugs widely used to treat estrogen-mediated conditions including breast cancer. Little is known about the role of this enzyme in drug metabolism or in drug interactions. Since this lack of knowledge has been an impediment to optimal therapy, it is important to understand these roles of aromatase. Therefore, a comprehensive series of studies was carried out to characterize its ability to metabolize drugs and its susceptibility to inhibition by xenobiotics. The overall objective of this work was to better understand the interactions of small molecules with aromatase and to use this new knowledge to predict aromatase-mediated drug interactions and anticipate novel molecular structures that interact with the enzyme. Aromatase was shown to be a drug metabolizing enzyme able to metabolize methadone both in vitro (Km of 314 μM) and in vivo (22% of methadone clearance). A number of novel aromatase inhibitors that employ diverse kinetic mechanisms were identified. These include a potent competitive inhibitor: norendoxifen (Ki of 35 nM), two non-competitive inhibitors: endoxifen (Ki of 4.0 μM) and N-desmethyl-tamoxifen (Ki of 15.9 μM), a mechanism-based inhibitor: methadone (KI of 40.6 ± 2.8 μM; kinact of 0.061 ± 0.001 min-1), and a stereoselective inhibitor: naringenin (IC50s of 2.8 μM for (R)-enatiomer and 1.4 μM for (S)-enatiomer). Through investigation of the structure-potency relationships so discovered, a series of new biochemical structures to be exploited as aromatase inhibitors were identified. These studies have identified new roles for aromatase as a catalyst for methadone metabolism and as a mediator of the effects of tamoxifen by demonstrating that a number of its metabolites can act as aromatase inhibitors. This work also provides a new mechanistic framework for the design of novel aromatase inhibitors that can be used in breast cancer. Overall, the data suggest ways to more consistently treat breast cancer with current medications, to better anticipate drug interactions, and therefore to improve the quality of life of patients in ways that minimize side effects, while optimizing therapeutic benefits, in each person treated.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.