O uso de esteróides androgénicos-anabolizantes em química terapêutica e o seu ilícito no mercado de venda paralelo

Monografia realizada no âmbito da unidade de Estágio Curricular do Mestrado Integrado em Ciências Farmacêuticas, apresentada à Faculdade de Farmácia da Universidade de CoimbraOs androgénios-anabolizantes são derivados sintéticos da testosterona introduzidos na terapêutica com o objetivo de exercerem ações anabólicas, com efeitos androgénicos reduzidos. Estes exercem os seus efeitos através do recetor androgénio, expresso em diferentes tecidos, ativando vias genómicas e não-genómicas. As modificações introduzidas na estrutura da testosterona e de esteróides sintéticos deverão ter em conta que existem certos grupos funcionais essenciais para a ligação do esteróide ao recetor androgénio, tais como o grupo 3-ceto, o 17-OH e substituições com pequenos grupos nas posições 7 e 17. Em química terapêutica, os androgénios-anabolizantes apresentam aplicações clínicas na caquexia associada a doenças crónicas, na terapêutica adjuvante da quimioterapia, na terapêutica hormonal de substituição em homens com hipogonadismo e em idosos, entre outras condições fisiopatológicas. No entanto, em algumas situações, a eficácia e a segurança ainda se encontra por demonstrar, quer em termos de melhoria das capacidades físicas, quer em relação ao aumento da qualidade de vida destes doentes. As doenças desmielinizantes como a esclerose múltipla e a prevenção e tratamento da doença de Alzheimer, parecem ser novas aplicações terapêuticas possíveis para os androgénios-anabolizantes. Na maioria dos países, os esteróides encontram-se à margem da lei, sendo o controlo da sua importação, do seu fabrico e da sua venda ao público deficitário. No desporto, o abuso de anabolizantes tem sido cada vez mais frequente, sendo atualmente um fenómeno global. As suas propriedades ergogénicas são reconhecidas por atletas, bodybuilders e powerlifters. O culto do corpo associado à desinformação e a falta de orientação por profissionais de saúde criam, assim, condições favoráveis à toma de anabolizantes, tornando-se o seu abuso um problema de saúde pública. Além disto, estes são usados com outras drogas ilícitas, produzindo um agravamento dos seus riscos e efeitos adversos. Deste modo, verifica-se a necessidade urgente de programas de prevenção e educação em escolas e universidades, podendo o farmacêutico ser um dos seus principais intervenientes. Apesar de controlados mundialmente pelas autoridades antidopagem, com os mais diversos métodos analíticos de deteção, estes continuam a ser usados no doping desportivo.Anabolic androgenic steroids are synthetic derivates of testosterone introduced in therapeutics in order to enhance anabolic actions with reduced androgenic effects. They mediate their effects through genomic activation of the androgenic receptor and nongenomic signaling pathways in different tissues. Testosterone and synthetic steroids structure modifications should consider the existence of a variety of functional groups that are crucial for the binding to the androgen receptor as 3-keto, 17-OH and small group substitutions in 7 and 17 positions. In clinical chemistry, the anabolic androgenic steroids have numerous therapeutic applications, as the treatment of cachexia associated with chronic diseases states, as adjuvant in chemotherapy, in the androgen replacement therapy of hypogonadism and in elderly men as well as other pathophisiological conditions. Nevertheless, in some clinical situations, their efficacy and safety still needs to be demonstrated in terms of improved physical condition and quality of life of the patients. New data establish the efficacy of androgens as remyelinating agents providing the preclinical rational for novel therapeutic use of anabolic androgenic steroids in males with multiple sclerosis and as a promising strategy to delay and perhaps prevent Alzheimer´s disease. In most countries, steroids are not subjects to laws and the control of parallel importation, manufacture and sale to the public are inadequate. In sport, the abuse of anabolic steroids has been increasingly common, becoming currently a global phenomenon. Their ergogenic effects are recognized by athletes, bodybuilders and powerlifters. Therefore, the body cult associated with misinformation and lack of guidance by health professionals create favorable conditions for the use of anabolic steroids, becoming their abuse a public health issue. Moreover, they are abused with other illicit drugs, producing the aggravation of their risks and adverse effects. In this respect, there is an urgent need for prevention and education programs in high schools and universities, allowing the pharmacist intervention become one of the best keys of the process. In spite of being controlled by worldwide anti-doping authorities, with the most diverse analytical methods for detection, anabolic androgenic steroids continue to be used in sport doping

In Vitro Androgen Bioassays as a Detection Method for Designer Androgens

Androgens are the class of sex steroids responsible for male sexual characteristics, including increased muscle mass and decreased fat mass. Illicit use of androgen doping can be an attractive option for those looking to enhance sporting performance and/or physical appearance. The use of in vitro bioassays to detect androgens, especially designer or proandrogens, is becoming increasingly important in combating androgen doping associated with nutritional supplements. The nutritional sports supplement market has grown rapidly throughout the past decade. Many of these supplements contain androgens, designer androgens or proandrogens. Many designer or proandrogens cannot be detected by the standard highly-sensitive screening methods such as gas chromatography-mass spectrometry because their chemical structure is unknown. However, in vitro androgen bioassays can detect designer and proandrogens as these assays are not reliant on knowing the chemical structure but instead are based on androgen receptor activation. For these reasons, it may be advantageous to use routine androgen bioassay screening of nutraceutical samples to help curb the increasing problem of androgen doping

ASSESSMENT OF ENDOCRINE ACTIVE SUBSTANCES USINGIN SILICO AND REPORTER ASSAYS IN CELL AND MICE

In the last two decades, there have been growing scientific concern, public debate, and media attention over the possible deleterious effects in humans and wildlife that may result from exposure to substances that have the potential to interfere with the endocrine system. Endocrine disrupting compounds (EDCs) encompass a variety of substance classes, including natural and synthetic hormones, plant constituents, pesticides, substances used in industry and in consumer products, pollutant. It is well documented that EDCs targets are mainly the nuclear receptors (NRs) such as the sexual hormones estrogen receptors ERs. ER is involved in a broad spectrum of physiological processes in different organs and tissues as well as in several diseases, such as breast and endometrial cancer, osteoporosis, and prostate hypertrophy, neurodegenerative diseases or in immune system activation. The regulatory agencies for the protection of human health and wildlife have been issuing the necessity to investigate and clarify the mode of action of these exogenous substances by the development of alternative toxicological methods such as in silico model and in vitro testing in order to predict the toxicity of these EDCs. This research had the ambitious aim to develop an integrated toxicological strategy based on the combination of available information of endocrine disrupting activity retrieved from the scientific literature, in silico model, imaging methodologies applied to reporter systems in vitro and in vivo and ex vivo to predict among a set of chemicals those with an endocrine disrupting activity or ability to activate other toxicological pathways such as inflammation and oxidative stress measured in the male reproductive organs and in the genital and abdominal area of mice. The selected molecules range from known (DES) to suspected (BPA) endocrine disruptors and included both synthetic (DEHP) and natural (genistein) compounds. The first step used was in silico analysis with evaluation of the possible binding of selected substances to the estrogen alpha receptor to support the hypothesis that their hormonal activity occurred through a receptorial mechanism. This approach is commonly used and is also part of the first level of investigation suggested by EFSA/ECHA in the recognition of EDCs. The computational methodology estimated different values of affinity of each ligand to hER Ligand Binding Domain (LBD). The use of two different approaches (XP GLIDE SCORE and MMGBSA dG Bind) also allowed for solvation to be taken into account. That meant that MMGBSA protocol considered both the interactions of some water molecules with the LBD and the solvent-ligand ones. The estradiol showed the best affinity values in both approaches as being the endogenous hormone was able to contrast the solvation effect. The dissociation constant (Ki) values calculated from the XP GLIDE SCORE fitted well with the Ki experimentally determined in vitro binding assay by other research groups. Thus, the computed Ki has been chosen as parameter for the prediction of putative endocrine disruptor activity. However, the lack of correlation between the (computed and experimental) Ki and in vivo experimental observed ED activity (from literature data) for all chemicals (only estradiol and zearalenone have similar affinity to ER and literature evidences of ED activity; genistein has a good Ki values but not ED activity such as BPA and methoxychlor), did not allow a prioritization of the investigated chemicals for ED activity through the results of their affinity. In the next step, to check if the receptor binding well correlated with the hormonal activity, the ER Reporter gene assay was performed, based on the ability of a compound to stimulate ER-dependent transcriptional activity in genetically engineered mammalian cells. The cell lines are MCF-7 cells which express human endogenous ER\u3b1. The cells are transformed (transfected) by introducing vectors containing DNA sequences for the receptor, along with EREs linked to a reporter gene, and the reporter gene itself. The reporter gene used in human cancer cells usually codes for luciferase (CALUX, chemically activated luciferase expression). In the transactivation the EDCs show their estrogenic potency calculated as EC50, in respect to the positive control, 17\u3b2-estradiol. This system has enabled us to evaluate the kinetic and the biological consequences of cellular activation in the same cell monolayer by bioluminescence imaging of photon emissions that were pictures of activated ER status at 6 and 48 hours after the initial treatments. From this in vitro assay three factors were taken into consideration, power, efficacy and trend over time. The in vitro dynamic ER activation showed that for some chemicals (genistein, BPA, methoxychlor), the potency (EC50) and the efficacy (fold induction) changed over time, but not for others (estradiol, zearalenone and DES). Considering that estradiol, zearalenone and DES certainly have an activity in the animal and in man as endocrine disruptors, the duration of effect parameter combined with power and efficacy were likely to be associated in predicting the activity. Together with receptor affinity and the ability to induce a biological response, it also seemed relevant how long the response was lasting. By consequence, the combination of the variation of the potency response and the efficacy, \u201cnormalized\u201d respect to the efficacy values quantify at 48 hours, was successfully used in discriminating positive and negative compounds for their endocrine disrupting activity. By means of this analysis 17\u3b2-estradiol, DES and zearalenone were put at the top of the list (also supported by their known ED activity), genistein resulted to represent a putative threshold of no-concern for ED effect, in supporting published data, while methoxychlor and BPA were definitely not considered a priority in terms of ED activity. This in vitro classification fitted well with the in silico outcomes, since the strongest estrogen receptor binders were ranked in the first positions (17\u3b2-estradiol, DES and zearalenone). Besides, was no possible to calculate EC50s for 4-nonylphenol, DEHP and vinclozolin, not making possible to classify them as ED, totally in agreement with in silico results and in line with literature data (vinclozolin is mainly an androgen antagonist). The third step of our stepwise approach was intended to verify in vivo the interaction of selected chemicals with the estrogen receptor and in addition the activation of their pathways triggering primary harmful effects. We used three reporter mice designed in order to evaluate the effect of selected compounds to activate ER and causing oxidative stress and inflammation. We have chosen to test zearalenone (well-known endocrine disruptor and clearly identified as such by our in silico-in vitro approach), and BPA for which there are controversial data in the literature and that our approach has negatively classified as ED. In our experiments zearalenone showed to be active on ER pathway in the abdominal area and significantly activated the inflammatory pathway in the genitals (in this specific case in the prostate, result of ex vivo bioluminescence analysis). These results were perfectly in line with the literature reports, in which prostate inflammation and metaphase were detected in both mice and rats. Bisphenol A did not produce a significant activation in both the areas and in the ex vivo analysis, again in agreement with in silico/in vitro results. We used a combination of innovative approaches that led to a conclusion that in silico screening cannot be used as a stand-alone procedure due to its intrinsic lack of biological meaning, although it can be successfully used as a first prioritizing step in a tier approach. The second mandatory check for the in silico positive hits should be an in vitro evaluation procedure, in which the affinity of the positive hits is measured through a reference cellular assay. Our results showed that integrating the time variable in the evaluation of the potency, the tested compounds could be classified as ED or no-ED. The in vivo experiment highlighted that a potent estrogenic compound, as zearalenone, could also raise concern for the activation of other toxicological pathway such as the inflammatory ones. We are aware that this indication of procedure must be evaluated and validated on dozens of molecules whose in vivo activity is already known before arriving at its use to predict the possible activity of ED of unknown molecules, but we think that this approach deserves to be implemented

Unravelling The Role Of Androgens In Polycystic Ovary Syndrome

Polycystic Ovary Syndrome (PCOS) is a multifaceted hormonal disorder which affects 5-15% of reproductive-aged women worldwide. While classically recognised as an ovarian disorder, PCOS is associated with a variety of reproductive, endocrine and metabolic features including ovulatory dysfunction, infertility, hyperandrogenism, obesity, and an increased risk of type 2 diabetes mellitus and cardiovascular disease. The most consistently present of these is hyperandrogenism – supraphysiological levels of androgens such as testosterone (T) and dihydrotestosterone (DHT). Typically thought of as male steroid hormones, androgens have been shown to play important role in the maintenance of normal female reproductive function. Despite the high prevalence of hyperandrogenism amongst patients, the role of androgens in the etiology and pathogenesis of PCOS has yet to be determined. The aim of this work was to unravel the association between excess androgen exposure and the development and advancement of the PCOS phenotype in mouse model of androgen-induced PCOS. The first study contained within this work (Chapter Three) provides the first comprehensive characterization of a range of reproductive, endocrine and metabolic traits associated with PCOS in four distinct mouse models: a model of prenatal androgenisation utilising the potent non-aromatizable androgen DHT administered during days 16-18 of gestation, and three diverse models of postnatal androgen exposure employing a long-term treatment with either DHT, the proandrogen dehydroepiandrosterone (DHEA), or letrozole (an aromatase inhibitor) for 90 days beginning at 3 weeks of age. Prenatal androgenisation produced some reproductive and endocrine traits, but failed to induce the metabolic abnormalities seen in PCOS. DHEA treatment did not reproduce any features associated with PCOS while treatment with letrozole produced few PCOS-like characteristics and some aberrant changes not typical of the syndrome. Additionally, letrozole treatment did not reproduce any metabolic attributes of PCOS. On the other hand, postnatal exposure to excess androgen, by way of DHT treatment, produced a breadth of reproductive, endocrine and metabolic traits that mimic those seen in human PCOS. This study revealed that a treatment regime of long-term postnatal exposure to DHT reproduced the strongest PCOS-like phenotype in our mice and provides a robust animal model in which to study the pathogenesis of PCOS. The second study (Chapter Four) aimed to explore the involvement of genomic androgen receptor (AR)-mediated actions in the development of these PCOS traits. As a prenatally androgenised mouse model of PCOS had previously been reported to exhibit impaired neuroendocrine hypothalamic feedback of the hypothalamic-pituitary-gonadal (HPG) axis, we took this opportunity to utilise mice from our own prenatal model to investigate the neuroendocrine regulation of the HPG axis in PCOS in addition to the effects of AR inactivation on the PCOS phenotype. PCOS was induced in wild-type (WT) and androgen receptor knockout (ARKO) mice using DHT administered on days 16-18 of gestation. A subset of these mice were also exposed to 17β-estradiol for 7 days prior to collection, via a subdermal implant, to investigate the impaired estradiol negative feedback on the hypothalamus. WT mice with DHT-induced PCOS displayed several reproductive abnormalities including aberrant cycling and ovulatory dysfunction in addition to adipocyte hypertrophy and hepatic steatosis. However, diestrus serum luteinising hormone and follicle stimulating hormone, and estradiol-induced negative feedback as well as hypothalamic expression of several neuropeptides were unaffected by DHT treatment in WT mice. Mice both homozygous and heterozygous for the global inactivation of the AR (ARKO), did not display any PCOS traits when exposed to excess androgens during prenatal life. This study showed the importance of AR signalling in the development of PCOS and revealed that even AR haplosufficiency is adequate to prevent induction of PCOS by prenatal hyperandrogenism. Finally, the third study (Chapter Five) aimed to shed further light on the AR-mediated androgen actions in PCOS with a focus on identifying the tissue-specific targets of these actions. Employing our postnatal model of PCOS induction, this study investigates the effects of: 1) global loss of AR signalling (ARKO), 2) neuronal knockout (NeurARKO) and 3) granulosa cell-specific AR inactivation (GCARKO) on the development of the PCOS phenotype induced by exposure to exogenous DHT. As in our prenatal model, ARKO mice were fully protected from all DHT-induced features of PCOS. Neuron-specific AR signaling was required for the development of a variety of reproductive and metabolic traits including classic polycystic ovaries, dysfunctional ovulation, obesity and dyslipidemia. In contrast, loss of AR signalling in granulosa cells did not impede the pathogenesis of PCOS-like features in GCARKO mice. To further examine the role of extra-ovarian AR signalling in PCOS, reciprocal ovary transplants were carried out in WT and ARKO mice. Results from ARKO hosts with transplanted WT ovaries revealed that excess androgen exposure requires functional extra-ovarian, and not intra-ovarian, AR signalling in order to produce features of PCOS. This study provides strong evidence that neuroendocrine genomic AR signaling is an important mediator in the development of PCOS. The studies contained within this thesis are the first to provide a comprehensive analysis of a mouse model of PCOS encompassing a breadth of reproductive, endocrine and metabolic features. This work has identified the optimal model in which to study this complex, multifactorial condition which affects a significant number of women worldwide. Additionally, our results have shown that the effects of androgens on the pathogenesis of PCOS are mediated via the androgen receptor in a dose-dependent manner such that two functional copies are required for DHT to reproduce features of PCOS in the mouse. Finally, in a crucial study to investigate the locus of androgen actions we have revealed the previously overlooked importance of extra-ovarian neuroendocrine androgen action in the origins and progression of PCOS, despite it being thought of primarily as an ovarian disorder. Overall, these studies have provided valuable insights into both the role of androgens in Polycystic Ovary Syndrome and potential new targets for the development of mechanism-based treatments of this disorder

Cellular bioassays as a clinical tool for monitoring sex steroids

Sex steroid production needs to be maintained within the physiological set point range, deviations outside of the normal range can be a symptom of- and risk factor for endocrine-related diseases (such as polycystic ovarian syndrome (PCOS), hypogonadism, breast- and prostate cancer). Testosterone is the dominant male sex steroid and estradiol (E2) is the most abundant and potent female sex steroid. Testosterone and E2 are mainly synthesised in gonads of both sexes. Testosterone and E2 induce a cellular response by diffusing into cells and binding to their respective nuclear receptor (androgen- (AR) and estrogen receptor (ER)), which upon activation, operate as transcription factors and regulators of gene transcription. It is clinically important to have an accurate measure of detection for testosterone and E2. The current clinical measures, which are immunoassay-based techniques, are under scrutiny due to their poor sensitivity and reproducibility, especially at low concentrations. Furthermore, although testosterone and E2 are the most prominent endogenously synthesised sex steroids, they are not the only molecules capable of activating their intracellular cognate receptor. Other endogenously synthesised androgenic and estrogenic precursor molecules to testosterone and E2 can also activate AR and ER, respectively. Furthermore, studies have shown that exogenous (anti-) estrogens and androgens sourced from synthetic and plant origin have potential to activate or inhibit AR and ERs. A novel technique to identify all estrogenic and androgenic molecules within a sample is reporter gene bioassays (RGBs). RGBs are a cell-based technique that can measure the level of ER or AR activation by a test substance. Cells can be transfected with an expression plasmid to over express AR or ER/ and a reporter gene plasmid that harbours the response element specific to the receptor, which is upstream of a reporter gene. Alternatively, a cell line with endogenous AR or ER/ is transfected with a reporter gene plasmid only. Upon activation of AR or ER/, the receptor binds to its response element on the reporter plasmid to initiate gene transcription of the reporter gene, which is translated into an enzyme, such as luciferase. The reporter gene is measured following addition of a substrate to quantify the level of light emission, which is proportional to the level of receptor activation. Via this mechanism, all molecules that are capable of activating the receptor can be monitored. The overarching aim of this study was to determine if RGBs can be used to measure clinically relevant samples. ER- and AR-RGBs hosted in T47D and HEK293 cells, respectively, were optimised to measure serum and plasma samples. The net serum estrogenicity and androgenicity measurement was compared with the data of the clinical standard immunoassay to determine if additional information could be provided. A major part of this PhD was the establishment of a clinical study, whereby serum from post-menopausal females was obtained for the purpose of measuring in the ER- and AR-RGBs. All patients recruited to the study started letrozole treatment and the aim of this clinical study was to determine if changes in ER- and AR bioactivity could be measured after the onset of treatment. Letrozole is an aromatase inhibitor that blocks the conversion of androgens into estrogens. Therefore, both ER- and AR-RGBs were utilised in a clinical setting to determine their clinical efficacy by comparing to the results of testosterone and E2 immunoassay data. Overall, both ER- and AR-RGBs provided a readout for all serum and plasma samples tested, whereas only some samples had a detectable level of testosterone and/or E2 by immunoassay. This study shows that information to companion quantitative measures of E2 and testosterone can be derived from ER- and AR-RGBs