Herein, we describe the design and synthesis of novel inhibitors of 17β-hydroxysteroid dehydrogenase type 3 which convert androstenedione into testosterone, which is then converted into dihydrotestosterone (DHT). This isozyme has been implicated in the growth of prostate cancer. Using an in silico pharmacophore model initial targets were planned, based around a diphenylether hydrophobic head linked to a 4-substituted piperidine ring. Over 45 compounds were synthesised and many show significant biological activity when evaluated in a 17β-HSD type 3 biological assay. The most potent compound in this series is 1-(4-[2-(4-chloro-phenoxy[-phenylamino]-piperidin)1-yl) ethanone with an IC₅₀ of 700 nM. The amine linked compounds are significantly more active than the amide equivalents. Synthesis of the amine-linked compounds was problematic and led to the development of a novel and general microwave assisted procedure for the reductive amination of anilines, enabling aromatic amine-linked compounds to be synthesised in excellent yields. A series of benzylamine linked inhibitors was also prepared. Over 30 analogues were synthesised and several show very promising biological activity. The most active compound is N-(2-([2-(4-Chloro-phenoxy)-phenylamino]-methyl)-phenyl)-acetamide, which exhibits an IC₅₀ of 900 nM. The synthesis of compounds with a benzophenone linked hydrophobic head group led to an unexpected product. X-ray crystallography was used to determine the structure, as a quinoline derivative. This led to optimisation of a novel modification of the Friedländer synthesis of quinolines. The potent inhibitors synthesised are selective over 17β-HASD Types 1 and 2. One inhibitor also shows potentially interesting activity against the leukaemia cell line CCRF-CEM, in the NCI screening, with a GI₅₀ of 10 nM.

Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

"December 2009" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Thesis advisor: David A. Wilson. Includes bibliographical references.

Background: An important role has been suggested for tissue-specific glucocorticoid metabolism in the development of obesity and its complications. 11ß hydroxysteroid dehydrogenase 1 (11ßHSD1) is an enzyme that catalyzes the interconversion of biologically inactive cortisone to active cortisol, thereby regulating its access to glucocorticoid receptors in target tissues. Indeed, an unfavorable metabolic outcome has been associated with increased 11ßHSD1 gene expression and activity in adipose tissue and liver in humans and rodents. Cortisol is an important regulator of phosphoenolpyruvate carboxykinase (PEPCK) a key enzyme in gluconeogenesis and lipid metabolism. In rodents, overexpression of PEPCK in adipose tissue leads to adiposity and increased fatty acid re-esterification. In human obesity, PEPCK has been positively associated with body fat, total cholesterol levels, and plasma triglycerides. However, few studies have addressed the putative reversibility of peripheral cortisol levels and disturbed fatty acid homeostasis that may accompany weight loss. The aim of this thesis was to investigate alterations in peripheral glucocorticoid metabolism in the context of obesity, and putative modulations of glucocorticoid metabolism in the context of weight changes in humans and rodents. Materials &amp; Methods: 11ßHSD1 expression/activity in different adipose tissue depots and liver, the expression of genes involved in adipogenesis and fatty acid homeostasis, and serum levels of adipose tissue-derived adipokines were investigated in severely obese women before and after surgically induced weight loss. The same parameters were measured in female Sprague-Dawley rats fed on high-fat and control diets. Results: In severely obese women, 11ßHSD1 expression was higher in subcutaneous adipose tissue (SAT), while 11ßHSD1 activity and PEPCK expression were higher in the omental depot. In a multivariate analysis, SAT 11ßHSD1 activity was an independent predictor for central fat accumulation. Hepatic 11ßHSD1 activity and levels of intra-abdominal fat storage correlated negatively, while 11ßHSD1 correlated positively with PEPCK in adipose tissue and liver. Weight loss after gastric bypass surgery was followed by significant and metabolically beneficial reductions in subcutaneous 11ßHSD1 and leptin gene expression, as well as reduced circulating leptin and increased adiponectin levels. In contrast, PEPCK gene expression did not change with weight loss. In rats, a high-fat diet did not affect body weight, but was associated with increased serum leptin and decreased adiponectin levels. Short-term, high-fat diet feeding resulted in the up-regulation of SAT 11ßHSD1 expression, while chronic feeding led to its significant down-regulation (compared with the control diet and short-term, high-fat feeding). Interestingly, hepatic 11ßHSD1 expression was constantly downregulated in rats that were fed a high-fat diet. Conclusions: Severe obesity in women was accompanied by a metabolically adverse increase of 11ßHSD1 in adipose tissue, with a concomitant decrease in the liver. Subcutaneous 11ßHSD1 was an independent predictor for central fat accumulation. As weight loss was followed by significant down-regulation of subcutaneous 11ßHSD1, we suggest that up-regulation of this enzyme was a consequence, rather than a cause of obesity. In rodents, a high-fat diet induced dynamic changes in 11ßHSD1 in SAT and liver, both being down-regulated after chronic high-fat feeding without altered weight. In summary, weight changes and alterations in fat and liver glucocorticoid metabolism are closely linked. Moreover, a high-fat diet significantly influences 11ßHSD1 expression/activity in adipose tissue and liver without affecting body weight.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12834号 / 論農博第2798号 / 新制||農||1026(附属図書館) / 学位論文||H26||N4857(農学部図書室) / 31372 / (主査)教授 栗原 達夫, 教授 植田 和光, 教授 平竹 潤 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18924号 / 薬科博第38号 / 新制||薬||5(附属図書館) / 31875 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 岡村 均, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Whilst, several phase II drugs as well as endogenous & exogenous steroids/steroid metabolites have been identified as inducers of gene transcription, the cellular transcription factors controlling the expression of AKR1C1 are incompletely elucidated. Herein, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene that controls its transcription. The 5’ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5’-flanking, ~3.0 kb region of the human AKR1C1 gene identified the region between -128 to -88 as the minimal proximal promoter essential for basal transcription of AKR1C1 in human ovarian (2008 & 2008/C13*), lung (H23 & A549) and liver carcinoma (HepG2) cells. Antioxidant response elements (ARE) have been shown to modulate the transcription ofv genes coding for phase II drug metabolizing enzymes. Cloning of the ARE upstream of the AKR1C1 proximal promoter resulted in increased transcription in human lung adenocarcinoma and liver hepatoblastoma cells but not in human ovarian carcinoma cells. Further, ARE increased the induction of the AKR1C1 gene in response to treatment with phase II drug inducers. However, ARE did not induce the transcription of AKR1C1 gene promoter in the presence of cisplatin in any of the cell lines. A computational analysis utilizing the Alibaba 2.0 on the proximal AKR1C1 gene promoter region was performed to identify potential transcription factor binding sites. Based on this analysis, a set of potential, putative transcription factor binding sites for Oct1, Sp1, Cp-1/NF-Y, CEBP, p40X, USF, NF1 and AP-2 were identified in the region -180 to -88 of the AKR1C1 gene promoter. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were involved in controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at -109ATTGG-105 of the AKR1C1 gene. Chromatin immunoprecipitation (ChIP) analysis confirmed the in vivo association between NF-Y and human AKR1C1 gene promoter in human ovarian, lung and liver carcinoma cells. Further, ectopic expression of NF-Y’s increased the AKR1C1 gene transcription, whereas expression of a dominant-negative NF-YA or knockdown of NF-YA by siRNA transfection, decreased the AKR1C1 gene transcription. A 2-fold increase in AKR1C1 transcription was observed specifically in cisplatin-treated 2008 cells that was CCAAT box-dependent. These results indicate that NF-Y regulates basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells and cisplatin-induced transcription in human ovarian carcinoma cells. / Pathology

Das Enzym 11beta-HSD1 stellt im intrazellulären Glucocorticoidstoffwechsel eine wichtige Prärezeptorkontrolle dar. Es reguliert die intrazelluläre Cortisolkonzentration durch die enzymatische Umwandlung des aus dem Blutkreislauf aufgenommenen und hormonell inaktiven Cortisons zum aktiven Cortisol. Die Bedeutung einer erhöhten 11beta-HSD1 Expression und Aktivität bei der Entstehung von Übergewicht und Insulinresistenz wurde bisher vorwiegend in Leber und Fettgewebe untersucht und nachgewiesen. Wenig erforscht sind die Funktionen der 11beta-HSD1 im Muskelgewebe. In dieser Arbeit wurde die Funktion und Regulation der 11beta-HSD1 im Skelettmuskel mithilfe der murinen Skelettmuskelzelllinie C2C12 und primärer humaner Myoblasten untersucht. Es konnte demonstriert werden, dass die 11beta-HSD1 in Abhängigkeit des Differenzierungsgrades exprimiert wird und als Oxo-Reduktase aktiv ist, sowie selbst einen Regulator der Differenzierung darstellt. Es zeigte sich ein Feed-Forward-Mechanismus des Cortisons, das die 11beta-HSD1 in den Skelettmuskelzellen akut und chronisch induzierte, sowie eine gleichzeitige Veränderung der GRalpha- und MRalpha-Expressionen gegenregulatorisch zur 11beta-HSD1. Die Daten aus der Mauszelllinie konnten zum größten Teil in primären humanen Myoblasten bestätigt werden. Zudem konnten mehrere Transkriptionsfaktoren wie CREB, Myogenin und MEF-2c identifiziert werden, die in den verschiedenen Phasen der Differenzierung unterschiedliche Relevanz für die Regulation der 11beta-HSD1 Promotoraktivität hatten. Des Weiteren wurden die Proteolyserate und die Expression der E3-Ubiquitin-Ligasen Atrogin-1 und MuRF-1 11beta-HSD1-abhängig durch Cortison induziert. Trotz alledem führte eine Langzeit-Stimulation mit Cortison zu einer 11beta-HSD1-abhängigen Induktion der Differenzierung mit einer Veränderung der Muskelfasertypen in Richtung langsam-zuckender Muskelfasern, was eine Bedeutung für das klinische Bild der glucocorticoid-induzierten Muskelatrophie haben kann. / The enzyme 11beta-HSD1 functions as an important pre-receptor control of intracellular glucocorticoid action regulating the intracellular cortisol concentration by enzymatic conversion of the hormonal inactive cortisone up-taken from blood circulation to the active cortisol. A pivotal role of an increased 11beta-HSD1 expression and activity for the development of overweight and insulin resistance has been analysed and demonstrated particularly in liver and adipose tissue. However, the functions of 11beta-HSD1 in skeletal muscle tissue are rarely investigated. For analysis of function and regulation of the 11beta-HSD1 in skeletal muscle the murine skeletal muscle cell line C2C12 as well as primary human myoblasts from healthy volunteers were used. 11beta-HSD1 was shown to be expressed and functionally active as oxo-reductase in human and murine skeletal muscle cells dependent on the differentiation but as well to function as a regulator of differentiation itself. The stimulation experiments revealed a feed-forward-mechanism of cortisone that induced 11beta-HSD1 acutely and chronically. Concurrently, GRalpha and MRalpha were expressed contra-regulatory to 11beta-HSD1. For the most part these data were confirmed in human primary myoblasts. Several transcription factors as CREB, Myogenin and MEF-2c were identified having different relevance for regulation of 11beta-HSD1 promoter activity during the different phases of differentiation. Furthermore, treatment with cortisone increased protein degradation and expression of the two E3-ubiquitin-ligases Atrogin-1 and MuRF-1 in an 11beta-HSD1-dependent way. Nonetheless, a long-term stimulation by cortisone revealed an 11beta-HSD1-dependent induction of differentiation accompanied by modification of muscle fiber type composition towards slow-twitch muscle fibers that may play a role for the clinical picture of glucocorticoid-induced muscle atrophy.

Background Obesity and associated metabolic dysregulation are linked to impaired cognitive function and alterations in brain structure, which increases the risk of age-related dementia. Increased glucocorticoid (GC) exposure may be a potential mediator of these negative effects on the brain. Methods and results In paper 1, we tested the relationship between cortisol levels, brain morphology and cognitive function in 200 women and men. Salivary cortisol levels were negatively related to cortical surface areas in prefrontal brain regions in both sexes. In participants with type 2 diabetes, high salivary cortisol levels were associated with lower memory performance. In paper 2, we tested in 70 overweight women the effects on tissue-specific GC metabolism of a Paleolithic diet or a diet following the Nordic nutrition recommendations. The 24-month interventions led to decreased expression of the GC-activating enzyme 11βHSD1 in adipose tissue, interpreted as a normalization of an obesity-related disturbance in GC metabolism. Furthermore, GC metabolism by 5α-reductase increased substantially after 2 years, an unexpected and novel result. The outcomes did not differ by diet. In paper 3, 20 women included in paper 2 were examined with functional magnetic resonance imaging (fMRI) while performing a memory task at baseline and after 6 months. Memory performance improved and functional brain responses increased in the hippocampus. Once again, the results were similar in both diet groups. In paper 4, 24 overweight participants with type 2 diabetes were examined with fMRI, using the same memory test as in paper 3, at baseline and after 12 weeks of intervention with a Paleolithic diet with or without exercise training. Functional brain response increased in the hippocampus, but memory was not improved. The addition of physical exercise did not alter the results. Conclusion Cortisol levels are linked to prefrontal brain structure and, at least in type 2 diabetes, lower memory performance. Furthermore, the dysregulated GC metabolism in obesity can be reversed by long-term diet- induced weight loss. Finally, dietary interventions with associated metabolic improvements alter functional brain responses during memory testing, including increased activation of the hippocampus. Whether these changes are linked to alterations in GC exposure and mediate improved cognition requires further study.

Background: Cardiovascular disease (CVD) is the leading cause of death among women in Sweden. The risk of CVD increases rapidly after the menopause. A major contributing factor may be the redistribution of adipose tissue, from the peripheral to central depots, associated with menopause. This change in body composition is commonly attributed to declining estrogen levels but may also be affected by tissue-specific alterations in exposure to other steroid hormones, notably glucocorticoids – mainly cortisol in humans. Indeed, adipose tissue-specific overexpression of the glucocorticoid-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) induces central obesity, insulin resistance and hypertension in mice. Interestingly, estrogen may regulate this enzyme. The aim of this thesis was to investigate putative links between estrogen and glucocorticoid activation by 11βHSD1. Materials and Methods: 11βHSD1 expression and/or activity in adipose tissue and liver, and adipose estrogen receptor α and β (ERα and ERβ) gene expression, were investigated in lean pre- and postmenopausal women and ovariectomized rodents with and without estrogen supplementation. In lean women measures of 11βHSD1 were correlated to risk markers for CVD. The association between adipose 11βHSD1 and ER mRNA expression was investigated in both lean women and rats and in an additional cohort of obese premenopausal women. In vitro experiments with adipocyte cell lines were used to explore possible pathways for estrogen regulation of 11βHSD1. Results: Subcutaneous adipose tissue transcript levels and hepatic activity of 11βHSD1 were higher in postmenopausal vs. premenopausal women. In rodents, estrogen treatment to ovariectomized rats decreased visceral adipose tissue 11βHSD1, resulting in a shift towards higher subcutaneous (vs. visceral) 11βHSD1 mRNA expression/activity. Increased adipose and hepatic 11βHSD1 were associated with increased blood pressure and a disadvantageous blood lipid profile in humans. We found significant positive associations between 11βHSD1 and ERβ transcript levels in adipose tissue. The in vitro experiments showed upregulation of 11βHSD1 mRNA expression and activity with estrogen or ERβ-agonist treatment at low (corresponding to physiological) concentrations. Conclusions: Our studies show for the first time increased local tissue glucocorticoid activation with menopause/age in women. This may contribute to an increased risk of CVD. Estrogen treatment in rodents induces a shift in 11βHSD1 activity towards the subcutaneous adipose tissue depots, which may direct fat accumulation to this metabolically “safer” depot. The in vitro studies suggest that low-dose estrogen treatment upregulates 11βHSD1 via ERβ. In summary, estrogen - glucocorticoid metabolism interactions may be key in the development of menopause-related metabolic dysfunction and in part mediate the beneficial effects of postmenopausal estrogen treatment on body fat distribution.