Metabolism of synthetic steroids by 11ß-hydroxysteroid dehydrogenases - in vivo and in vitro studies

1\. Titelblatt 2\. Inhaltsverzeichnis 3\. Einleitung 1 4\. Problemstellung 19 5\. Material_und_Methoden 21 6\. Ergebnisse 39 7\. Diskussion 61 8\. Zusammenfassung 91 9\. Abkürzungsverzeichnis 94 10\. Literaturverzeichnis 96 11\. Strukturformeln_der_verwendeten_Steroide 12\. Lebenslauf 13\. DanksagungenDie Isoenzyme der 11ß-Hydroxysteroid-Dehydrogenase (11ß-HSD) spielen eine Schlüsselrolle für die Modulation der Wirkung von Glucocorticoiden. Die in vivo als Reduktase fungierende 11ß-HSD1 ist mit dem Glucocorticoidrezeptor kolokalisiert und zeigt eine hohe Expression in Leber und visceralem Fettgewebe. Die 11ß-HSD2 wird hauptsächlich in Mineralocorticoid-Zielgeweben exprimiert, wo sie als Oxidase den Mineralocorticoidrezeptor vor der Okkupation durch endogene Glucocorticoide schützt. In dieser Arbeit wurde die Aktivität der 11ß-HSD1 und der 11ß-HSD2 gegenüber verschiedenen - häufig klinisch eingesetzten - synthetischen Steroiden untersucht. Untersuchungen an transfizierten CHO-Zellen: An mit der 11ß-HSD1 transfizierten Chinese hamster ovarial (CHO) - Zellen konnten bekannte in vivo-Charakteristika des Isoenzyms beobachtet werden. Die Reduktaseaktivität der 11ß-HSD1 wurde durch eine 2-Methylgruppe stark, durch eine 2-Chlorgruppe vollständig gehemmt. Eine verstärkte 11ß-Reduktion ließ sich bei 9\- und 6-Fluorosteroiden (Fluorocortisol und Fluocortolon) sowie bei Prednisolon (1-Dehydrokonfiguration) beobachten. Die first pass-Aktivierung in der Leber scheint daher bei Prednison effektiver abzulaufen als bei Cortison. Die 11ß- HSD2 der transfizierten Zellen zeigte die charakteristische hohe Substrataffinität des Enzyms und fungierte gegenüber unfluorierten Steroiden ausschließlich als 11ß-Oxidase. Die 11ß-Oxidation der 11ß-HSD2 war vermindert bei Steroiden mit einer 6-Methylgruppe (Methylprednisolon), 16-Methylenkonfiguration (Prednyliden), 16-Methylgruppe und 9-Fluorierung (Dexamethason). Aus klinischer Sicht sind 9-Fluorosteroide daher für den Einsatz in Geweben mit hoher 11ß-HSD2 Expression besonders geeignet. Im Vergleich zu dem weithin verwendeten Prednisolon sollten 11-OH-Steroide mit geringerer 11-Oxidation durch die 11ß-HSD2 (z.B. 6-Methylprednisolon, Deflazacort oder Prednyliden) aus pharmakokinetischer Sicht für eine renale Immunsuppression vorteilhafter sein. In vivo Versuche an gesunden Probanden In vitro wird die Reduktaseaktivität der 11ß-HSD1 unter dem Einfluß von Glucocorticoiden stimuliert. In dieser Arbeit untersuchten wir den in vivo Effekt einer fünftägigen Gabe von 30mg Prednisolon auf die Aktivität der 11ß- HSD1. Unter Prednisolon zeigte sich eine signifikant stärkere Umwandlung von Cortison zu Cortisol sowie ein signifikant höherer Cortisol/Cortison-Quotient im Serum nach oraler Cortisongabe. Eine Induktion der 11ß-HSD1 durch Glucocorticoide scheint daher die ACTH-Antwort des Körpers in Stressituationen zu verstärken. Die Entwicklung selektiver Inhibitoren der 11ß-HSD1 ist von pharmakologischem Interesse zur Therapie von Insulinresistenz und Adipositas. Wir überprüften mit Chenodesoxycholsäure eine potentielle Substanz auf ihre Wirksamkeit in vivo. Es konnte jedoch kein Effekt auf die Reduktaseaktivität der 11ß-HSD1 nachgewiesen werden. Höher selektive und potentere Inhibitoren des Enzyms können vermutlich erst nach Aufklärung der Tertiärstruktur beider Isoenzyme der 11ß-HSD entwickelt werden. Beide von uns etablierten Bioassays eignen sich in sehr guter Weise für die klinische Testung der Aktivität der Isoenzyme der 11ß-HSD. Somit eröffnen die von uns etablierten in vitro - und in vivo - Systeme neue Einblicke in Funktion und Regulation der physiologisch und pathophysiologisch wichtigen 11ß-HSD-Enzyme.The 11ß-hydroxysteroid dehydrogenases (11ß-HSD´s) convert cortisol to its inactive metabolite cortisone and vice versa. 11ß-HSD type 1 (11ß-HSD1) functions as a reductase in vivo, regulating cortisol access to the glucocorticoid receptor. In contrast, 11ß-HSD2 only mediates oxidation of natural glucocorticoids, protecting the mineralocorticoid receptor from high cortisol concentrations. Although 11ß-HSD activities are important determinants for the efficacy of synthetic MCs and GCs as well, corresponding pharmacokinetic data are scanty. Therefore, we characterized 11ß-HSD profiles for a wide range of steroids often used in clinical practice. In vitro measurements: 11ß-HSD1 and 11ß-HSD2 were selectively examined in Chinese hamster ovarian cells stably transfected with 11ß-HSD1 or 11ß-HSD2 expression vectors. Reduction by 11ß-HSD1 was diminished by 16-methyl, 16ß-methyl, 2-methyl, and 2-chlor substitution, whereas it was increased by the 1-dehydro configuration in prednisone, resulting in higher hepatic first pass activation of prednisone compared with cortisone. Oxidation of steroids by 11ß-HSD2 was diminished if they are fluorinated in position 6, 9 (e.g. in dexamethasone) or methylated at 2 or 6 (in methylprednisolone) or 16 or 16ß, by a methylene group at 16 (in prednylidene), methyloxazoline at 16, 17 (in deflazacort), or a 2-chlor configuration. Whereas the methyl groups also decreased reductase activity (steric effects), fluorination increased reductase activity (negative inductive effect). This may explain the strong MC activity of 9-fluorocortisol and should be considered in GC therapy directed to 11ß-HSD2-expressing tissues (kidney, colon, and placentofetal unit). 11ß- HSD2 oxidation of prednisolone is more effective than that of cortisol, explaining the reduced MC activity of prednisolone compared with cortisol. In vivo measurements: As glucocorticoid mediated enhanced acitivity of 11ß-HSD1 seems to play a role in stress adaptation, we furthermore investigated the effect of a short term prednisolone therapy on 11ß-HSD1 activity in healthy males. Reduction of orally ingested cortisone to cortisol (representing activity of 11ß-HSD1) was significantly enhanced after prednisolone therapy. Due to increased conversion of inactive cortisone to active cortisol, the liver seems to be an important cortisol production site in stress situations, besides the adrenals. Recent work shows that obese humans and rodents have increased 11beta-HSD1 activity selectively in adipose tissue. Selective Inhibition of the enzyme may offer new treatment options in metabolic disorders such as diabetes mellitus or syndrome X. As chenodesoxycholic acid selectively inhibits activity of 11ß-HSD1 in vitro, we measured the influence of the substance on reduction of orally ingested cortisone to cortisol, representing activity of 11ß-HSD1. Unfortunately, chenodesoxycholic acid � given in therapeutical dose -did not alter in vivo activity of the enzyme. Newer and more selective 11HSD1 inhibitors are in development. Nevertheless, therapeutic inhibition of 11beta-HSD1 reductase activity in patients with obesity and the metabolic syndrome, as well as in glaucoma , osteoporosis and cognitive dysfunction, remains an exciting prospect. In summary, both, the in vitro as well as the in vivo systems used in this study help to delight physiological, pathophysiological and therapeutical aspects of the 11ß- hydroxysteroid-dehydrogenase system

Cost-effectiveness of Tyrosine Kinase Inhibitor Treatment Strategies for Chronic Myeloid Leukemia in Chronic Phase After Generic Entry of Imatinib in the United States

Background: We analyzed the cost-effectiveness of treating incident chronic myeloid leukemia in chronic phase (CML-CP) with generic imatinib when it becomes available in United States in 2016. In the year following generic entry, imatinib's price is expected to drop 70% to 90%. We hypothesized that initiating treatment with generic imatinib in these patients and then switching to the other tyrosine-kinase inhibitors (TKIs), dasatinib or nilotinib, because of intolerance or lack of effectiveness ("imatinib-first") would be cost-effective compared with the current standard of care: "physicians' choice" of initiating treatment with any one of the three TKIs. Methods: We constructed Markov models to compare the five-year cost-effectiveness of imatinib-first vs physician's choice from a US commercial payer perspective, assuming 3% annual discounting ($US 2013). The models' clinical endpoint was five-year overall survival taken from a systematic review of clinical trial results. Per-person spending on incident CML-CP treatment overall care components was estimated using Truven's MarketScan claims data. The main outcome of the models was cost per quality-adjusted life-year (QALY). We interpreted outcomes based on a willingness-to-pay threshold of$100 000/QALY. A panel of European LeukemiaNet experts oversaw the study's conduct. Results: Both strategies met the threshold. Imatinib-first ($277 401, 3.87 QALYs) offered patients a 0.10 decrement in QALYs at a savings of$88 343 over five years to payers compared with physician's choice ($365 744, 3.97 QALYs). The imatinibfirst incremental cost-effectiveness ratio was approximately$883 730/QALY. The results were robust to multiple sensitivity analyses. Conclusion: When imatinib loses patent protection and its price declines, its use will be the cost-effective initial treatment strategy for CML-CP

Perioperative Pembrolizumab for Early-Stage Non-Small-Cell Lung Cancer

BACKGROUND Among patients with resectable early-stage non-small-cell lung cancer (NSCLC), a perioperative approach that includes both neoadjuvant and adjuvant immune checkpoint inhibition may provide benefit beyond either approach alone.METHODS We conducted a randomized, double-blind, phase 3 trial to evaluate perioperative pembrolizumab in patients with early-stage NSCLC. Participants with resectable stage II, IIIA, or IIIB (N2 stage) NSCLC were assigned in a 1:1 ratio to receive neoadjuvant pembrolizumab (200 mg) or placebo once every 3 weeks, each of which was given with cisplatin-based chemotherapy for 4 cycles, followed by surgery and adjuvant pembrolizumab (200 mg) or placebo once every 3 weeks for up to 13 cycles. The dual primary end points were event-free survival (the time from randomization to the first occurrence of local progression that precluded the planned surgery, unresectable tumor, progression or recurrence, or death) and overall survival. Secondary end points included major pathological response, pathological complete response, and safety.RESULTS A total of 397 participants were assigned to the pembrolizumab group, and 400 to the placebo group. At the prespecified first interim analysis, the median follow-up was 25.2 months. Event-free survival at 24 months was 62.4% in the pembrolizumab group and 40.6% in the placebo group (hazard ratio for progression, recurrence, or death, 0.58; 95% confidence interval [CI], 0.46 to 0.72; P<0.001). The estimated 24-month overall survival was 80.9% in the pembrolizumab group and 77.6% in the placebo group (P = 0.02, which did not meet the significance criterion). A major pathological response occurred in 30.2% of the participants in the pembrolizumab group and in 11.0% of those in the placebo group (difference, 19.2 percentage points; 95% CI, 13.9 to 24.7; P<0.0001; threshold, P = 0.0001), and a pathological complete response occurred in 18.1% and 4.0%, respectively (difference, 14.2 percentage points; 95% CI, 10.1 to 18.7; P<0.0001; threshold, P = 0.0001). Across all treatment phases, 44.9% of the participants in the pembrolizumab group and 37.3% of those in the placebo group had treatment-related adverse events of grade 3 or higher, including 1.0% and 0.8%, respectively, who had grade 5 events.CONCLUSIONS Among patients with resectable, early-stage NSCLC, neoadjuvant pembrolizumab plus chemotherapy followed by resection and adjuvant pembrolizumab significantly improved event-free survival, major pathological response, and pathological complete response as compared with neoadjuvant chemotherapy alone followed by surgery. Overall survival did not differ significantly between the groups in this analysis

Prognostic value of end-of-induction PET response after first-line immunochemotherapy for follicular lymphoma (GALLIUM): secondary analysis of a randomised, phase 3 trial

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