Hamburg, Univ., Diss., 2005. / Erscheinungsjahr an der Haupttitelstelle: 2004. Computerdatei im Fernzugriff.

Hamburg, Universiẗat, Diss., 2005. / Erscheinungsjahr an der Haupttitelstelle: 2004.

類胰島素生長因子1(IGF1)是一種負責代謝、細胞生長、身體發展的多肽激素。微衛星和單核苷酸多態性(SNP), 與循環IGF1水平顯著相關。然而，目前沒有研究指出微衛星和SNPs的綜合影響，且這些遺傳變異對IGF1的調控機制仍是未知。本研究的假設是，微衛星和SNPs在啟動子區域可能有相互作用從而調控IGF1水平。因此，本研究的目的是找出影響IGF1表達的主要元素，並研究每個基因變異的作用。 / 在這項研究中，整個IGF1的基因，包括上游和下游的5萬鹼基對(bp)，可分為4個單體型區段，而IGF1的調控區在第3和第4個區段。與其它重復序列的微衛星相比，有21個重復序列的微衛星(IGF1上游969bp)與一套獨特的SNPs有關連。此外，有19個重復序列的微衛星有較低的循環IGF1。 / 功能性細胞分析進一步分析在生長激素(GH)依賴模型和GH獨立模型中，每個基因變異的角色。在GH獨立模型中，常見的單體型之間有不同的轉錄活性。與以前的研究結果相一致的是，有19個重複序列的單倍型轉錄活性最低。當單倍型為C-T-T，啓動子的轉錄活性受微衛星長度影響，較長的單體型有較低的轉錄活性。微衛星的長度效應或倚賴功能性SNP 1411C> T（rs35767）和叉頭蛋白A3（FOXA3）。以前研究發現在不同基因調控中，一個只結合C等位點並含有CCAAT /增強子結合蛋白delta（CEBPD）的轉錄激活複合物與FOXA3並存。因此，CEBPD可能與FOXA3相互作用從而調控IGF1的表達。而微衛星長度可能通過調節上游CEBPD轉錄複雜和下游FOXA3的相互作用從而影響IGF1的表達。單倍型T-C-A可能採取另一種調控機制，該機制或許被長約178鹼基對，含有“CA“部分的片段調控。GH依賴模型是模擬幼年時期IGF1的表達。在這個階段中，常見的單體型之間有不同的轉錄活性，但每個基因變異的調節作用均不強。 / 總括而言，IGF1的表達主要是由微衛星和SNPs組成的單體型調控。在幼年和成年，常見的單體型之間有差別顯著的轉錄活動。然而，GH獨立模型和GH依賴模型的調控機制是不同的。 / Insulin-like growth factor 1 (IGF1) is a polypeptide hormone responsible for metabolism, cell growth, and somatic development. Microsatellite and SNPs have been demonstrated to be significantly associated with circulating IGF1 level. However, no studies have ever investigated the combined effects of microsatellite and SNPs, and regulatory mechanisms of IGF1 expression by these genetic variants are yet unknown. The hypotheses of this study were that the microsatellite and SNPs may have certain regulatory functions in the promoter region, and interact with each other in the regulation. Therefore, the objectives were to identify the primary regulatory element in the regulation of IGF1 expression and to investigate the role of each genetic variant. / In this study, the whole IGF1 gene, including 50kb upstream and downstream, was divided into four haplotype blocks, in which the regulatory region of IGF1 lied in haploblock 3 and 4. Results of high-resolution melting analysis showed that a microsatellite (969bp upstream) with 21 repeats was associated with a different set of SNPs, compared to microsatellite with other repeat numbers. Also, haplotype with 19 CA repeats was significantly associated with a lower level of circulating IGF1. / Functional cellular assays were performed to further analyze the roles of each genetic variant in growth hormone (GH)-independent and GH-dependent models. In GH-independent model, it was found that common haplotypes showed differential transcriptional activities, and, consistent with previous findings, haplotype with 19 repeats was the least activated. On the background of haplotype C-T-T, transcriptional activity was regulated by microsatellite length, in which the haplotype with a longer microsatellite length tended to have a lower transcriptional activity. Further analysis showed that the microsatellite length effect depended on a functional polymorphism -1411C>T (rs35767) and forkhead box A3 (FOXA3), whose binding sites were several base pairs upstream of IGF1 transcription start site. Telgmann et al found a transcription activator complex containing CCAAT/enhancer binding protein delta (CEBPD) bound exclusively to the C allele and CEBPD often coexisted with FOXA3 in the regulation of various genes. Therefore, in the activation of IGF1, microsatellite length might regulate the interaction between the upstream CEBPD transcription complex and the downstream FOXA3. Haplotype T-C-A showed a yet unknown regulatory mechanism of IGF1 expression, which might be accounted for by the “C-A“ portion. In GH-dependent model, common haplotypes also showed differential transcriptional activities. However, further analysis revealed that the regulatory effects of each genetic variant alone (microsatellite or SNPs) were not strong. / To conclude, haplotype effect, which was contributed by both microsatellite and SNPs, played an important role in the regulation of IGF1 expression. Common haplotypes showed significantly differential transcriptional activities. However, the regulatory mechanisms were different in GH-independent model and GH-dependent model. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Yu Holly. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-140). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / LIST OF ABBREVIATIONS --- p.vi / LIST OF FIGURES --- p.viii / LIST OF TABLES --- p.x / PUBLICATIONS --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION TO INSULIN-LIKE GROWTH FACTOR 1 (IGF1) --- p.1 / Chapter 1.1 --- Production of IGF1 --- p.1 / Chapter 1.2 --- Other factors affecting IGF1 level --- p.5 / Chapter 1.2.1 --- Nutritional status --- p.5 / Chapter 1.2.2 --- Ethnicity --- p.5 / Chapter 1.2.3 --- Age --- p.8 / Chapter 1.2.4 --- Gender --- p.8 / Chapter 1.2.5 --- IGFBP --- p.9 / Chapter 1.2.6 --- Other growth factors --- p.10 / Chapter 1.3 --- Cellular functions of IGF1 --- p.10 / Chapter 1.4 --- Physiological functions of IGF1 --- p.13 / Chapter 1.4.1 --- Metabolism --- p.14 / Chapter 1.4.2 --- Somatic growth --- p.17 / Chapter 1.4.3 --- Longevity --- p.18 / Chapter CHAPTER 2 --- PATHOLOGY OF IGF1 --- p.20 / Chapter 2.1 --- IGF1 and cancer predisposition --- p.20 / Chapter 2.1.1 --- Evidences in genetic studies --- p.21 / Chapter 2.1.2 --- Evidences in lifestyle factors --- p.21 / Chapter 2.1.3 --- Evidences from population studies --- p.22 / Chapter 2.1.4 --- Miscellaneous evidence --- p.22 / Chapter 2.2 --- IGF1 and diabetes mellitus (DM) --- p.23 / Chapter 2.3 --- IGF1 and other diseases --- p.24 / Chapter CHAPTER 3 --- HYPOTHESES AND AIMS OF THE STUDY --- p.26 / Chapter 3.1 --- Hypotheses of the study --- p.26 / Chapter 3.2 --- Aims of the study --- p.26 / Chapter CHAPTER 4 --- RELATIONSHIP BETWEEN GENETIC VARIANTS AND IGF1 EXPRESSION --- p.28 / Chapter 4.1 --- Introduction --- p.28 / Chapter 4.2 --- Materials and methods --- p.31 / Chapter 4.2.1 --- Study subjects --- p.31 / Chapter 4.2.2 --- tagSNP selection and haplotype block construction --- p.32 / Chapter 4.2.3 --- Genescan analysis of the CA repeat microsatellite --- p.32 / Chapter 4.2.4 --- Genotyping assay of tagSNPs --- p.34 / Chapter 4.2.5 --- Statistical analysis --- p.36 / Chapter 4.3 --- Results --- p.37 / Chapter 4.3.1 --- Characteristics of the subjects --- p.37 / Chapter 4.3.2 --- Determination of haplotype blocks --- p.38 / Chapter 4.3.3 --- Selection of tagSNPs --- p.41 / Chapter 4.3.4 --- Genotyping analysis of tagSNPs --- p.43 / Chapter 4.3.5 --- Genescan analysis of -969bp CA repeat microsatellite --- p.46 / Chapter 4.3.6 --- Phased haplotype consisting of SNP / SNP and microsatellite --- p.48 / Chapter 4.3.7 --- Correlation between haplotypes in IGF1 promoter and circulating IGF1 level --- p.50 / Chapter 4.4 --- Discussion --- p.53 / Chapter CHAPTER 5 --- Transcriptional regulation of GENETIC VARIANTS IN different haplotypeS --- p.57 / Chapter 5.1 --- Introduction --- p.57 / Chapter 5.1.1 --- IGF1 gene structure --- p.57 / Chapter 5.1.2 --- Regulatory elements in IGF1 promoter --- p.58 / Chapter 5.1.3 --- Functional variant -1411C>T (rs35767) in IGF1 promoter --- p.60 / Chapter 5.1.5 --- Objectives of the study --- p.62 / Chapter 5.2 --- Materials and methods --- p.64 / Chapter 5.2.1 --- Comparative genomics --- p.64 / Chapter 5.2.2 --- Study subjects --- p.64 / Chapter 5.2.3 --- tagSNP selection and genotyping assay --- p.64 / Chapter 5.2.4 --- Primers and standard polymerase chain reaction (PCR) --- p.65 / Chapter 5.2.5 --- Enzyme digestion --- p.68 / Chapter 5.2.6 --- Ligation --- p.68 / Chapter 5.2.7 --- Transformation of DNA ligation products --- p.68 / Chapter 5.2.8 --- Preparation of E.coli supercompetent cells --- p.69 / Chapter 5.2.9 --- Construction of plasmids --- p.70 / Chapter 5.2.10 --- Cell lines --- p.71 / Chapter 5.2.11 --- Nucleic acid extraction --- p.72 / Chapter 5.2.12 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.73 / Chapter 5.2.13 --- Transient transfection --- p.73 / Chapter 5.2.14 --- Luciferase reporter assay --- p.73 / Chapter 5.2.15 --- Optimization of a saturated luciferase reporter system --- p.74 / Chapter 5.2.16 --- Electrophoretic mobility shift assay (EMSA) --- p.74 / Chapter 5.2.17 --- Western blot analysis --- p.74 / Chapter 5.2.18 --- Prediction of putative functional SNPs --- p.76 / Chapter 5.2.19 --- Statistical analysis --- p.77 / Chapter 5.3 --- Results --- p.77 / Chapter 5.3.1 --- Evolutionarily conserved region (ECR) --- p.77 / Chapter 5.3.2 --- Frequency distribution of haplotypes of IGF1 promoter in the Chinese population --- p.79 / Chapter 5.3.3 --- Optimization of luciferase reporter system --- p.81 / Chapter 5.3.3.1 --- Gene expression level of different cell lines --- p.81 / Chapter 5.3.3.2 --- Cell line selection --- p.81 / Chapter 5.3.3.3 --- Saturation of expression plasmids in the luciferase reporter system --- p.83 / Chapter 5.3.3.4 --- Western blot analysis of gene expression level after transfection --- p.86 / Chapter 5.3.4 --- Possible functional SNPs in IGF1 regulatory region beyond ECR --- p.89 / Chapter 5.3.4.1 --- In silico analysis of putative functional SNPs --- p.89 / Chapter 5.3.4.2 --- Binding capacity of possible functional SNPs --- p.92 / Chapter 5.3.5 --- Transcriptional activities of common haplotypes and their derivatives --- p.95 / Chapter 5.3.5.1 --- GH-independent (GH-) model --- p.95 / Chapter 5.3.5.1.1 --- Common haplotypes --- p.95 / Chapter 5.3.5.1.2 --- Effect of microsatellite length on transcriptional activity of IGF1 promoter --- p.97 / Chapter 5.3.5.1.3 --- Effect of SNP on transcriptional activity of IGF1 promoter --- p.99 / Chapter 5.3.5.1.4 --- Summary --- p.101 / Chapter 5.3.5.2 --- GH-dependent (GH+) model --- p.101 / Chapter 5.3.5.2.1 --- Common haplotypes --- p.101 / Chapter 5.3.5.2.2 --- Effect of microsatellite length on transcriptional activity of IGF1 promoter --- p.103 / Chapter 5.3.5.2.3 --- Effect of SNP on transcriptional activity of IGF1 promoter --- p.105 / Chapter 5.3.5.2.4 --- Summary --- p.106 / Chapter 5.3.6 --- Putative mechanism of the interaction between microsatellite and SNPs --- p.106 / Chapter 5.3.6.1 --- Microsatellite length effect in C-T-T haplotype relied on rs35767 (-1411C>T) --- p.107 / Chapter 5.3.6.2 --- The interaction of SNP and microsatellite was dependent on FOXA3 --- p.110 / Chapter 5.3.6.3 --- Summary --- p.112 / Chapter 5.3.7 --- Serial deletion of IGF1 promoter fragment --- p.112 / Chapter 5.4 --- Discussion --- p.116 / Chapter 5.4.1 --- Distal regulatory mechanism of IGF1 expression --- p.116 / Chapter 5.4.2 --- Localized regulatory mechanism of IGF1 expression --- p.117 / Chapter CHAPTER 6 --- CONCLUSIONS AND FUTURE STUDIES --- p.125 / Chapter 6.1. --- Conclusions --- p.125 / Chapter 6.2. --- Future studies --- p.126 / Reference --- p.127

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references (p. 56-64).

No description available.

No description available.

No description available.

No description available.

acase@tulane.edu / The goal of the following experiments was to determine the cellular mechanisms through which estrogen receptor activity is maintained in hippocampal cells following termination of ovarian function. Aim 1 determined that kinase signaling contributes to the maintenance of estrogen receptor activity in the hippocampus of ovariectomized mice in addition to local synthesis of brain derived “neuroestrogens”. Inhibition of both the mitogen activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K) cascades with intracerebroventricular infusion of specific kinase inhibitors reduced estrogen response element (ERE)-dependent gene expression in the hippocampus of ovariectomized mice. Aim 2 determined that neuroestrogen synthesis, MAPK signaling, and PI3K signaling interact to regulate the transcriptional output of estrogen receptors in response to insulin like growth factor-1 receptor (IGF-1R) activation in the Neuro-2A cell culture model. Rapid IGF-1R-dependent MAPK signaling promotes, while PI3K signaling inhibits, IGF-1R-dependent activation of endogenous estrogen receptors in Neuro-2A cells. Long-term IGF-1R stimulation reduces ERE-dependent gene expression in part through phosphorylation of estrogen receptor alpha (ERα). Rapid IGF-1R-dependent activation but not long-term repression of estrogen receptor activity in Neuro-2A cells requires neuroestrogen synthesis. Aim 3 determined that exposure to 40 days of continuous unopposed estradiol at the time of ovariectomy results in lasting enhancement of estrogen receptor activity in the hippocampus and lasting enhancement of hippocampus dependent memory in female mice beyond the period of short-term estradiol exposure. Together these three aims determine that neuroestrogen synthesis and kinase signaling interact to actively maintain estrogen receptor signaling in neuronal cells and these autonomous neuronal mechanisms of estrogen receptor activation have functional consequences on cognition long after cessation of ovarian function. / 1 / Kevin J Pollard

SUMMARY Insulin-like growth factor I (IGF-I) is a polypeptide with a molecular weight of 7.649 kDa and an anabolic potential. Thereby, IGF-I has a promising therapeutic value e.g. in muscle wasting diseases such as sarcopenia. IGF-I is mainly secreted by the liver in response to growth hormone (GH) stimulation and is rather ubiquitously found within all tissues. The effects of IGF-I are mediated by its respective IGF-I transmembrane tyrosine kinase receptor triggering the stimulation of protein synthesis, glucose uptake and the regulation of cell growth. The actions of IGF-I are modulated by six IGF binding proteins binding and transporting IGF-I in a binary or ternary complex to tissues and receptors and modulating the binding of IGF-I to its receptor. The nature of the formed complexes impacts IGF-I`s half-life, modulating the half-life between 10 minutes (free IGF-I) to 12 - 15 hours when presented in a ternary complex with IGF binding protein 3 and an acid labile subunit (ALS). Therefore, sustained drug delivery systems of free IGF-I are superficially seen as interesting for the development of controlled release profiles, as the rate of absorption is apparently and easily set slower by simple formulation as compared to the rapid rate of elimination. Thereby, one would conclude, the formulation scientist can rapidly develop systems for which the pharmacokinetics of IGF-I are dominated by the formulation release kinetics. However, the in vivo situation is more complex and as mentioned (vide supra), the half-life may easily be prolonged up to hours providing proper IGF-I complexation takes place upon systemic uptake. These and other aspects are reviewed in Chapter I, within which we introduce IGF-I as a promising therapeutic agent detailing its structure and involved receptors along with the resulting signaling pathways. We summarize the control of IGF-I pharmacokinetics in nature within the context of its complex system of 6 binding proteins to control half-life and tissue distribution. Furthermore, we describe IGF-I variants with modulated properties in vivo and originated from alternative splicing. These insights were translated into sophisticated IGF-I delivery systems for therapeutic use. Aside from safety aspects, the challenges and requirements of an effective IGF-I therapy are discussed. Localized and systemic IGF-I delivery strategies, different routes of administration as well as liquid and solid IGF-I formulations are reviewed. Effective targeting of IGF-I by protein decoration is outlined and consequently this chapter provides an interesting guidance for successful IGF-I-delivery. In Chapter II, we firstly outline the stability of IGF-I in liquid formulations with the intention to deliver the biologic through the lung and the impact of buffer type, sodium chloride concentration and pH value on IGF-I stability is presented. IGF-I integrity was preserved in histidine buffer over 4 months at room temperature, but methionine 59 oxidation (Met(o)) along with reducible dimer and trimer formation was observed in an acidic environment (pH 4.5) and using acetate buffer. Strong aggregation resulted in a complete loss of IGF-I bioactivity, whereas the potency was partly maintained in samples showing a slight aggregation and complete IGF-I oxidation. Atomization by air-jet or vibrating-mesh nebulizers yielded in limited Met(o) formation and no aggregation. The results of IGF-I nebulization experiments regarding aerosol output rate, mass median aerodynamic diameter and fine particle fraction were comparable with 0.9% sodium chloride reference, approving the applicability of liquid IGF-I formulations for pulmonary delivery. In Chapter III we escalated the development to solid delivery systems designed for alveolar landing upon inhalation and by deploying trehalose and the newly introduced for pulmonary application silk-fibroin as carriers. Microparticles were produced using nano spray drying following analyses including IGF-I integrity, IGF-I release profiles and aerodynamic properties. In vitro transport kinetics of IGF-I across pulmonary Calu-3 epithelia were suggesting similar permeability as compared to IGF-I’s cognate protein, insulin that has already been successfully administered pulmonary in clinical settings. These in vivo results were translated to an ex vivo human lung lobe model. This work showed the feasibility of pulmonary IGF-I delivery and the advantageous diversification of excipients for pulmonary formulations using silk-fibroin. Chapter IV focuses on an innovative strategy for safe and controllable IGF-I delivery. In that chapter we escalated the development to novel IGF-I analogues. The intention was to provide a versatile biologic into which galenical properties can be engineered through chemical synthesis, e.g. by site directed coupling of polymers to IGF-I. For this purpose we genetically engineered two IGF-I variants containing an unnatural amino acid at two positions, respectively, thereby integrating alkyne functions into the primary sequence of the protein. These allowed linking IGF-I with other molecules in a site specific manner, i.e. via a copper catalyzed azide-alkyne Huisgen cycloaddition (click reaction). In this chapter we mainly introduce the two IGF-I variants, detail the delivery concept and describe the optimization of the expression conditions of the IGF-I variants. In conclusion, we span from simple liquid formulations for aerolization through solid systems for tailored for maximal alveolar landing to novel engineered IGF-I analogues. Thereby, three strategies for advanced IGF-I delivery were addressed and opportunities and limitations of each were outlined. Evidence was provided that sufficiently stable and easy to manufacture formulations can be developed as typically required for first in man studies. Interestingly, solid systems – typically introduced in later stages of pharmaceutical development – were quite promising. By use of silk-fibroin as a new IGF-I carrier for pulmonary administration, a new application was established for this excipient. The demonstrated success using the ex vivo human lung lobe model provided substantial confidence that pulmonary IGF-I delivery is possible in man. Finally, this work describes the expression of two IGF-I variants containing two unnatural amino acids to implement an innovative strategy for IGF-I delivery. This genetic engineering approach was providing the fundament for novel IGF-I analogues. Ideally, the biologic is structurally modified by covalently linked moieties for the control of pharmacokinetics or for targeted delivery, e.g. into sarcopenic muscles. One future scenario is dicussed in the ‘conclusion and outlook’ section for which IGF-I is tagged to a protease sensitive linker peptide and this linker peptide in return is coupled to a polyethylenglykole (PEG) polymer (required to prolong the half-life). Some proteases may serve as proxy for sarcopenia such that protease upregulation in compromised muscle tissues drives cleavage of IGF-I from the PEG. Thereby, IGF-I is released at the seat of the disease while systemic side effects are minimized. / ZUSAMMEMFASSUNG Insulin-like growth factor I (IGF-I) ist ein 7.6 kDa großes Polypeptid, das eine anabole Wirkung besitzt und dadurch ein vielversprechendes Therapeutikum in Muskelerkrankungen wie z.B. Sarkopenie darstellt. IGF-I wird hauptsächlich von der Leber gebildet und infolge der Stimulation des Wachstumshormons Somatropin sezerniert. In fast jedem Gewebe des Körpers kommt IGF-I vor. Die Wirkungen von IGF-I werden über eigene Rezeptoren, die an die Zellmembran gebunden sind, die Rezeptor-Tyrosinkinasen, ausgeführt. Zu den Wirkungen gehören unter anderem die Stimulation der Proteinsynthese, die Aufnahme von Glucose in die Zellen und die Regulierung des Zellwachstums. Die Effekte von IGF-I werden von 6 IGF- Bindungsproteinen (IGFBP 1-6) gesteuert, indem IGF-I in einem binären oder ternären Komplex zu den Geweben transportiert oder auch die Bindung von IGF-I an den Rezeptor verhindert werden kann. Die sich bildenden Komplexe haben auch einen Einfluss auf die Halbwertszeit (HWZ) von IGF-I, da für ungebundenes IGF-I eine HWZ von ca. 10 Minuten festgestellt werden konnte, aber IGF-I, gebunden in einem ternären Komplex mit dem Bindungsprotein 3 und der säurelabilen Untereinheit (ALS) eine erhöhte HWZ von 12 – 15 Stunden aufweist. Deswegen sind „sustained drug delivery“ Systeme von ungebundenem IGF-I auf den ersten Blick interessant für die Entwicklung von kontrollierten Freisetungsprofilen, da die Absorptionsgeschwindigkeit offensichtlich und problemlos durch triviale Formulierung verlangsamt werden kann im Vergleich zu der schnellen Eliminationsgeschwindigkeit. Deshalb könnte man daraus schließen, dass ein Formulierungsexperte recht schnell Systeme entwickeln kann, in denen die Freisetzungskinetik der Formulierung über die pharmakokinetischen Eigenschaften von IGF-I dominiert. Jedoch ist die in vivo Situation wesentlich komplexer und wie oben bereits erwähnt, könnte die Halbwertszeit problemlos bis zu mehreren Stunden verlängert werden, sofern geeignete Komplexbildung von IGF-I nach systemischer Aufnahme erfolgt. Diese und weitere Aspekte werden in Kapitel I beschrieben. Außerdem stellen wir IGF-I als wertvolles Therapeutikum vor, beschreiben dessen Struktur, die beteiligten Rezeptoren und die daraus resultierenden Signalwege. Wir fassen die Kontrolle der Pharmakokinetik von IGF-I in der Natur zusammen, im Rahmen von einem komplexen System aus 6 Bindungsproteinen, die die Halbwertszeit und die Gewebeverteilung steuern. Außerdem beschreiben wir IGF-I Varianten, die veränderte Eigenschaften in vivo aufweisen und durch alternatives Spleißen entstanden sind. Diese Erkenntnisse werden in hochentwickelte „IGF-I delivery“ Systeme für den therapeutischen Gebrauch umgesetzt. Neben Sicherheitsaspekten werden die Herausforderungen und Anforderungen einer effektiven IGF-I Therapie diskutiert. Darüber hinaus wird über lokale und systemische „IGF-I delivery“ Strategien, verschiedene Verabreichungswege sowie flüssige und feste IGF-I Formulierungen berichtet. Ebenso wird die wirkungsvolle IGF-I Freisetzung am Zielort durch Ausschmückung des Proteins beschrieben und dementsprechend liefert dieses Kapitel eine interessante Orientierungshilfe für eine erfolgreiche IGF-I Therapie. Im Kapitel II untersuchen wir die Stabilität von IGF-I in flüssigen Formulierungen zur pulmonalen Anwendung bezüglich Puffersystem, Natriumchlorid Konzentration und pH Wert. Die IGF-I Integrität wurde im Histidin Puffer über 4 Monate bei Raumtemperatur aufrechterhalten. Allerdings wurde bei Verwendung eines Acetat Puffers pH 4.5, Oxidation am Methionin 59 (Met(o)) sowie die Entstehung von reduzierbaren Dimeren und Trimeren beobachtet. Starke Aggregation führte zum vollständigen Verlust der IGF-I Bioaktivität, während die Wirkung in Proben aufrechterhalten werden konnte, in denen eine geringe Aggregation, aber deutliche Oxidation festgestellt wurde. Nach der Verneblung der flüssigen IGF-I Formulierung im Histidin-Puffer pH 6.5 mit einem Druckluftvernebler und einem Schwingmembranvernebler wurde jeweils eine leichte Bildung von Met(o), aber keine Aggregatbildung ermittelt. Die Ergebnisse der IGF-I Verneblungsexperimente waren vergleichbar mit den Referenzwerten einer isotonischen Kochsalzlösung bezüglich der Abgabeleistung, dem massenbezogenen medianen aerodynamischen Durchmesser und dem Feinpartikel Anteil. Hierdurch wurde gezeigt, dass sich flüssige IGF-I Formulierungen zur pulmonalen Anwendung eignen. Im Kapitel III berichten wir von einer Weiterentwicklung zu festen IGF-I Formulierungen für die pulmonale Route unter Verwendung von Trehalose und Seidenfibroin als neues Trägermaterial für die pulmonale Applikation. Mikropartikel wurden durch Nanosprühtrocknung hergestellt und anschließend auf IGF-I Integrität, IGF-I Freisetzung und dem aerodynamischen Durchmesser untersucht. Die Kinetik des in vitro Transportes von IGF-I durch Calu-3 Lungenepithelzellen war vergleichbar zur Durchgängigkeit von Insulin, das bereits erfolgreich pulmonal verabreicht wurde. Dieser Erfolg wurden auch ex vivo in einem menschlichen Lungenlappen Model bestätigt. In der Arbeit wird somit gezeigt, dass IGF-I zur pulmonalen Anwendung geeignet ist und die Verwendung von Seidenfibroin eine nützliche Erweiterung zu den bisher eingesetzten Trägermaterialien darstellt. Das Kapitel IV konzentriert sich auf eine innovative Strategie, um IGF-I sicher und kontrollierbar zu verabreichen. In diesem Kapitel erweitern wir die Entwicklung zu neuartigen IGF-I Varianten. Wir streben damit an ein vielseitiges Biologikum zu entwickeln, dessen Eigenschaften durch chemische Reaktionen verändert werden können wie zum Beispiel die spezifische Verknüpfung mit Polymeren. Zu diesem Zweck erzeugten wir gentechnisch zwei IGF-I Varianten, die jeweils an zwei Positionen eine unnatürliche Aminosäure aufweisen und führten dadurch Alkine Gruppen in die Primärstruktur der Proteine ein. Diese Vorgehensweise ermöglicht es nun IGF-I mit anderen Molekülen positionsspezifisch zu verbinden wie zum Beispiel durch die kupferkatalysierte Azid-Alkin-Cycloaddition (Click – Reaktion). In diesem Kapitel stellen wir hauptsächlich die zwei IGF-I Varianten vor, beschreiben ausführlich das Konzept der IGF-I Zustellung und erklären die Vorgehensweise zur Optimierung der Expressionsbedingungen der IGF-I Varianten. Abschließend lässt sich sagen, dass sich diese Arbeit über einfach flüssige Formulierungen zur Verneblung, feste Formulierung mit guten aerodynamischen Eigenschaften zur Erreichung der Alveolen und neuartig entwickelte IGF-I Varianten erstreckt. Hierzu werden drei Strategien zur modernen IGF-I Gabe thematisiert und sowohl die Möglichkeiten als auch die Grenzen der jeweiligen Therapie erörtert. Wir haben den Nachweis erbracht, dass ausreichend stabile und leicht herzustellende Formulierungen entwickelt werden können, die üblicherweise für „First-In-Man“ Studien benötigt werden. Interessanterweise stellten sich die festen Formulierungen, die eigentlich in den späteren Phasen der pharmazeutischen Entwicklung eingeführt werden, als sehr vielversprechend heraus. Durch den Einsatz von Seidenfibroin als neuen Träger zur pulmonalen Anwendung haben wir einen neuen Verwendungszweck für Seidenfibroin etabliert. Der erfolgreiche Versuch ex vivo am menschlichen Lungenlappen Model liefert die feste Überzeugung, dass es möglich ist, IGF-I im Menschen pulmonal anzuwenden. Letztendlich, beschreibt die Arbeit die Expression von zwei IGF-I Varianten, die zwei unnatürliche Aminosäuren aufweisen, um eine neuartige Strategie zur IGF-I Verabreichung umzusetzen. Dieser gentechnische Ansatz liefert die Grundlage für neue IGF-I Varianten. Idealerweise, wird das Biopharmazeutikum strukturell durch kovalent gebundene Reste verändert, um die pharmakokinetischen Eigenschaften zu steuern oder um zielgenaue Wirkstoffabgabe zu erreichen zum Beispiel in den sarkopenischen Muskeln. Ein Zukunftsszenarium wird im Abschnitt „Conclusion and Outlook“ diskutiert, in dem IGF-I mit einem Protease empfindlichen Linker versehen wird, der wiederum mit einem Polyethylenglykol (PEG) Polymer verknüpft ist. Der PEG Rest wird benötigt, um die Hablbwertszeit von IGF-I zu erhöhen. Einige Proteasen könnten als Stellvertreter für Sarkopenie dienen, so dass die Hochregulierung der Proteasen in gefährdeten Muskelgeweben zur Spaltung von IGF-I und dem PEG Rest führt. Dadurch wird IGF-I am Ursprung der Erkrankungen freigesetzt, während die systemischen Nebenwirkungen weitgehend vermindert sind.