Oral uygulama amacıyla insülin taşıyıcı sistemlerin hazırlanması, karakterizasyonu ve salım çalışmaları
Küçük Resim Yok
Tarih
2015
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Uluslararası Diyabet Federasyonunun 2013 yılı verilerine göre tüm dünyada 382 milyon kişi diyabet hastasıdır ve her sene dünya sağlık harcamalarının 548 milyon dolarlık kısmı diyabete harcanmaktadır. İnsüline bağımlı diyabet hastaları her gün tekrarlayan dozlarda enjeksiyon uygulamasına maruz kalmaktadır. Biyoteknolojideki gelişmeler birçok proteinin terapötik amaçlı kullanımının önünü açmıştır. Diabetes Mellitus tedavisinde kullanılan insülin bunlardan bir tanesidir. Protein yapısındaki bir ilaç korunmadığı sürece mide ve bağırsaktaki proteaz etkinlikleri, midedeki asidik pH, mukus, proteaz içeren mikrovilluslar gibi fiziksel bariyerler nedeniyle oral biyoyararlanımı %1-2 'den daha düşüktür. Bu nedenle proteinlerin oral uygulanmasını sağmak için farklı formülasyon stratejileri geliştirilmiştir. Bu tezde, insülin taşınmasında oral olarak uygulanabilecek bir mikroemülsiyon sistemi geliştirilmesi hedeflenmiştir. Bu amaçla yapılan ön formülasyon denemeleri sonucunda yağ olarak Plurol Oleik, sürfaktan olarak Tween 80 ve Lesitin, kosürfaktan olarak etanol içeren S/Y mikroemülsiyon sistemi geliştirilmiştir. Rekombinant insülin solüsyonu (100 IU/mL) formülasyona iç faz olarak ilave edilmiştir. Hazırlanan insülin taşıyıcı mikroemülsiyon sistemini daha kararlı hale getirmek ve insülini mide asiditesi ve enzimlerinden korumak için formülasyonun iç fazına farklı oranlarda kitozan ve sodyum tripolifosfat çözeltileri ilave edilmiştir. Elde elde edilen formülasyonların karakterizasyonları yapılmış, fizikokimyasal özellikleri incelenmiştir. Stabilite çalışmalarında +4?C' de 6 ay boyunca stabil kaldıkları gözlenmiştir. Sirküler dikroizm ile yapılan analizlerde insülinin konformasyonel stabilitesinin kitozan ve sodyum tripolifosfat ilavesinden ve hazırlama koşullarınmdan etkilenmediği gösterilmiştir. Ayrıca, formülasyonların farklı pH değerlerinde mide ve bağırsak vasatında gösterdikleri salım özellikleri incelenmiştir. Buna göre midede insülini en çok koruyan ve bağırsakta en yüksek oranda insülin salımı sağlayan formülasyon serisi ile çalışmalara devam edilmiştir. Bu formülasyonlar ile L929 fare fibroblast hücre hattında toksisite testleri yapılmıştır ve bu hücreler üzerine olan toksik dozlar saptanmıştır. Wistar Albino sıçanlar ile in vivo hayvan deneyleri gerçekleştirilmiştir. Çalışmada, sıçanlara intragastrik olarak strandart insülin çözeltisi, iç fazda sadece insülin içeren mikroemülsiyon formülasyonu ve kitozan ile sodyum tripolifosfat içeren optimum mikroemülsiyon formülasyonu uygulanmıştır. Geliştirilen optimum formülasyon, uygulamayı takiben 1. saatten 8. saate kadar etkin bir kan glukoz düzeyi düşüşü sağlamıştır. Kan glukoz düzeyinde elde edilen bu düşüş uygulamadan 8 saat sonrasına kadar diğer gruplara göre anlamlı bulunmuştur. Sonuç olarak, tez kapsamında geliştirilen ve iç fazında insülin, kitozan ve sodyum tripolifosfat solüsyonlarını içeren S/Y mikroemülsiyon sistemleri insülinin oral yolla uygulanması için alternatif bir dozaj formu olarak önerilmektedir
According to the International Diabetes Federation's data in 2013, 382 million people around the world are suffering from Diabetes Mellitus and 548 million dollars of the world's health expenditure is spent on diabetes each year. Patients with insulin dependent Diabetes Mellitus are exposed to repeatedly injection administration each day. Developments in biotechnology has opened the opportunity for therapeutic use of many proteins. Insulin, which is used in the treatment of diabetes mellitus, is one of them. Oral bioavailability of an unprotected protein drug is less than 1-2% due to the gastric and intestinal protease activity and physical barriers like acidic gastric pH, mucus, and protease-containing microvilli. For this reason, versatile formulation strategies are needed in order to provide oral applicability to proteins. In our thesis, it was aimed to develop microemulsion system which is, applicable for oral insulin delivery. For this purpose, in the preformulation experiments a water-in-oil microemulsion system which is composed of Plurol Oleique as the oil, Tween80 and Lecithin as surfactants, etanol as co-surfactant, was developed. A solution of recombinant insulin (100 IU/mL) was added as the internal water phase. Different ratios of chitosan and sodium tripolyphosphate were examined for the inner phase of the microemulsion to obtain more stable system, with higher protective function against acidic and enzymatic degradation. The obtained formulations were characterized and their physicochemical properties were investigated. According to stability studies, formulations were stable at +4?C for 6 months. Circular dichroism analyses revealed that conformational stability of insulin is not affected by the preparation conditions and the presence of chitosan and/or sodium tripolyphosphate. Furthermore, release properties of formulations were investigated under different pH levels, to simulate gastric and intestinal conditions. According to these studies, further experiments were carried out with the formulations which provided greater gastric protection and higher intestinal insulin release. Toxicity studies on L929 mouse fibroblast cells were performed with these formulations and toxic doses on these cells were determined. In vivo experiments were carried out with Wistar Albino rats. In these experiments standard insulin solution, microemulsion containing only insulin and optimal microemulsion which contains chitosan and sodium tripolyphosphate were administered intragastrically to the animals. The optimal formulation provided effective reduction on blood glucose level from the 1st to the 8th hour after administration. The reduction on blood glucose level was statistically significant at the 8th hour, as compared to the other groups. In conclusion, the developed water-in-oil microemulsion system containing insulin, chitosan and sodium tripolyphosphate in the inner phase may be suggested as an alternative dosage forms for oral insulin delivery.
According to the International Diabetes Federation's data in 2013, 382 million people around the world are suffering from Diabetes Mellitus and 548 million dollars of the world's health expenditure is spent on diabetes each year. Patients with insulin dependent Diabetes Mellitus are exposed to repeatedly injection administration each day. Developments in biotechnology has opened the opportunity for therapeutic use of many proteins. Insulin, which is used in the treatment of diabetes mellitus, is one of them. Oral bioavailability of an unprotected protein drug is less than 1-2% due to the gastric and intestinal protease activity and physical barriers like acidic gastric pH, mucus, and protease-containing microvilli. For this reason, versatile formulation strategies are needed in order to provide oral applicability to proteins. In our thesis, it was aimed to develop microemulsion system which is, applicable for oral insulin delivery. For this purpose, in the preformulation experiments a water-in-oil microemulsion system which is composed of Plurol Oleique as the oil, Tween80 and Lecithin as surfactants, etanol as co-surfactant, was developed. A solution of recombinant insulin (100 IU/mL) was added as the internal water phase. Different ratios of chitosan and sodium tripolyphosphate were examined for the inner phase of the microemulsion to obtain more stable system, with higher protective function against acidic and enzymatic degradation. The obtained formulations were characterized and their physicochemical properties were investigated. According to stability studies, formulations were stable at +4?C for 6 months. Circular dichroism analyses revealed that conformational stability of insulin is not affected by the preparation conditions and the presence of chitosan and/or sodium tripolyphosphate. Furthermore, release properties of formulations were investigated under different pH levels, to simulate gastric and intestinal conditions. According to these studies, further experiments were carried out with the formulations which provided greater gastric protection and higher intestinal insulin release. Toxicity studies on L929 mouse fibroblast cells were performed with these formulations and toxic doses on these cells were determined. In vivo experiments were carried out with Wistar Albino rats. In these experiments standard insulin solution, microemulsion containing only insulin and optimal microemulsion which contains chitosan and sodium tripolyphosphate were administered intragastrically to the animals. The optimal formulation provided effective reduction on blood glucose level from the 1st to the 8th hour after administration. The reduction on blood glucose level was statistically significant at the 8th hour, as compared to the other groups. In conclusion, the developed water-in-oil microemulsion system containing insulin, chitosan and sodium tripolyphosphate in the inner phase may be suggested as an alternative dosage forms for oral insulin delivery.
Açıklama
Anahtar Kelimeler
Biyoteknoloji, Biotechnology
