A novel soluble supramolecular system for sustained rh-GH delivery

Abstract Methoxy-poly(ethylene glycol)s bearing a terminal cholanic moiety (mPEG 5kDa –cholane, mPEG 10kDa –cholane and mPEG 20kDa –cholane) were physically combined with recombinant human growth hormone (rh-GH) to obtain supramolecular assemblies for sustained hormone delivery. The association constants (Ka) calculated by Scatchard analysis of size exclusion chromatography (SEC) data were in the order of 10 5  M −1 . The complete rh-GH association with mPEG 5kDa –cholane, mPEG 10kDa –cholane and mPEG 20kDa –cholane was achieved with 7.5 ± 1.1, 3.9 ± 0.4 and 2.6 ± 0.4 w/w % rh-GH/mPEG–cholane, respectively. Isothermal titration calorimetry (ITC) yielded association constants similar to that calculated by SEC and showed that rh-GH has 21–25 binding sites for mPEG–cholane, regardless the polymer molecular weight. Dialysis studies showed that the mPEG–cholane association strongly delays the protein release; 80–90% of the associated rh-GH was released in 200 h. However, during the first 8 h the protein formulations obtained with mPEG 10kDa –cholane and mPEG 20kDa –cholane showed a burst release of 8 and 28%, respectively. Circular dichroism (CD) analyses showed that the mPEG 5kDa –cholane association does not alter the secondary structure of the protein. Furthermore, mPEG 5kDa –cholane was found to enhance both the enzymatic and physical stability of rh-GH. In vivo pharmacokinetic and pharmacodynamic studies were performed by subcutaneous administration of rh-GH and rh-GH/mPEG 5kDa –cholane to normal and hypophysectomised rats. The study showed that mPEG 5kDa –cholane decreases the maximal concentration in the blood but prolongs the body exposure of the protein, which resulted in 55% bioavailability increase. Finally, rh-GH formulated with mPEG 5kDa –cholane yielded prolonged weight increase of hypophysectomised rats as compared to rh-GH in buffer or formulated with mPEG 5kDa –OH. After the second administration the weight of the animals treated with rh-GH formulated with mPEG 5kDa –cholane was about 2 times higher than that obtained with equal dose of non-formulated rh-GH.