A novel soluble supramolecular system for sustained rh-GH delivery

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 8h 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.