PURPOSE:This work evaluated the feasibility of combining insulin PEGylation with pH responsive hydrogels for oral insulin delivery.METHODS:A mono-substituted PEG-insulin conjugate was synthesized and purified. The site of conjugation was determined by MALDI-TOF MS. Uptake and release of PEGylated insulin was performed in complexation hydrogels to simulate oral dosing. The bioactivity of the conjugate and PK/PD profile was measured in vivo in rats.RESULTS:PEGylation was confirmed to be specifically located at the amino terminus of the B-chain of insulin. Higher loading efficiency was achieved with PEGylated insulin than regular human insulin in pH responsive hydrogels. The release of PEGylated insulin was lower than that of human insulin at all pH levels considered. Full retention of bioactivity of the PEG-insulin conjugate was confirmed by intravenous dosing while subcutaneous dosing exhibited a relative hypoglycemic effect 127.8% that of human insulin.CONCLUSIONS:Polyethylene glycol conjugated specifically to the amino terminus of the B-chain of insulin maintained the bioactivity of the protein and significantly extended the duration of the hypoglycemic effect. Used in combination with pH responsive hydrogels, PEGylated insulin has significant potential for oral delivery.

Environmentally responsive hydrogels composed of poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) have shown promise for oral insulin delivery due to their pH responsive complexation behavior. A series of hydrogel formulations were polymerized with varying amounts of crosslinker and varying monomer volume fraction. The mesh size of the network depended primarily on pH, varying from 8.0 to 27.2 nm. insulin loading efficiency varied directly with crosslink density, ranging from 42.7 to 84.9% of available insulin loaded into the hydrogels. The release of insulin was performed with each polymer formulation at 5 pH levels ranging from 2.7 to 6.8. insulin release was less than 20% for all formulations tested with insulin for the duration of the 3 hour release study for all pH levels considered except when the pH was 6.8, at which point the release occurred as a burst. Loading studies performed with insulin glargine, an insulin analog with an increased pI, showed the same trends as native insulin. However, the release of insulin glargine only occurred at a pH level above that of the pI of the protein. These results indicate that hydrogen bonds and ionic interactions between the protein and P(MAA-g-EG) may strongly influence its loading and release behavior in vitro.

Hydrogels comprised of poly(methacrylic acid) grafted with poly(ethylene glycol) (P(MAA-g-EG)) were characterized and examined for their potential as oral insulin carriers. Insulin loaded polymer (ILP) samples were made using two different polymer formulations. The values for the effective molecular weight between crosslinks, M¯e, and the network mesh size, ξ, were characterized and increased with increasing pH levels for both formulations. Insulin uptake studies indicated a high insulin loading efficiency for all samples tested, however release was dependent on the amount of insulin loaded. The effect of total polymer dosing was investigated by in situ administration in isolated ileal segments in rats. All ILP samples induced a hypoglycemic effect and an increase in insulin levels, proving that insulin was still biologically active. Insulin dosing amounts were varied by (i) maintaining a constant insulin fraction within an ILP sample while changing the amount of ILP and (ii) by varying the insulin fraction while dosing with the same amount of ILP. The total insulin absorption was dependent on both the amount of the polymer present and the concentration of insulin within an ILP sample, with a maximum relative bioavailability of 8.0%.

The insulin-transport enhancing effects of a pH-sensitive poly((methacrylic acid)-grafted-poly(ethylene glycol)) hydrogel system were studied using Caco-2 monolayers as an in vitro model of intestinal transport. Further, the ability of the hydrogel system to protect entrapped proteins through the upper gastrointestinal tract via digestion in simulated gastric and simulated intestinal fluids with digestive enzymes was confirmed. Caco-2 cell monolayers were exposed to a series of formulations including insulin alone, the polymer in insulin solution, insulin-loaded polymer (ILP) and ILP previously subjected to simulated digestive fluids with enzymes. These studies demonstrated greatly increased insulin transport for the ILP samples when compared with insulin alone and insulin in the presence of polymer, P-app = 12.7 x 10(-8) cm/s and 6.61 x 10(-8) cm/s versus 0.07 x 10(-8) cm/s and 0.06 x 10(-8) cm/s, respectively. While enhanced transport with the ILP was observed, the largest changes in TEER values did not coincide with the highest amounts of insulin transport, this suggests that the paracellular route may not be the sole mechanism of transport. Further, as the Caco-2 cell line has been demonstrated to possess the insulin receptor, active transport or a mixed mechanism cannot be ruled out.