Extending Residence Time and Stability of Peptides by Protected Graft Copolymer (PGC) Excipient: GLP-1 Example

Abstract

Purpose The purpose of this study is to determine whether a Protected Graft Copolymer (PGC) containing fatty acid can be used as a stabilizing excipient for GLP-1 and whether PGC/GLP-1 given once a week can be an effective treatment for diabetes. Methods To create a PGC excipient, polylysine was grafted with methoxypolyethyleneglycol and fatty acid at the epsilon amino groups. We performed evaluation of 1) the binding of excipient to GLP-1, 2) the DPP IV sensitivity of GLP-1 formulated with PGC as the excipient, 3) the in vitro bio-activity of excipient-formulated GLP-1, 4) the in vivo pharmacokinetics of excipient-formulated GLP-1, and 5) the efficacy of the excipient-formulated GLP-1 in diabetic rats. Results We showed reproducible synthesis of PGC excipient, showed high affinity binding of PGC to GLP-1, slowed protease degradation of excipient-formulated GLP-1, and showed that excipient-formulated GLP-1 induced calcium influx in INS cells. Excipient-formulated GLP-1 stays in the blood for at least 4 days. When excipient-formulated GLP-1 was given subcutaneously once a week to diabetic ZDF rats, a significant reduction of HbA1c compared to control was observed. The reduction is similar to diabetic ZDF rats given exendin twice a day. Conclusions PGC can be an ideal in vivo stabilizing excipient for biologically labile peptides.

Protected Graft Copolymer Excipient Leads to a Higher Acute Maximum Tolerated Dose and Extends Residence Time of Vasoactive Intestinal Peptide Significantly Better than Sterically Stabilized Micelles

“…We have previously demonstrated that PGC reaches the blood stream after SC injection (4). Since the T max is reached much later, AUC is much larger and MRT is much longer for PGC-VIP, compared to SSM-VIP and unformulated VIP, it reasonable to expect that PGC-VIP is slowly entering the blood stream from the SC injection site as an intact complex, similar to PGC-GLP-1 (4), thereby protecting VIP in the circulation. We also showed here that serum of mice injected with PGC-VIP contain 15-times more VIP in the form of high molecular weight structures that can be filtered out with a 100 kDa MWCO filter, compared to serum from mice injected with unformulated VIP.…”

“…PGC was synthesized as described by Castillo et al (4). Specifically, a 20PLL solution was prepared by dissolving 0.46 g 20 kDa Poly-l-lysine (20PLL, SAFC, St. Louis MO) in 10 ml of 0.23 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Fisher Scientific, Waltham MA) in water.…”

Peptide Drug Release Behavior from Biodegradable Temperature-Responsive Injectable Hydrogels Exhibiting Irreversible Gelation

“…As shown in Figure 4, although F(P1) reverts to the sol state completely by Day 30, 100% cumulative release is not attained. The GLP-1 molecule can adsorb onto the polymer chains by hydrophobic interactions [23], and GLP-1 may be unstable due to spontaneous hydrolysis; consequently, 100% of the GLP-1 molecules could not be detected by reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. On the other hand, the release of GLP-1 from the F(P1/D+PA 40 ) hydrogel became very slow after Day 2.…”

Omeprazole and PGC-Formulated Heparin Binding Epidermal Growth Factor Normalizes Fasting Blood Glucose and Suppresses Insulitis in Multiple Low Dose Streptozotocin Diabetes Model

“…The PGC used for HBEGF was made by linking a chondroitin sulfate (mw=31–54 kiloDalton Pfaltz&Bauer, Waterbury, CT) backbone with multiple amino-terminated polyethylene glycol (amino PEG, mw=5 kiloDalton; Laysan Bio Inc. Arab, AL) chains at a weight ratio of 1:2 (chondroitin sulfate:PEG) using a N-hydroxysuccinimide and 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride facilitated reaction as previously described (8), see Fig. 2.…”

“…PGC for gastrin was synthesized in a similar manner but with PEI (branched polyethyleneimine; mw=10 kiloDalton, Polysciences, Inc. Warrington, PA) as the backbone instead of chondroitin sulfate. The backbone amino groups of PEI were linked to, in order: 1) multiple carboxyl terminated PEG (Laysan Bio, Inc., Arab, AL) up to 44% amino saturation, 2) stearic acid up to 86% saturation, and 3) Iodomethane up to 100% saturation, using the same facilitated reaction as above for the first and second reactions, as previously described (8). …”