Extending Insulin Action in Vivo by Conjugation to Carboxymethyl Dextran

Abstract

The biochemical and pharmacological properties of bioactive peptides and proteins can be altered by conjugation with polymers. This report describes site-specific attachment of insulin to activated carboxyl groups of carboxymethyl dextran (CMD, MW=51000) through the GlyA1 insulin amino group. On average, three or four insulin molecules were grafted to a CMD linear chain. Coupled insulin molecules were properly folded, and the bioactivity of conjugated insulin in the blood glucose depression assay was 9.6 IU/mg, which was only 2.6 times less than that for native insulin. The cell growth study indicated that the CMD-insulin conjugate was as mitogenic as insulin on vascular smooth muscle cells, whereas the starting CMD polymer was not. The insulin receptor binding constant of the conjugate (3.6 x 10[9] M[-1]) compared well with that of native insulin (7.6 x 10[9] M[-1]), indicating that the CMD chain does not present any major constraints to binding. Plasma clearance of CMD-insulin obeyed a two-compartment pharmacokinetic (PK) model with a CMD-insulin conjugate plasma elimination half-life of 114.1 min, which was significantly longer than that of soluble Zn-insulin (12.4 min). In contrast, pharmacodynamic (PD) profiles (blood glucose lowering effects) after intravenous (iv) administration of the conjugate or insulin in rats were not different. Subcutaneous (sc) administration of the conjugate resulted in a significantly prolonged plasma profile with a noncompartmental PK parameter mean residence time (MRT) of 103.5 min which was significantly longer than that of soluble Zn-insulin (40.5 min). This was reflected in the protracted PD effect of sc administered conjugate with time needed to reach minimum glucose concentration Tnadir of 95.7 min, which was significantly longer than that of insulin (62 min). We conclude that the conjugation of insulin to CMD leads to a bioactive conjugate with a delayed sc PD profile showing prolonged response, resembling intermediate acting insulin preparations.

Half‐Life Extension of Biopharmaceuticals using Chemical Methods: Alternatives to PEGylation

“…Other examples include dextran conjugates of asparaginase, carboxypeptidase G 2 , and hemoglobin, respectively, as well as CMD‐insulin conjugates …”

Glucose-responsive insulin release: Analysis of mechanisms, formulations, and evaluation criteria

“…For example, native insulin was typically released when the insulin releasing mechanism involves matrix swelling, matrix disassembly or degradation. In the case of glucose binding competition induced insulin release, the working insulin released was typically an insulin modified with polymers, such as dextran-insulin conjugate, [93] or small molecules, such as succinyl amidophenyl glucopyranoside [32]. It is particularly important to examine the bioactivity of the modified insulin.…”

“…It is particularly important to examine the bioactivity of the modified insulin. Reports show that conjugating dextran to insulin loses 60–90% of native insulin bioactivity [93,94]. Thus, a significantly higher molar dose of conjugated insulin has to be delivered than unmodified native insulin in achieving comparable glucose suppressive effect.…”

Polymers for Protein Conjugation

“…Investigators have demonstrated a direct correlation between the M W of the coupled dextran and the increase in the conjugate's half-life [52]. Other examples of protein-dextran conjugates include: uricase [54], superoxide dismutase [55], insulin [56] and hemoglobin [57,58]. Readers can refer to Mehva's review for an exhaustive overview of all dextran conjugates, including those with low M W s [43].…”