عنوان

Polymeric Materials to Improve the Stability and Delivery of Insulin and Glucagon

Number

TL4hw3n4mb

.Language of Text, Soundtrack etc

انگلیسی

Title Proper

Polymeric Materials to Improve the Stability and Delivery of Insulin and Glucagon

General Material Designation

[Thesis]

First Statement of Responsibility

Messina, Kathryn Margaret Mansfield

Subsequent Statement of Responsibility

Maynard, Heather D

Date of Publication, Distribution, etc.

2020

Body granting the degree

Maynard, Heather D

Text preceding or following the note

2020

Text of Note

Proteins and peptides have become an important class of therapeutics due to their high specificity and general biocompatibility. However, using proteins and peptides as drugs has several intrinsic challenges. These macromolecules are generally cleared rapidly due to renal filtration and enzymatic degradation following administration. Issues of instability during storage due to their chemical composition and specific three-dimensional confirmations necessitate that most protein and peptide therapeutics be stored in the refrigerator or as a lyophilized powder. Additionally, they must be administered through injection and are not responsive to intrinsic biological signals, unlike endogenous processes. Thus, it is important to develop methods to improve characteristics of protein and peptide therapeutics to improve their stability, pharmacokinetics, and delivery in response to biological stimuli. Chapter Two describes the investigation of the insulin stabilization properties of trehalose glycopolymer as excipient and conjugate. Addition of a styrenyl backbone trehalose polymer excipient stabilized insulin against aggregation induced by exposure of insulin to heat or mechanical agitation. Conjugation of the trehalose polymer to insulin was achieved by reductive amination and the stability assays were repeated with the conjugate, showing results like the excipient. The conjugation site was identified as GlyA1 and LysB29 by indirect characterization through acid-cleavage of the polymer. While conjugation prolonged the half-life in mice, addition of trehalose polymer excipient did not alter protein pharmacokinetics. The mechanism of insulin stabilization was investigated with a methacrylate backbone trehalose polymer excipient, showing presence of the polymer inhibits both fibrillation and deamidation. Chapter Three continues the development of a new strategy for site-specific conjugation of a trehalose polymer to insulin for improved stability and bioactivity. Conditions for AGET ATRP under mild, aqueous conditions were optimized. A site-specific insulin macroinitiator was prepared targeting modification at LysB29 utilizing its higher nucleophilicity over the other possible amine conjugation sites and purifying to isolate the desired species. Trehalose monomer was polymerized directly from this site-specific macroinitiator resulting in a conjugate with improved heat stability. A lower dosage of the site-specific conjugate compared to the nonspecific conjugate was needed to achieve the same change in blood glucose. Chapter Four details the synthesis of blood triggered self-immolative linkers designed for use as spacers for rapid-acting insulin-trehalose glycopolymer conjugates. Linkers triggered by serum albumin through base-catalyzed β-elimination were first prepared and triggering was characterized. During conjugation, the first linker underwent premature triggering from the primary amines of insulin and the exposed amine catalyzed further triggering. The second linker design underwent base-catalyzed self-immolation over the course of 20 h. Linkers that could be triggered by the thiol concentration in the blood were then synthesized and evaluated. Both aliphatic and aromatic linkers underwent rapid self-immolation with small molecules across the disulfide under relevant glutathione concentrations. Conjugation with trehalose glycopolymer slowed the kinetics of the self-immolation, likely because of the increase in steric bulk. Chapter Five describes optimization of the background release of insulin from a trehalose glycopolymer hydrogel for improved stability and glucose-responsive delivery of the protein. Several strategies were used to decrease the background release. Two methods to increase the binding affinity of the boronic acid to polyols was used to strengthen the hydrogel network. The influence of pore size/crosslink density was also explored. Finally, incorporation of comonomers for electrostatic attraction to insulin resulted in the lowest background release of insulin without glucose after optimization of gelation procedure. Chapter Six introduces glucose-responsive materials for regulation of glucagon delivery. Glucose-responsive nanogels are prepared by precipitation polymerization and post-polymerization modification with phenylboronic acid as glucose-sensing unit. Nanogels were thermo- and glucose-responsive through incorporation of thermoresponsive pNIPAM or pPEGMA with glucose acting as an additive that alters the hydration of the polymers. Native glucagon was found to degrade during the loading of the nanogels, so a more stable soluble analog was used that improved to loading.

Whitley, Melissa

Messina, Kathryn Margaret Mansfield

UCLA

Electronic name

p

[Thesis]

276903

a

Y