Browsing by Subject "Controlled release"

Over 30 million people in the United States suffer from type 2 diabetes (T2D), and this figure is rapidly increasing. Currently available glucose-lowering drugs largely treat the symptoms of diabetes and not the underlying pathology, leaving one third of diabetes patients with improperly managed disease. Thus, there exists an urgent need for novel drugs that slow T2D progression while posing a minimal burden on the patient.

The metabolic regulatory factor fibroblast growth factor 21 (FGF21) is under investigation as a T2D therapeutic due to its favorable effects on glycemic control and body weight. However, the feasibility of native FGF21 as a drug candidate is impeded by its rapid in vivo clearance and by costly production methods associated with poor protein solubility. To address these issues, FGF21 was recombinantly expressed in E. coli as a fusion with an elastin-like polypeptide (ELP), a repetitive peptide polymer with reversible thermal phase behavior. Below their transition temperature (Tt), ELPs exist as soluble unimers, while above their Tt, they aggregate into an insoluble coacervate. The thermal responsiveness of the ELP was retained when genetically fused to FGF21, with several notably positive impacts for the synthesis and efficacy of this protein drug. First, the ELP fusion partner acted as a solubility enhancer, yielding 50 mg/L of active FGF21 protein from the soluble cell lysate fraction in shaker flask culture, and eliminating the need for protein refolding. Second, the phase transition behavior of the ELP was exploited for chromatography-free FGF21 purification. Third, the Tt of the ELP was tuned to below body temperature, such that the phase transition was initiated solely by body heat. Indeed, in vivo injection of the fusion resulted in an immiscible viscous phase in the subcutaneous (s.c.) space that dissolved at a steady rate, temporally releasing fusion unimers into circulation. The injectable FGF21 drug depot was tested in diabetic ob/ob mice, and conferred dose-dependent glucose-and weight-lowering effects that were sustained out to 5 days following a single s.c. injection.

Once an optimized ELP-based FGF21 delivery strategy was established, the fusion concept was applied to a combination therapy to afford even greater metabolic benefits, while providing controlled release properties exclusive to the ELP platform. Recent evidence supports the development of combination drug treatments that incorporate complementary mechanisms of action to more effectively treat T2D. Thus, we developed a unimolecular dual agonist by combining the incretin glucagon-like peptide-1 (GLP1) with FGF21, hypothesizing that this agent would merge the insulinotropic and anorectic effects of GLP1 with the enhanced insulin sensitivity and energy expenditure associated with FGF21 signaling. The dual agonist was designed as a single polypeptide fusion, with GLP1 located at the N terminus and FGF21 at the C terminus. This orientation allowed each peptide to activate its endogenous receptor, while the linear architecture enabled facile synthesis in a bacterial expression system. An ELP was fused between GLP1 and FGF21 to serve as both a flexible linker and a depot-forming delivery scaffold. Indeed, a single s.c. injection of GLP1-ELP-FGF21 into diabetic db/db mice resulted in potent metabolic effects that were sustained at least 7 days, indicating formation of an ELP depot with a highly controlled rate of drug release. Furthermore, dual agonist treatment outperformed a long-acting GLP1 analog in restoring glycemic control and inducing weight loss, supporting the rationale for a GLP1/FGF21 combination therapy.

With a significant proportion of T2D patients failing to properly manage their disease, there is an urgent need for novel drug and drug combinations that effectively target disease pathophysiology, while posing a minimal burden on the patient. Meanwhile, the vast – and global – prevalence of metabolic disease argues for cost-effective and scalable manufacturing methods for new drugs. An ELP-based approach to therapeutics precisely addresses these needs by providing a streamlined method for production, as well as an innovative strategy for drug delivery to reduce the frequency of administration and thereby promote patient compliance. Furthermore, the ELP platform can be utilized to unite distinct drugs into one multi-functioning molecule to more effectively treat diabetes, altogether simplifying and improving metabolic disease management.

Novel drug delivery methods aims to improve the therapeutic efficacy by enhancing the molecule’s pharmacokinetic profile as well as increasing its accumulation at the target size while reducing the amount at off-target sites. The results of effective drug delivery can be economical – reducing the amount of wasted drug – as well as therapeutic, by minimizing side effects and simplifying dosing regimens. While most methods achieve first-order release at best, zero-order release is the ultimate goal of controlled release systems, wherein the drug is released at a constant rate that is independent of concentration. Towards this end, we have developed a system that achieves near zero-order release kinetics for the delivery of glucagon-like peptide-1 (GLP-1) for the treatment of type 2 diabetes. GLP-1’s activity is glucose dependent, potentiating insulin release only when glucose levels are high. Thus, although type 2 diabetes is a complicated chronic condition to treat, maintaining constant levels of GLP-1 in circulation at all times would ensure that insulin is released only when the body needs it.

We have achieved impressive delivery of this peptide, which stands up to other FDA approved formulations, by recombinant fusion to an elastin-like polypeptide (ELP). The ELP, a thermally sensitive biopolymer, was engineered to undergo a soluble to insoluble phase transition upon injection to form a slowly releasing drug depot. By optimizing the biopolymer’s tunable parameters – molecular weight and transition temperature (Tt) – we were able to demonstrate circulation times of up to 10 days in mice and 17 days in monkeys. The optimized pharmacokinetics, showing near zero-order kinetics, leads to 10 days of glycemic control in three different mouse models of diabetes, as well as to reduction of glycosylated hemoglobin levels and weight gain in ob/ob mice treated once weekly for 8 weeks. Furthermore, we saw no obvious injection site reactions in mice and low immunogenicity in monkeys. The fusion had equivalent if not superior performance compared to clinically used formulations of this drug class, Bydureon and Trulicity, which are both FDA approved for once-weekly injection. Thus, this optimized GLP1 formulation has the potential to enhance therapeutic outcomes by eliminating peak-and-valley pharmacokinetics and by improving overall safety and tolerability.

We have also begun the design and synthesis of next-generation injectable depots. Using molecular and synthetic biology tools, we are developing a unimolecular dual incretin, that would display GLP-1 and its sister peptide, glucose-dependent insulinotropic peptide (GIP) on the same molecule. Like GLP-1, GIP is also released post-prandially to augment insulin secretion upon binding its receptor on the pancreatic β-cell. We hypothesize that a unimolecular dual agonist will have synergistic effects in vivo due to avidity enhanced by its bivalency, but also due to the crosslinking of receptors and potential changes in downstream signaling bias. We have successfully synthesized a unimolecular dual agonist by expressing individual fusions (GLP1-ELP and GIP-ELP) that bear the substrate sequence for Sortase A, a bacterial enzyme that can perform site-specific native peptide ligation of the substrate sequence and a chemically modified oligoglycine, which acts as a nucleophile. Using Sortase A, we can functionalize each fusions with a bioorthogonal azide group and then link the two peptides with a homobifunctional crosslinker. While in vitro testing of the molecule is in preliminary stages, the design principles that we have established for injectable depots, combined with the methods for synthesizing a unimolecular dual agonist, should be broadly applicable for improving the pharmacological performance of a number of peptide and protein therapeutics.