Affinity-Directed Dynamics of Host-Guest Motifs for Pharmacokinetic Modulation via Supramolecular PEGylation

Abstract

Proteins are an impactful class of therapeutics but can exhibit suboptimal therapeutic performance, arising from poor control over the timescale of clearance. Covalent PEGylation is one established strategy to extend circulation time but often at the cost of reduced activity and increased immunogenicity. Supramolecular PEGylation may afford similar benefits without necessitating that the protein be permanently modified with a polymer. Here, we show that insulin pharmacokinetics can be modulated by tuning the affinity-directed dynamics of a host-guest motif used to non-covalently endow insulin with a poly(ethylene glycol) (PEG) chain. When administered subcutaneously, supramolecular PEGylation with higher binding affinities extends the time of total insulin exposure systemically. Pharmacokinetic modeling reveals that the extension in the duration of exposure arises specifically from decreased absorption from the subcutaneous depot governed directly by the affinity and dynamics of host-guest exchange. The lifetime of the supramolecular interaction thus dictates the rate of absorption, with negligible impact attributed to association of the PEG upon rapid dilution of the supramolecular complex in circulation. This modular approach to supramolecular PEGylation offers a powerful tool to tune protein pharmacokinetics in response to the needs of different disease applications.

Figure 1.. Schematic of insulin absorption.

Covalent PEGylation permanently alters the hydrodynamic size of the insulin necessitating uptake from the subcutaneous space through lymphatic vessels and driving increased circulation time once in the blood. In contrast, supramolecular PEGylation, using host–guest binding with cucurbit[7]uril, results in an insulin/PEG complex with a larger hydrodynamic radius that is not readily absorbed from the subcutaneous space into the blood. However, as dynamic exchange of the host–guest binding occurs, some insulin will become available for absorption into the blood where it is unhindered by the bulky PEG chain.

Figure 2.. Schematic of PEGylated insulin formulations.

To assess the effect of host–guest binding affinity on the pharmacokinetics of dynamically PEGylated insulin, two non-covalently PEGylated insulin systems were tested. (A) The N-terminal B1 phenylalanine of insulin can bind to cucurbit[7]uril (CB[7]) with a K_eq~10⁶ M⁻¹. A PEG_20k-CB[7] can thus be used to non-covalently PEGylate the phenylalanine on insulin. (B) To test host–guest motifs with higher binding affinity, a covalent insulin-CB[7] conjugate was combined with a guest-linked PEG_20k. To depict the conjugation between CB[7] and insulin, the chemical structures of CB[7], bicyclo[6.1.0]nonyne (BCN), and the PEG linker used are shown (left). PEG_20k-xylylenediamine (K_eq~10⁹ M⁻¹) or PEG_20k-O-adamantane (K_eq~10¹⁰ M⁻¹) were selected as guests. Bond lifetime (τ), is inversely related to the dissociation rate k_off and was calculated based on the following relationships and assumptions: K_eq = k_on/k_off; k_on~10⁷; τ = 1/k_off. See Table S1 for a complete list of values.

Figure 3:. Synthesis of insulin conjugates.

(A) Scheme showing insulin-CB[7] and insulin-PEG_20k synthesis. Insulin was functionalized with bicyclo[6.1.0]nonyne (BCN) using a N-hydroxysuccinimide (NHS)-ester reaction followed by a copper-free click reaction between BCN and azide-modified CB[7] or azide-PEG_20k, resulting in the final insulin conjugates. (B) MALDI-MS was used to verify formation of the intermediate (Insulin-BCN, expected m/z=6217.11) and final product (Insulin-CB[7], expected m/z=7491.29). MALDI-MS spectra were baseline corrected, with the minimum intensity value in the spectra subtracted from all other values.

Figure 4:. Pharmacokinetics and pharmacodynamics in diabetic rats.

Fasted diabetic rats were administered one of five insulin formulations subcutaneously: (i) insulin (2 U/kg), (ii) insulin & CB[7]-PEG_20k (10 U/kg), (iii) insulin-CB[7] & Xyl-PEG_20k (10 U/kg), (iv) insulin-CB[7] & O-Ada-PEG_20k (10 U/kg), (v) insulin-PEG_20k (10 U/kg). Following administration, rats were given access to food. (A) Change in blood glucose levels following insulin administration. (B) Pharmacokinetics in terms of serum insulin concentrations following insulin administration. (C) Pharmacokinetics for each rat was normalized individually, and normalized values were averaged for insulin concentration for each treatment group. (D) Time to reach peak insulin concentrations. (E) Duration of exposure, defined as time to depletion of 25% of insulin peak concentration. Error bars indicate means ± SEM with n = 4 for all groups.

Figure 5:. Modeling of formulation pharmacokinetics.

(A) Pharmacokinetic scheme showing a three-compartment model. After injection, insulin will transition dynamically between a PEGylated state (not readily absorbed) and a free state where it is available for absorption into the blood. Available insulin will be absorbed into the blood from the subcutaneous space and then cleared from the blood. (B) Normalized pharmacokinetics in diabetic rats modeled using a least-squares fit to determine k₁, and k₂, with k₃ based on the elimination half-life calculated from intravenous injection experiments. Data is shown as mean ± SEM. (C) k₁ (fitted parameter from experimental data) plotted versus k_off (calculated based on host–guest binding affinity). A semi-log line was fit to the data using GraphPad Prism 9 best fit: Y=0.1735*log(X)+0.1761, Adjusted R²=0.9995.