Many drugs and drug candidates are suboptimal because of short duration of action. For example, peptides and proteins often have serum half-lives of only minutes to hours. One solution to this problem involves conjugation to circulating carriers, such as PEG, that retard kidney filtration and hence increase plasma half-life of the attached drug. We recently reported an approach to half-life extension that uses sets of self-cleaving linkers to attach drugs to macromolecular carriers. The linkers undergo β-eliminative cleavage to release the native drug with predictable half-lives ranging from a few hours to over 1 y; however, half-life extension becomes limited by the renal elimination rate of the circulating carrier. An approach to overcoming this constraint is to use noncirculating, biodegradable s.c. implants as drug carriers that are stable throughout the duration of drug release. Here, we use β-eliminative linkers to both tether drugs to and cross-link PEG hydrogels, and demonstrate tunable drug release and hydrogel erosion rates over a very wide range. By using one β-eliminative linker to tether a drug to the hydrogel, and another β-eliminative linker with a longer half-life to control polymer degradation, the system can be coordinated to release the drug before the gel undergoes complete erosion. The practical utility is illustrated by a PEG hydrogel-exenatide conjugate that should allow once-a-month administration, and results indicate that the technology may serve as a generic platform for tunable ultralong half-life extension of potent therapeutics.click chemistry | regenerative medicine | tetra-PEG C onjugation of drugs to macromolecular carriers is a proven strategy for improving pharmacokinetics. In one approach, the drug is covalently attached to a long-lived circulating macromolecule-such as PEG-through a linker that is slowly cleaved to release the native drug (1, 2). We have recently reported such conjugation linkers that self-cleave by a nonenzymatic β-elimination reaction in a highly predictable manner, and with half-lives of cleavage spanning from hours to over a year (3). In this approach, a macromolecular carrier is attached to a linker that is attached to a drug or prodrug via a carbamate group (1; Scheme 1); the β-carbon has an acidic carbon-hydrogen bond (C-H) and also contains an electron-withdrawing "modulator" (Mod) that controls the pK a of that C-H. Upon hydroxide ion-catalyzed proton removal to give 2, a rapid β-elimination occurs to cleave the linkercarbamate bond and release the free drug or prodrug and a substituted alkene 3. The rate of drug release is proportional to the acidity of the proton, and that is controlled by the chemical nature of the modulator; thus, the rate of drug release is controlled by the modulator. It was shown that both in vitro and in vivo cleavages were linearly correlated with electron-withdrawing effects of the modulators and, unlike ester bonds commonly used in releasable linkers (2), the β-elimination reaction was not catalyzed by general bases or ser...

We have developed a unique long-acting drug-delivery system for the GLP-1 agonist exenatide. The peptide was covalently attached to Tetra-PEG hydrogel microspheres by a cleavable β-eliminative linker; upon s.c. injection, the exenatide is slowly released at a rate dictated by the linker. A second β-eliminative linker with a slower cleavage rate was incorporated in polymer cross-links to trigger gel degradation after drug release. The uniform 40 μm microspheres were fabricated using a flow-focusing microfluidic device and in situ polymerization within droplets. The exenatide-laden microspheres were injected subcutaneously into the rat, and serum exenatide measured over a one-month period. Pharmacokinetic analysis showed a t1/2,β of released exenatide of about 7 days which represents over a 300-fold half-life extension in the rat and exceeds the half-life of any currently approved long-acting GLP-1 agonist. Hydrogel-exenatide conjugates gave an excellent Level A in vitro-in vivo correlation of release rates of the peptide from the gel, and indicated that exenatide release was 3-fold faster in vivo than in vitro. Pharmacokinetic simulations indicate that the hydrogel-exenatide microspheres should support weekly or biweekly subcutaneous dosing in humans. The rare ability to modify in vivo pharmacokinetics by the chemical nature of the linker indicates that an even longer acting exenatide is feasible.

We have developed an approach to prepare drug-releasing Tetra-PEG hydrogels with exactly four cross-links per monomer. The gels contain two cleavable β-eliminative linkers: one for drug attachment that releases the drug at a predictable rate, and one with a longer half-life placed in each cross-link to control biodegradation. Thus, the system can be optimized to release the drug before significant gel degradation occurs. The synthetic approach involves placing a heterobifunctional connector at each end of a four-arm PEG prepolymer; four unique end-groups of the resultant eight-arm prepolymer are used to tether a linker-drug, and the other four are used for polymerization with a second four-arm PEG. Three different orthogonal reactions that form stable triazoles, diazines, or oximes have been used for tethering the drug to the PEG and for cross-linking the polymer. Three formats for preparing hydrogel-drug conjugates are described that either polymerize preformed PEG-drug conjugates or attach the drug postpolymerization. Degradation of drug-containing hydrogels proceeds as expected for homogeneous Tetra-PEG gels with minimal degradation occurring in early phases and sharp, predictable reverse gelation times. The minimal early degradation allows design of gels that show almost complete drug release before significant gel-drug fragments are released.

The purpose of this work was to develop equipment and procedures for large‐scale aseptic production of injectable microsphere (MS) drug conjugates. The two major challenges were (a) to prepare sufficient amounts of MSs for clinical trials, and (b) to prepare the MS‐drug product under aseptic conditions. The approach was to prepare the MS‐drug conjugate in two stages. Stage 1 was the preparation of monodisperse tetra‐PEG amine derivatized MSs (amino‐MS) from two soluble PEG prepolymers under low to no bioburden conditions. To accomplish this, custom‐engineered equipment compatible with both aqueous and organic solvents was fabricated for parallel microfluidic preparation of amino‐MS. The system was capable of preparing up to ∼2 L of high quality 50 μm diameter amino‐MS per day. Stage 2 was the sterilization of the starting amino‐MS and aseptic production of the MS‐drug conjugate. The amino‐MS were first sterilized by autoclaving then transferred to a custom‐engineered autoclave‐sterilized washer‐reactor. This apparatus allowed for activation of the amino‐MS and attachment of a linker‐drug under aseptic conditions to give the sterile MS‐drug conjugate drug substance. The final drug product was produced by addition of excipients to form a homogeneous suspension. The entire process is exemplified by an engineering production run of a sterile MS‐peptide drug product.

Three N-alkoxyamines were synthesized for use in nitroxide-mediated radical polymerization. Upon thermolysis, they generate new acyclic a-hydrogen nitroxides: one adamantyl substituted and two diol-containing nitroxides. The initiators were tested in polymerization reactions in direct comparison with the initiator derived from the nitroxide TIPNO.Nitroxide-mediated 'living' free radical polymerization (NMRP) has become a very attractive method for the controlled polymerization of olefins, as monomers bearing a wide variety of functionality can be tolerated under the polymerization conditions. The resulting polymers generally display good control over both molecular weight and polydispersity, and the 'living' nitroxide endcap allows for the preparation of nanoscopic materials with highly designed architecture. 1 Other 'living' radical techniques such as Atom Transfer Radical Polymerization (ATRP) mediated by a metal complex, 2 and Reversible Addition Fragmentation Transfer (RAFT) mediated by a thiocarbonyl intermediate 3 have also been developed. ATRP has the advantage that it can be run at lower temperatures, and it functions well with methacrylates, but amine containing monomers sometimes coordinate to the metal catalyst, interfering with the polymerization process. Polymers made by ATRP contain traces of metal, derived from the metal catalyst. RAFT polymerization is effective with a wider range of monomers, including electron rich vinyl acetates, which are not good substrates for NMRP and ATRP. However sulfur-containing impurities may lead to undesirable colored polymers prepared by the RAFT process.Thus all three methods constitute valuable options in the methodologies available for producing designed polymers using free radical intermediates, yet there is room for improvement. With NMRP, an important achievement would be developing a system that would allow polymerizations to be carried out at temperatures lower than the 105-125 °C that are typically employed. The ability to polymerize electron rich olefins in a controlled manner would further extend the versatility of NMRP. For all of these goals, the characteristics of the nitroxide end-cap are key to improving the polymerization profile. Previously, we 4 and the group of Tordo 5 have introduced a-hydrogen nitroxides TIPNO 1 and SG-1 2, which are effective in the polymerization of styrenes and a variety of electron poor olefin monomers. Very recently a number of cyclic 6 and acyclic 7 new nitroxides have been introduced for improved efficacy in NMRP. Herein we present work on initiators based on several new acyclic a-hydrogen nitroxides for NMRP, in which the N-tert-butyl has been modified.