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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.

Intratumoral (IT) therapy is a powerful method of controlling tumor growth, but a major unsolved problem is the rapidity that injected drugs exit tumors, limiting on-target exposure and efficacy. We have developed a generic long acting IT delivery system in which a drug is covalently tethered to hydrogel microspheres (MS) by a cleavable linker; upon injection the conjugate forms a depot that slowly releases the drug and “bathes” the tumor for long periods. We established technology to measure tissue pharmacokinetics and studied MSs attached to SN-38, a topoisomerase 1 inhibitor. When MS ~ SN-38 was injected locally, tissues showed high levels of SN-38 with a long half-life of ~ 1 week. IT MS ~ SN-38 was ~ tenfold more efficacious as an anti-tumor agent than systemic SN-38. We also propose and provide an example that long-acting IT therapy might enable safe use of two drugs with overlapping toxicities. Here, long-acting IT MS ~ SN-38 is delivered with concurrent systemic PARP inhibitor. The tumor is exposed to both drugs whereas other tissues are exposed only to the systemic drug; synergistic anti-tumor activity supported the validity of this approach. We propose use of this approach to increase efficacy and reduce toxicities of combinations of immune checkpoint inhibitors such as αCTLA-4 and αPD-1.

The purpose of this work was to develop technology for facile, large‐scale production of tetra‐polyethylene glycol (PEG) hydrogel amino‐microspheres to serve as intermediates for injectable microsphere‐drug conjugates produced under cGMP guidelines. Here we developed equipment and procedures utilizing tubular cross‐flow membrane emulsification to produce the amino‐microspheres. The equipment comprised a polyether ether ketone tube containing 1000 evenly spaced pores encased in a cylindrical stainless steel jacket. The dispersed phase – an aqueous solution of two polymerizable tetra‐PEG prepolymers – is delivered into the outer jacket of the assembly by a pulse dampened pump, and the continuous phase – decane and surfactant – is delivered into the bore of the microporous tube from a pressurized tank. As the dispersed phase is pressed through the pores, the continuous phase induces the detachment of small, uniform droplets of ˜60 μm diameter at the mouths of the pores. After collection of the emulsified droplets, they are allowed to self‐polymerize and are then sieved to remove aberrantly small and large particles. The polymerized amino‐microspheres – obtained in ˜70% yield – are well within acceptance specifications, and show excellent injectability. Using this approach, we could prepare about 30 L of swollen amino‐microspheres per day which could be forwarded to production of microsphere‐drug substance under cGMP guidelines. This work was to develop technology for large‐scale production of amino‐microspheres – intermediates for production of injectable microsphere‐drug conjugates. Here we developed equipment and procedures utilizing tubular cross‐flow membrane emulsification to produce the amino‐microspheres. Using this approach, we could prepare about 30 L of swollen amino‐microspheres per day, by a simple cost effective process.

Sterilization of degradable polymeric biomaterials intended for injection presents a formidable challenge. Often, either the polymer backbone or labile crosslinks controlling degradation are adversely affected by commonly used sterilization methods. The purpose of this work was to develop an approach to sterilize tetra‐polyethylene glycol hydrogel microspheres (MSs) with β‐eliminative crosslinks that are destined to be carriers for drug delivery. The approach taken was to acidify the medium to compensate for the base‐catalyzed cleavage of linkers at high temperatures. We determined that rates of linker cleavage at pH 4 or below were sufficiently slow as to allow autoclaving and showed that precursor amine‐derivatized MSs could withstand autoclaving at pH 4 for at least four cycles of 20 minutes each at 121°C. Thus, amine‐MSs need not be prepared aseptically, but instead can be prepared in a low bioburden environment, and then sterilized by autoclaving before drug attachment. Sterilization of degradable polymeric biomaterials intended for injection presents a formidable challenge due to degradation by commonly used sterilization methods. The purpose of this work was to develop an autoclave method to sterilize tetra‐PEG hydrogel microspheres with beta‐eliminative crosslinks that are destined to be carriers for drug delivery.

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. The purpose of this work was to develop equipment and procedures for large‐scale aseptic production of injectable microsphere (MS) drug conjugates. The approach was to prepare the MS‐drug conjugate in two stages. Stage 1 was the preparation of amine derivatized microspheres (amino‐MS) under low to no bioburden conditions. Stage 2 was the autoclave sterilization of the starting amino‐MS and aseptic production of the MS‐drug conjugate.

The degradation of Tetra-PEG hydrogels containing β-eliminative crosslinks has been studied in order to provide an in vitro-in vivo correlation for the use of these hydrogels in our chemically controlled drug delivery system. We measured time-dependent gel mass loss and ultrasound volume changes of 13 subcutaneously implanted Tetra-PEG hydrogel microspheres having degradation times ranging from ~3 to 250 days. Applying a previously developed model of Tetra-PEG hydrogel degradation, the mass changes correlate well with the in vitro rates of crosslink cleavage and hydrogel degelation. These results allow prediction of in vivo biodegradation properties of these hydrogels based on readily obtained in vitro rates, despite having degradation times that span 2 orders of magnitude. These results support the optimization of drug-releasing hydrogels and their development into long-acting therapeutics. The use of ultrasound volume measurements further provides a noninvasive technique for monitoring hydrogel degradation in the subcutaneous space.

Polymeric microparticles can serve as carriers or sensors to instruct or characterize tissue biology. However, incorporating microparticles into tissues for in vitro assays remains a challenge. We exploit three-dimensional cell-patterning technologies and directed epithelial self-organization to deliver microparticles to the lumen of reconstituted human intestinal microtissues. We also develop a novel pH-sensitive microsensor that can measure the luminal pH of reconstituted epithelial microtissues. These studies offer a novel approach for investigating luminal microenvironments and drug-delivery across epithelial barriers.

Intratumoral (IT) therapy is a powerful method of controlling tumor growth, but a major unsolved problem is the rapidity that injected drugs exit tumors, limiting on-target exposure and efficacy. We have developed a generic long acting IT delivery-system in which a drug is covalently tethered to hydrogel microspheres (MS) by a cleavable linker; upon injection the conjugate forms a depot that slowly releases the drug and bathes the tumor for long periods. We established technology to measure tissue pharmacokinetics and studied MSs attached to SN-38, a topoisomerase 1 inhibitor. When MS~SN-38 was injected locally, tissues showed high levels of SN-38 with a long half-life of ~1 week. IT MS~SN-38 was ~10-fold more efficacious as an anti-tumor agent than systemic SN-38. We also propose and provide an example that long-acting IT therapy might enable safe use of two drugs with overlapping toxicities. Here, long-acting IT MS~SN-38 is delivered with concurrent systemic PARP inhibitor. The tumor is exposed to both drugs whereas other tissues are exposed only to the systemic drug; synergistic anti-tumor activity supported the validity of this approach. We propose use of this approach to increase efficacy and reduce toxicities of combinations of immune checkpoint inhibitors such as αCTLA4 and αPD-1.Competing Interest StatementAll authors manuscript except D. Charych hold options or shares in ProLynx.