Probing the luminal microenvironment of reconstituted epithelial microtissues.

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

Development of cell-active irreversible inhibitors of the human centrosomal kinase Nek2

A structure-based design strategy was used to transform a promiscuous oxindole kinase inhibitor scaffold into the first cysteine-reactive, irreversible inhibitors of the human centrosomal kinase Nek2. These compounds achieve selective irreversible inhibition of Nek2 through alkylation of a key active-site cysteine (Cys22). This cysteine is found in the glycine-rich loop of a small subset (2%) of human kinases. Many of the irreversible Nek2 inhibitors that we developed turned out to also be potent (nM to pM affinity) inhibitors of the mitotic regulator Cdk1. Consistent with inhibition of Cdk1, these compounds rapidly triggered mitotic exit without cell division when added to cells arrested in mitosis. The induction of this dominant Cdk1-mediated phenotype limits the usefulness of these compounds with respect to studying the mitotic function of Nek2. However, our medicinal chemistry efforts led to the discovery of the propynamide oxindole compound 2 (JH295). Compound 2 contains a key ethyl group that destabilizes binding to Cdk1, resulting in 2,000-fold loss of potency, while retaining nanomolar potency toward Nek2. At concentrations that give full inhibition of Nek2 in cells, compound 2 does not prevent mitotic progression or cause major defects in spindle assembly. These results suggest that compound 2 does not inhibit the mitotic regulatory kinases Cdk1, Aurora B, or Plk1 in cells.The development of several chemical and biological tools for the study of the cellular roles of Nek2 is also presented. These include new leads into Nek2 inhibitor scaffolds, an in vitro kinase assay to measure the binding affinity and alkylation efficiency of irreversible inhibitors, Nek2 active site-directed probes, cell-based Nek2 inhibition assays, cell lines expressing an inhibitor-resistant Nek2 mutant, and control compounds designed to tease out nonspecific effects of the active Nek2 inhibitors in cells

Hydrogel drug delivery system with predictable and tunable drug release and degradation rates

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

Biodegradable Tetra-PEG Hydrogels as Carriers for a Releasable Drug Delivery System

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

Subcutaneously Administered Self-Cleaving Hydrogel–Octreotide Conjugates Provide Very Long-Acting Octreotide

We developed a long-acting drug-delivery system that supports subcutaneous administration of the peptidic somatostatin agonist octreotidea blockbuster drug used to treat acromegaly and neuroendocrine tumors. The current once-a-month polymer-encapsulated octreotide, Sandostatin LAR, requires a painful intragluteal injection through a large needle by a health-care professional. To overcome such shortcomings, Tetra-PEG hydrogel microspheres were covalently attached to the α-amine of d-Phe¹ or the ε-amine of Lys⁵ of octreotide by a self-cleaving β-eliminative linker; upon subcutaneous injection in the rat using a small-bore needle, octreotide was slowly released. The released drug from the ε-octreotide conjugate showed a remarkably long serum half-life that exceeded two months. The α-octreotide conjugate had a half-life of ∼2 weeks, and showed an excellent correlation of in vitro and in vivo drug release. Pharmacokinetic models indicate these microspheres should support once-weekly to once-monthly self-administered subcutaneous dosing in humans. The hydrogel–octreotide conjugate shows the favorable pharmacokinetics of Sandostatin LAR without its drawbacks

Hydrogel Drug Delivery System Using Self-Cleaving Covalent Linkers for Once-a-Week Administration of Exenatide

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 <i>t</i>_1/2,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

In Vitro-In Vivo Correlation for the Degradation of Tetra-PEG Hydrogel Microspheres with Tunable β-Eliminative Crosslink Cleavage Rates

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