Pharmacokinetics and Absorption, Distribution, Metabolism and Excretion of RGLS4326 in Mouse and Monkey, an Anti-miR-17 Oligonucleotide for the Treatment of Polycystic Kidney Disease.

RGLS4326 is a short oligonucleotide inhibitor of microRNA-17 (miR-17) that preferentially distributes to the kidney and displaces miR-17 from translationally active polysomes. Here, we present pharmacokinetics and absorption, distribution, metabolism, and excretion properties of RGLS4326 from mice and monkeys. RGLS4326 was absorbed rapidly after subcutaneous administration, distributed extensively to the kidney and liver, with preferential distribution to the kidney, and cleared rapidly from plasma by tissue uptake and renal excretion. Plasma exposure increased in a dose-proportional manner with no notable accumulation after repeat doses. Plasma protein binding of RGLS4326 across all species tested was between 79% and 96%. RGLS4326 predominantly distributed to the kidney with a long half-life (t1/2; t1/2 ranged from 8-11 days) and no marked (≤twofold) accumulation in kidney and liver after repeat doses. RGLS4326 was minimally metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species and underwent sequential hydrolysis from both 3' and 5' ends to produce chain-shortened metabolites. There were no human unique metabolites observed. Renal excretion was the major route of elimination of RGLS4326, and a significant fraction (50%-79%) of the dose was recovered intact in the urine of mice and monkeys across all dose levels. RGLS4326 is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, RGLS4326 exhibits low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Pharmacokinetics (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of RGLS4326 were characterized in vivo and in vitro. RGLS4326 shows similar PK and ADME properties across mice and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in preferential exposure of RGLS4326 to the intended target organ (kidney) to drive maximum target engagement. These studies support the interpretation of toxicology and efficacy studies and help characterize the disposition of RGLS4326 in humans.

Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.

2018 White Paper on Recent Issues in Bioanalysis: 'A global bioanalytical community perspective on last decade of incurred samples reanalysis (ISR)' (Part 1 - small molecule regulated bioanalysis, small molecule biomarkers, peptides & oligonucleotide bioanalysis).

The 2018 12th Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.

Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.

Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method--miRNA Polysome Shift Assay (miPSA)--for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited miRNA complexes. Active miRNAs bind target mRNAs in high molecular weight polysome complexes, while inhibited miRNAs are sterically blocked by anti-miRs from forming this interaction. These two states can be assessed by fractionating tissue or cell lysates using differential ultracentrifugation through sucrose gradients. Accordingly, anti-miR treatment causes a specific shift of cognate miRNA from heavy to light density fractions. The magnitude of this shift is dose-responsive and maintains a linear relationship with downstream target gene derepression while providing a substantially higher dynamic window for aiding drug discovery. In contrast, we found that the commonly used 'RT-interference' approach, which assumes that inhibited miRNA is undetectable by RT-qPCR, can yield unreliable results that poorly reflect the binding stoichiometry of anti-miR to miRNA. We also demonstrate that the miPSA has additional utility in assessing anti-miR cross-reactivity with miRNAs sharing similar seed sequences.

Nanoscale clustering of carbohydrate thiols in mixed self-assembled monolayers on gold.

Self-assembled monolayers (SAMs) bearing pendant carbohydrate functionality are frequently employed to tailor glycan-specific bioactivity onto gold substrates. The resulting glycoSAMs are valuable for interrogating glycan-mediated biological interactions via surface analytical techniques, microarrays, and label-free biosensors. GlycoSAM composition can be readily modified during assembly by using mixed solutions containing thiolated species, including carbohydrates, oligo(ethylene glycol) (OEG), and other inert moieties. This intrinsic tunability of the self-assembled system is frequently used to optimize bioavailability and antibiofouling properties of the resulting SAM. However, until now, our nanoscale understanding of the behavior of these mixed glycoSAMs has lacked detail. In this study, we examined the time-dependent clustering of mixed sugar + OEG glycoSAMs on ultraflat gold substrates. Composition and surface morphologic changes in the monolayers were analyzed by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. We provide evidence that the observed clustering is consistent with a phase separation process in which surface-bound glycans self-associate to form dense glycoclusters within the monolayer. These observations have significant implications for the construction of mixed glycoSAMs for use in biosensing and glycomics applications.

Metallosurfactants of bioinorganic interest: Coordination-induced self assembly.

This review covers selected surfactant ligands that undergo a change in aggregate morphology upon coordination of a metal ion, with a particular focus on coordination-induced micelle-to-vesicle transitions. The surfactants include microbially produced amphiphilic siderophores, as well as synthetic amphiphilic ligands. The mechanism of the metal-induced phase change is considered in light of the coordination chemistry of the metal ions, the nature of the ligands, and changes in molecular geometry that result from metal coordination. Of particular interest are biologically produced amphiphiles that coordinate transition metal ions and amphiphilic ligands of relevance to bioinorganic chemistry.

XAS study of a metal-induced phase transition by a microbial surfactant.

The metal-induced micelle-to-vesicle phase change that the ferric complex of the microbially produced amphiphile, marinobactin E (M(E)), undergoes has been investigated by X-ray diffraction (XRD) and X-ray absorption spectroscopy (XAS). Marinobactin E is one member of the suite of siderophores, marinobactins A-E, that are used by the source bacterium to facilitate iron acquisition. Fe(III)-M(E) undergoes a micelle-to-multilamellar vesicle transition in the presence of Cd(II) and Zn(II). XRD measurements indicate the interlamellar repeat distance of the Cd(II)- and Zn(II)-induced multilamellar vesicles is approximately 5.3 nm. XAS spectra of the sedimented Cd(II)- and Zn(II)-induced multilamellar vesicles suggests hexadentate coordination of Cd(II) and Zn(II) consisting of two monodentate carboxylate ligands and four water ligands. This coordination environment supports the hypothesis that Cd(II) and Zn(II) bridge the terminal carboxylate moiety of two Fe(III)-M(E) headgroups, pulling the headgroups together in an arrangement that favors vesicle formation over the formation of micelles. XAS spectra of the Fe(III) center in the sedimented Cd(II)- and Zn(II)-induced vesicles confirm the anticipated six-coordinate geometry of Fe(III) by the M(E) headgroup via the two hydroxamate groups and the alpha-hydroxy amide moiety.

Metal-dependent self-assembly of a microbial surfactant.

Small-angle neutron scattering (SANS), cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) were used to study the metal-dependent phase behavior of microbially produced surfactants-marinobactins B, D, and E (MB, MD, and ME). Marinobactins A-E are siderophores that facilitate Fe(III) acquisition by the source bacterium through the coordination of Fe(III) by the peptidic headgroup. All of the marinobactins have the same six amino acid headgroup but differ in the length and saturation of the monoalkyl fatty acid tail. Fe(III) coordinated to ME (Fe(III)-ME) was found to form micelles with a diameter of approximately 3.5 nm that underwent a supramolecular transformation to produce a monodisperse population of vesicles with an average diameter ranging from approximately 90 to 190 nm upon addition of Cd(II), Zn(II), or La(III). SANS profiles of the transition-metal-induced phase exhibit a Bragg peak at QB approximately 0.11-0.12 A-1 and were fit to a SANS model for multilamellar vesicles that have an interbilayer repeat distance of 2pi/QB approximately 5.6-5.0 nm. Cryo-TEM images of the Zn(II)-induced phase reveals the presence of approximately 100 nm diameter approximately spherical aggregates of uniform electron density. The temperature dependence of the Zn(II)-induced transformation was also investigated as a function of the length and degree of unsaturation of the Fe(III)-marinobactin fatty acid tail. The Cd(II)-, Zn(II)-, and La(III)-induced phase changes have features that are similar to those of the previously reported Fe(III)-induced micelle-to-vesicle transition, and this observation has opened questions regarding the role that Cd(II) and Zn(II) may play in bacterial iron uptake.

Micelle-to-vesicle transition of an iron-chelating microbial surfactant, marinobactin E.

Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) techniques have been applied to study the self-assembly processes of a microbially produced siderophore, marinobactin E (ME). ME is one of a series of marinobactins A-E that facilitate Fe(III) acquisition by the source bacterium through coordination of Fe(III) by the marinobactin headgroup. ME is a six-amino-acid peptide amphiphile appended by palmitic acid (C16), and differs only in the nature of the fatty acid moiety from the other marinobactins. Apo-ME (uncoordinated ME) assembles to form micelles with an average diameter of 4.0 nm. Upon coordination of one equivalent of Fe(III), the mean micellar diameter of Fe(III)-ME shrinks to approximately 2.8 nm. However, in the presence of excess Fe(III), Fe(III)-ME undergoes a micelle-to-vesicle transition (MVT). At a small excess of Fe(III) over Fe(III)-ME (i.e., <1.2 Fe(III)/ME), a fraction of the Fe(III)-ME micelles rearrange into approximately 200 nm diameter unilamellar vesicles. At even greater Fe(III)/ME ratios (e.g., 2-3) multilamellar aggregates begin to emerge, consistent with either multilamellar vesicles or lamellar stacks. The MVT exhibited by ME may represent a unique mechanism by which marine bacteria may detect and sequester iron required for growth.