PTG-100, an Oral α4ß7 Antagonist Peptide: Preclinical Development and Phase 1 and 2a Studies in Ulcerative Colitis.

BACKGROUND & AIMS: Oral therapies targeting the integrin α4ß7 may offer unique advantages for the treatment of inflammatory bowel disease. We characterized the oral α4ß7 antagonist peptide PTG-100 in preclinical models and established safety, pharmacokinetic/pharmacodynamic relationships, and efficacy in a phase 2a trial in patients with ulcerative colitis (UC). METHODS: In vitro studies measured binding properties of PTG-100. Mouse studies measured biomarkers and drug concentrations in blood and tissues. The phase 1 study involved healthy volunteers. In phase 2a, patients with moderate to severe active UC were randomized to receive PTG-100 (150, 300, or 900 mg) or placebo once daily for 12-weeks. RESULTS: PTG-100 potently and selectively blocks α4ß7. Oral dosing of PTG-100 in mice showed high levels of target engagement and exposure in gut-associated lymphoid tissues. In healthy volunteers, PTG-100 showed dose-dependent increases in plasma exposure and blood target engagement. Although this phase 2a study initially did not meet the primary endpoint, a blinded reread of the endoscopy videos by a third party indicated clinical efficacy in conjunction with histologic remission at doses correlating with less than 100% receptor occupancy in peripheral blood. CONCLUSIONS: PTG-100 showed local gastrointestinal tissue target engagement and inhibition of memory T-cell trafficking in mice. It was safe and well tolerated in phase 1 and 2 studies. Phase 2a data are consistent with biological and clinical response and showed a dose response reflecting similar activities in preclinical models and healthy individuals. These data suggest that local gut activity of an oral α4ß7 integrin antagonist, distinct from full target engagement in blood, are important for efficacy and the treatment of UC. (ClinicalTrials.gov, Number NCT02895100; EudraCT, Number 2016-003452-75).

Single- and Multiple-Dose Pharmacokinetics and Pharmacodynamics of PN-943, a Gastrointestinal-Restricted Oral Peptide Antagonist of α4ß7, in Healthy Volunteers.

PN-943 is an orally stable, gastrointestinal-restricted peptide that binds specifically to α4ß7 integrin on leukocytes, blocking leukocyte trafficking to and activation in the gut, inhibiting colon inflammation and reducing signs and symptoms of active ulcerative colitis. Two pharmacokinetic/pharmacodynamic studies were conducted in healthy volunteers. Study 1 was a first-in-human study with 40 male subjects receiving PN-943, 100 to 1400 mg or placebo, as single doses and 57 male subjects receiving PN-943, 100 to 1000 mg or placebo, as multiple doses. Study 2 was a randomized, crossover study comparing multiple doses of 450-mg PN-943 twice daily as a liquid solution and as an immediate-release tablet in 10 subjects. No subjects discontinued due to treatment-emergent adverse events. Consistent with the gastrointestinal-restricted nature of the peptide, systemic exposure was minimal; there was an approximate dose-proportional increase in area under the plasma concentration-time curve. There was minimal accumulation with once-daily dosing and an absence of time-dependent changes in pharmacokinetics. Administration of PN-943 after a high-fat meal reduced peak plasma concentration and area under the plasma concentration-time curve. There was minimal (<0.1%) urinary excretion of intact drug, and there was a dose-related increase in fecal excretion of intact PN-943. Dose-dependent increases in blood receptor occupancy and reduction in blood receptor expression were observed, supporting target engagement. Twice-daily dosing resulted in sustained receptor occupancy with low plasma fluctuations (143%). PN-943 was generally well tolerated following single and multiple oral doses with low systemic exposure. Twice-daily dosing resulted in sustained pharmacokinetics and pharmacodynamics, supporting further investigation in efficacy studies.

Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists.

A series of subtype selective sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists are disclosed. Our high-throughput screening campaign revealed hit 1 for which an increase in potency and mouse oral exposure was achieved with minor modifications to the chemical scaffold. In vivo efficacy revealed that at high doses compounds 12 and 15 inhibited tumor growth. Further optimization of our lead series led to the discovery of proline derivatives 37 (XL541) and 38 which had similar efficacy as our first generation analogues at significantly lower doses. Analogue 37 displayed excellent pharmacokinetics and oral exposure in multiple species.

Optical coding of mammalian cells using semiconductor quantum dots.

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.

In vitro selection as a powerful tool for the applied evolution of proteins and peptides.

New in vitro methods for the applied evolution of protein structure and function complement conventional cellular and phage-based methods. Strategies employing the direct physical linkage of genotype and phenotype, and the compartmental association of gene and product to select desired properties are discussed, and recent useful applications are described. Engineering of antibodies and other proteins, selection from cDNA libraries, and the creation of functional protein domains from completely random starting sequences illustrate the value of the in vitro approaches. Also discussed is an emerging new direction for in vitro display technology: the self-assembly of protein arrays.