NKTR-358: A novel regulatory T-cell stimulator that selectively stimulates expansion and suppressive function of regulatory T cells for the treatment of autoimmune and inflammatory diseases.

Impaired interleukin-2 (IL-2) production and regulatory T-cell dysfunction have been implicated as immunological mechanisms central to the pathogenesis of multiple autoimmune and inflammatory diseases. NKTR-358, a novel regulatory T-cell stimulator, is an investigational therapeutic that selectively restores regulatory T-cell homeostasis in these diseases. We investigated NKTR-358's selectivity for regulatory T-cells, receptor-binding properties, vo and pharmacodynamics, ability to suppress conventional T-cell proliferation in mice and non-human primates, and functional activity in a murine model of systemic lupus erythematosus.

NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy.

Background: NKTR-255 is a novel polyethylene glycol-conjugate of recombinant human interleukin-15 (rhIL-15), which was designed to retain all known receptor binding interactions of the IL-15 molecule. We explored the biologic and pharmacologic differences between endogenous IL-15 receptor α (IL-15Rα)-dependent (NKTR-255 and rhIL-15) and IL-15Rα-independent (precomplexed rhIL-15/IL-15Rα) cytokines.

Methods: In vitro pharmacological properties of rhIL-15, NKTR-255 and precomplex cytokines (rhIL-15/IL-15Rα and rhIL-15 N72D/IL-15Rα Fc) were investigated in receptor binding, signaling and cell function.

NKTR-181: A Novel Mu-Opioid Analgesic with Inherently Low Abuse Potential.

The increasing availability of prescription opioid analgesics for the treatment of pain has been paralleled by an epidemic of opioid misuse, diversion, and overdose. The development of abuse-deterrent formulations (ADFs) of conventional opioids such as oxycodone and morphine represents an advance in the field and has had a positive but insufficient impact, as most opioids are still prescribed in highly abusable, non-ADF forms, and abusers can tamper with ADF medications to liberate the abusable opioid within. The abuse liability of mu-opioid agonists appears to be dependent on their rapid rate of entry into the central nervous system (CNS), whereas analgesic activity appears to be a function of CNS exposure alone, suggesting that a new opioid agonist with an inherently low rate of influx across the blood-brain barrier could mediate analgesia with low abuse liability, regardless of formulation or route of administration.

Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy.

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug.

Prevention of hepatitis C virus infection and spread in human liver chimeric mice by an anti-CD81 monoclonal antibody.

Unlabelled: CD81 is a required receptor for hepatitis C virus (HCV) infection of human hepatocytes in vitro. We generated several high-affinity anti-human CD81 monoclonal antibodies (mAbs) that demonstrated potent, specific, and cross-genotype inhibition of HCV entry. One of these mAbs, K04, was administered to human liver chimeric mice before or after HCV infection to determine its ability to prevent HCV infection or spread of HCV infection, respectively.

AF-353, a novel, potent and orally bioavailable P2X3/P2X2/3 receptor antagonist.

Background And Purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist.

Experimental Approach: The antagonistic potencies (pIC(50)) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology.

In vitro assessment of intestinal IGF-I stability.

Insulin-like growth factor (IGF-I) is a 7648-Da polypeptide consisting of 70 amino acids. Clinically, IGF-I might be used in type II diabetes, which requires a life-long treatment. Therefore, delivery routes other than parenteral injections are highly desirable.

Molecular modeling of arginine-glycine-aspartic acid (RGD) analogs: relevance to transepithelial transport.

Purpose: The aim of this research is to model the effect of methylation on hydrogen bonding ability, surface area, polar surface area, volume, lipophilicity, charge, and cross-sectional diameters of a series of mono-, di-, and tri- methyl substituted analogs of arginine-glycine-aspartic acid (RGD) and compare these parameters to in vitro transport properties across Caco-2 monolayers.

Methods: Molecular modeling was used to investigate the structural parameters that may influence the transport properties of RGD and its methyl analogs at pH 7.4.

Nasal epithelial permeation of thymotrinan (TP3) versus thymocartin (TP4): competitive metabolism and self-enhancement.

Purpose: To investigate concentration dependent permeabilities and metabolism kinetics of thymotrinan (TP3) versus thymocartin (TP4) in nasal epithelium in vitro.

Methods: Excised bovine nasal mucosa was used as an in vitro model. Permeabilities were studied in a diffusion chamber, metabolism kinetics in a reflection kinetics set-up.

Validation of excised bovine nasal mucosa as in vitro model to study drug transport and metabolic pathways in nasal epithelium.

The present work aims at the validation of excised bovine nasal mucosa as an in vitro model to address transport and metabolism pathways relative to the nasal mucosal uptake of therapeutic peptides. Preservation of the viability of the excised tissue in the course of in vitro studies of up to 3 h was demonstrated by (i) positive viability staining, (ii) constant transepithelial electrical resistance (42 +/- 12 Omega cm(2)), (iii) constant rates of metabolic turnover, and (iv) linear permeation profiles of therapeutic peptides and (3)H-mannitol. Using 1-leucine-4-methoxy-2-naphthylamide as a model substrate, we observed no difference between bovine and human nasal aminopeptidase activity.

Translocation of human calcitonin in respiratory nasal epithelium is associated with self-assembly in lipid membrane.

We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability approximately 10(-)5 cm s-1). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [Nalpha-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) were synthesized. In presence of the endocytosis inhibitor cytochalasin D mucosal-to-serosal and serosal-to-mucosal hCT permeabilities were equal.

P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation.

A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model.

Caco-2 cell monolayers mimic intestinal absorptive epithelium and represent a very useful tool for studying transepithelial transport. The literature on Caco-2 cells is controversial regarding transepithelial resistance and permeabilities of different marker compounds across monolayers. This paper discusses probable causes for these discrepancies.

Flux measurements across Caco-2 monolayers may predict transport in human large intestinal tissue.

Confluent monolayers of Caco-2 cells, a human colonic carcinoma cell line, have been used extensively to predict intestinal absorption. A direct comparison of uptake characteristics, however, between cell monolayers and human tissue is missing in the literature. We have determined the flux for a series of small organic molecules, peptide and protein therapeutics, across Caco-2 monolayers and normal human colonic and rectal tissue in vitro to assess whether or not a predictive correlation of transport exists.

Transport of biologically active interferon-gamma across human skin in vitro.

Purpose: Several studies have suggested epidermal uptake of cytokines, such as interferons, can be facilitated using topical liposomal formulations. We have evaluated the in vitro transport of biologically active recombinant human interferon-gamma (rhIFN-gamma) into and through split-thickness human skin to assess this possibility.

Methods: Skin samples were exposed to rhIFN-gamma under various conditions involving hydrated and dry surface conditions in the presence and absence of liposomes.

Mechanism of dextran transport across rabbit intestinal tissue and a human colon cell-line (CACO-2).

The in vitro permeabilities of 14C labeled dextrans (10, 40, and 70 kD) were calculated from mass transport across Peyer's patches and non-patch tissues derived from rabbit jejunum, and a human colon cell line (Caco-2) grown as a monolayer on polycarbonate filters. Size distribution of dextrans did not change upon transport as judged from size exclusion chromatography. Permeabilities decreased in a size-dependent manner.

In vitro evaluation of dexamethasone-beta-D-glucuronide for colon-specific drug delivery.

Dexamethasone-beta-D-glucuronide is a potential prodrug for colonic delivery of the antiinflammatory corticosteroid dexamethasone. Previous studies [T. R.