CC-4066 therapy delivered to kidneys during cold storage and assessed with normothermic reperfusion is feasible and safe.

Introduction: Currently there is an urgent need to translate interventions that may be beneficial to marginal donor kidneys prior to transplant, to improve their quality from bench to bedside. This project investigated the effects of CC-4066, a potent dual inhibitor of cyclophilin proteins A and D, treatment during static cold storage (SCS) in a porcine model of renal ischemia-reperfusion injury (IRI) using Normothermic Reperfusion (NR). Materials and methods: Porcine kidneys and autologous blood were retrieved in pairs from a local abattoir (n = 7). One kidney from each pair was randomly allocated to treatment and one allocated to control and flushed with preservation solution containing CC-4066 or vehicle. After 7 h of SCS kidneys underwent 3 h Normothermic Reperfusion (NR) with autologous whole blood while perfusion characteristics and samples were collected. Results: Perfusion and metabolic parameters showed similar trends and no statistical differences were observed between the groups. IL-6 showed a significant increase over time but no significant difference between groups (p-value 0.009 and 0.14 respectively, two-way ANOVA). Oxygen consumption and lactate levels were similar between groups but there was increased vacuolation on histology in the control group. Conclusions: The addition of CC-4066 during SCS of kidneys is safe and feasible and has no adverse or detrimental effects on perfusion during assessment on NR. There was no difference in cytokine levels although there was a trend towards less vacuolation on histology in the treatment group.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle.

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.

Discovery of Potent Cyclophilin Inhibitors Based on the Structural Simplification of Sanglifehrin A.

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency. Replacing the C18-C21 diene unit of sanglifehrin with a styryl group led to potent compounds that displayed a novel binding mode in which the styrene moiety engaged in a π-stacking interaction with Arg55 of cyclophilin A (Cyp A), and the m-Tyr residue was displaced into solvent. This observation allowed further simplifications of the scaffold to generate new lead compounds in the search for orally bioavailable cyclophilin inhibitors.

Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency.

Mitochondrial complex I (CI) deficiency is the most prevalent defect in the respiratory chain in paediatric mitochondrial disease. This heterogeneous group of diseases includes serious or fatal neurological presentations such as Leigh syndrome and there are very limited evidence-based treatment options available. Here we describe that cell membrane-permeable prodrugs of the complex II substrate succinate increase ATP-linked mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres. Lactate accumulation in platelets due to rotenone-induced CI inhibition is reversed and rotenone-induced increase in lactate:pyruvate ratio in white blood cells is alleviated. Metabolomic analyses demonstrate delivery and metabolism of [(13)C]succinate. In Leigh syndrome patient fibroblasts, with a recessive NDUFS2 mutation, respiration and spare respiratory capacity are increased by prodrug administration. We conclude that prodrug-delivered succinate bypasses CI and supports electron transport, membrane potential and ATP production. This strategy offers a potential future therapy for metabolic decompensation due to mitochondrial CI dysfunction.

Cellular basis for bimatoprost effects on human conventional outflow.

PURPOSE: Bimatoprost is a widely used ocular hypotensive agent to treat glaucoma. It lowers intraocular pressure in humans by increasing both pressure-independent (uveoscleral) and pressure-dependent (conventional) aqueous humor outflow. The present study specifically examines bimatoprost effects on the cells that populate human outflow tissues. METHODS: The authors tested for prostamide receptor activation in primary cultures of human trabecular meshwork (TM), Schlemm's canal (SC), and ciliary smooth muscle (CSM) cells using cellular dielectric spectroscopy (CDS). RESULTS: The authors observed that bimatoprost produced an immediate and concentration-dependent increase in cell monolayer impedance for TM, SC, and CSM cells with EC(50) values of 4.3, 1.2, and 1.7 nM, respectively; corresponding to decreased cell contractility. Notably, in TM, SC, and CSM cells, bimatoprost was approximately equipotent to the selective FP receptor agonists fluprostenol and 17-phenyl PGF(2α). Bimatoprost effects were insensitive to cholera toxin and pertussis toxin but were abolished by phorbol 12-myristate 13-acetate pretreatment, suggesting Gq-involvement in cell signaling. The effects of bimatoprost on TM and SC cells were inhibited by the prostamide receptor antagonist AGN211334, with IC(50) values of 1.2 and 3.3 µM, respectively. Interestingly, AGN211334 behaved as an apparent inverse agonist in CDS assays involving TM cells but as a neutral prostamide antagonist with SC cells. CONCLUSIONS: Taken together, results suggest that bimatoprost specifically activates receptors in both cell types of the human conventional outflow pathway to modify intraocular pressure. However, only TM cell monolayers appear to have autocrine, or agonist-independent, receptor signaling that is sensitive to a prostamide receptor antagonist.

Bimatoprost, prostamide activity, and conventional drainage.

PURPOSE: Despite structural similarity with prostaglandin F(2 alpha), the ocular hypotensive agent bimatoprost (Lumigan; Allergan, Inc., Irvine, CA) shows unique pharmacology in vitro and functional activity in vivo. Unfortunately, the precise mechanisms that underlie bimatoprost's distinctive impact on aqueous humor dynamics are unclear. The purpose of the present study was to investigate the effects of bimatoprost and a novel prostamide-selective antagonist AGN 211334 on human conventional drainage. METHODS: Two model systems were used to test the consequences of bimatoprost and/or AGN 211334 treatment on conventional drainage. Human anterior segments in organ culture were perfused at a constant flow rate of 2.5 microL/min while pressure was recorded continuously. After stable baseline facilities were established, segments were treated with drug(s), and pressure was monitored for an additional 3 days. In parallel, the drugs' effects on hydraulic conductivity of human trabecular meshwork (TM) cell monolayers were evaluated. Pharmacological properties of AGN 211334 were characterized in isolated feline iris preparations in organ culture and heterologously expressed G-protein-coupled receptors were examined in vitro. RESULTS: Bimatoprost increased outflow facility by an average of 40% +/- 10% within 48 hours of treatment (n = 10, P < 0.001). Preincubation or coincubation with AGN 211334 significantly blunted bimatoprost's effects by 95% or 43%, respectively. Similar results were obtained in cell culture experiments in which bimatoprost increased hydraulic conductivity of TM cell monolayers by 78% +/- 25%. Pretreatment with AGN 211334 completely blocked bimatoprost's effects, while coincubation decreased its effects on average by 74%. In both models, AGN 211334 alone significantly decreased fluid flux across trabecular tissues and cells. CONCLUSIONS: The findings indicate that bimatoprost interacts with a prostamide receptor in the trabecular meshwork to increase outflow facility.