Liver-Selective Imidazolopyrazine Mitochondrial Uncoupler SHD865 Reverses Adiposity and Glucose Intolerance in Mice.

Excess body fat is a risk factor for metabolic diseases and is a leading preventable cause of morbidity and mortality worldwide. There is a strong need to find new treatments that decrease the burden of obesity and lower the risk of obesity-related comorbidities, including cardiovascular disease and type 2 diabetes. Pharmacologic mitochondrial uncouplers represent a potential treatment for obesity through their ability to increase nutrient oxidation. Herein, we report the in vitro and in vivo characterization of compound SHD865, the first compound to be studied in vivo in a newly discovered class of imidazolopyrazine mitochondrial uncouplers. SHD865 is a derivative of the furazanopyrazine uncoupler BAM15. SHD865 is a milder mitochondrial uncoupler than BAM15 that results in a lower maximal respiration rate. In a mouse model of diet-induced adiposity, 6-week treatment with SHD865 completely restored normal body composition and glucose tolerance to levels like those of chow-fed controls, without altering food intake. SHD865 treatment also corrected liver steatosis and plasma hyperlipidemia to normal levels comparable with chow-fed controls. SHD865 has maximal oral bioavailability in rats and slow clearance in human microsomes and hepatocytes. Collectively, these data identify the potential of imidazolopyrazine mitochondrial uncouplers as drug candidates for the treatment of obesity-related disorders.

A Synthetic Bottle-brush Polyelectrolyte Reduces Friction and Wear of Intact and Previously Worn Cartilage.

A poly(7-oxanorbornene-2-carboxylate) polymer containing pendent triethyleneglycol (TEG) chains of 2.8 MDa ("2.8M TEG") was synthesized and evaluated for long-term lubrication and wear reduction of ex vivo bovine cartilage as well as for synovitis in rats and dogs after intra-articular administration. Bovine cartilage surfaces were tested under torsional friction for 10,080 rotations while immersed in either saline, bovine synovial fluid (BSF), or 2.8M TEG. For each solution, coefficient of friction (µ), changes in surface roughness, and lost cartilage glycosaminoglycan were compared. To directly compare 2.8M TEG and BSF, additional samples were tested sequentially in BSF, BSF, 2.8M TEG, and then BSF. Finally, another set of samples were tested twice in saline to induce surface roughness and then tested in BSF, Synvisc, or 2.8M TEG to determine each treatment's effect on worn cartilage. Next, male Lewis rats were injected in one knee with 2.8M TEG or saline and evaluated for effects on gait, and female beagles were injected with either 2.8M TEG or saline in one knee, and their synovial tissues analyzed for inflammation by H&E staining. Treatment with 2.8M TEG lowers µ, lessens surface roughness, and minimizes glycosaminoglycan loss compared to saline. The 2.8M TEG also reduces µ compared to BSF in pairwise testing and on worn cartilage surfaces. Injection of 2.8M TEG in rat or beagle knees gives comparable effects to treatment with saline, and does not cause significant synovitis.

A synthetic polymeric biolubricant imparts chondroprotection in a rat meniscal tear model.

Intra-articular injection of hyaluronic acid (HA) is used to treat osteoarthritis (OA) as a viscosupplement, yet it only provides short-term benefit because HA is cleaved by hyaluronidase and cleared out of the joint after several days. Therefore, we developed a new polymer biolubricant based on poly-oxanorbornane carboxylate to enhance joint lubrication for a prolonged time. Rheological and biotribological studies of the biolubricant reveal viscoelastic properties and coefficient of friction equivalent and superior to that of healthy synovial fluid, respectively. Furthermore, in an ex vivo bovine cartilage plug model, the biolubricant exhibits superior long-term reduction of friction and wear prevention compared to saline and healthy synovial fluid. ISO 10993 biocompatibility tests demonstrate that the biolubricant polymer is non-toxic. In an in vivo rat medial meniscal tear OA model, where the performance of the leading HA viscosupplement (Synvisc-one®) is comparable to the saline control, treatment with the biolubricant affords significant chondroprotection compared to the saline control.

Synthesis and Preclinical Characterization of a Cationic Iodinated Imaging Contrast Agent (CA4+) and Its Use for Quantitative Computed Tomography of Ex Vivo Human Hip Cartilage.

Contrast agents that go beyond qualitative visualization and enable quantitative assessments of functional tissue performance represent the next generation of clinically useful imaging tools. An optimized and efficient large-scale synthesis of a cationic iodinated contrast agent (CA4+) is described for imaging articular cartilage. Contrast-enhanced CT (CECT) using CA4+ reveals significantly greater agent uptake of CA4+ in articular cartilage compared to that of similar anionic or nonionic agents, and CA4+ uptake follows Donnan equilibrium theory. The CA4+ CECT attenuation obtained from imaging ex vivo human hip cartilage correlates with the glycosaminoglycan content, equilibrium modulus, and coefficient of friction, which are key indicators of cartilage functional performance and osteoarthritis stage. Finally, preliminary toxicity studies in a rat model show no adverse events, and a pharmacokinetics study documents a peak plasma concentration 30 min after dosing, with the agent no longer present in vivo at 96 h via excretion in the urine.

A large-molecular-weight polyanion, synthesized via ring-opening metathesis polymerization, as a lubricant for human articular cartilage.

A large-molecular-weight polyanion is found to possess lubricating properties for cartilage. The polyanion, sodium poly(7-oxanorbornene-2-carboxylate), is synthesized by ring-opening metathesis polymerization of methyl 5-oxanorbornene-2-carboxylate. When dissolved in aqueous solution and applied to the surface of human cartilage it reduces the friction at the interface and acts as a lubricant. Its performance is similar to that of synovial fluid and superior to those of saline and Synvisc in an ex vivo human cartilage plug-on-plug model. The polymer is also not readily degraded by hyaluronidase or cytotoxic to human chondrocytes in vitro. As such, this polymer is a new type of viscosupplement, and the results provide insight into the design requirements for synthesizing highly efficacious synthetic biolubricants.

Diphosphonium ionic liquids as broad-spectrum antimicrobial agents.

PURPOSE: One of the most disturbing trends in recent years is the growth of resistant strains of bacteria with the simultaneous dearth of new antimicrobial agents. Thus, new antimicrobial agents for the use on the ocular surface are needed. METHODS: We synthesized a variety of ionic liquid compounds, which possess 2 positively charged phosphonium groups separated by 10 methylene units in a "bola"-type configuration. We tested these compounds for antimicrobial activity versus a variety of ocular pathogens, as well as their cytoxicity, in vitro in a corneal cell line and in vivo in mice. RESULTS: The ionic liquid Di-Hex C10 demonstrated broad in vitro antimicrobial activity at low micromolar concentrations versus gram-negative and gram-positive organisms, including methicillin-resistant Staphylococcus aureus strains and ocular fungal pathogens. Treatment with Di-Hex C10 resulted in bacterial killing in as little as 15 minutes in vitro. Di-Hex C10 showed little cytotoxicity at 1 µM versus a corneal epithelial cell line or at 10 µM in a mouse corneal wound model. We also show that this bis-phosphonium ionic liquid structure is a key because a comparable monophosphonium ionic liquid is cytotoxic to both bacteria and corneal epithelial cells. CONCLUSIONS: Here, we report the first use of dicationic bis-phosphonium ionic liquids as antimicrobial agents. Our data suggest that diphosphonium ionic liquids may represent a new class of broad-spectrum antimicrobial agents for the use on the ocular surface.

Synthesis and characterization of dendron cross-linked PEG hydrogels as corneal adhesives.

In pursuit of a wound-specific corneal adhesive, hydrogels formed by the reaction of propionaldehyde, butyraldehyde, or 2-oxoethyl succinate-functionalized poly(ethylene glycol) (PEG) with a peptide-based dendritic cross-linker (Lys(3)Cys(4)) were characterized. These macromers react within minutes of mixing to form transparent and elastic hydrogels with in vitro degradation times that range from hours to months based on the type of bonds formed during the cross-linking reaction, either thiazolidine or pseudoproline. The mechanical properties of these materials, determined via parallel plate rheology, were dependent on the polymer concentration, as was the hydrogel adhesive strength, which was determined by lap shear adhesive testing. In addition, these hydrogels were efficacious in closing ex vivo 4.1 mm central corneal lacerations: wounds closed with these hydrogel adhesives were able to withstand intraocular pressure values equivalent to, or in excess of, those obtained by closing the wounds with suturing.

A versatile reagent to synthesize diverse ionic liquids ranging from small molecules and dendrimers to functionalized proteins.

An ionic liquid "reagent" bearing a succinimidyl activated ester is reported that can be used to synthesize a variety of small molecule and macromolecular ionic liquids. In addition, the ionic liquid reagent was used to modify lysozyme, and the protein retained its structure and function after modification. This study describes a facile and reliable route to new ionic liquid compositions.

Acidic polysaccharide mimics via ring-opening metathesis polymerization.

An efficient and general synthetic strategy for the preparation of high-molecular-weight hydrophilic polymers bearing both carboxylic acid and hydroxyl pendant groups is described. Specifically, poly(5,6-dihydroxyoxanorbornane carboxylic acid) with molecular weight ranging from ∼100 000 to 5 000 000 g/mol was prepared by ring-opening metathesis polymerization of methyl 5-oxanorbornene-2-carboxylate in the presence of Grubbs catalyst II and subsequently modified to tune the hydrophobic/hydrophilic properties by the introduction of either hydroxyl or carboxylic acid functionalities. These polymers mimic the natural acidic polysaccharide alginate and form hydrogels with polylysine. These polymers belong to a class of carbohydrate-like polymers, which are of interest for investigating the relationships between chemical structure and rheological properties as well as for providing new synthetic polysaccharide substitutes for applications in the biotechnology and pharmaceutical industries.

In vitro sealing of clear corneal cataract incisions with a novel biodendrimer adhesive.

OBJECTIVE: To determine if a biodendrimer adhesive will seal a clear corneal cataract incision. DESIGN: An experimental study in which 2.75-mm clear corneal cataract incisions were made in 8 human donor eyes. The corneas were mounted on an artificial anterior chamber. Leaking pressure was determined in 6 corneas. These corneas were then treated with adhesive and leaking pressure was again measured in 4 of them. India ink was then applied to the 2 remaining treated and the 2 untreated corneas. Chamber pressure was cycled between 100 and 0 mm Hg. Optical coherence tomography was used to visualize the wound dynamics of a ninth cornea treated with adhesive, mounted, and pressure cycled in a similar fashion. RESULTS: The mean (SD) leaking pressure was 77 (14) mm Hg for the nonsealed wounds and 142 (22) mm Hg for the adhesive-sealed wounds. india ink entered the nonsealed wounds and anterior chamber when the intraocular pressure was cyclically raised and lowered, whereas no india ink entered the adhesive-sealed wounds. The optical coherence tomography-visualized corneal wound did gape under pressure cycling, but the adhesive remained intact and stretched to conform to the wound. CONCLUSIONS: Biodendrimer adhesives may be used to seal cataract wounds to prevent leakage and influx of fluid.

Real time imaging of supramolecular assembly formation via programmed nucleolipid recognition.

Supramolecular assembly formation resulting from molecular recognition between complementary nucleolipids has been visualized in real time at the micrometer scale.

Synthesis and properties of supramolecular ionic networks.

The synthesis of a supramolecular ionic network, its physical properties, and the use of this network property to form macroscopic porphyrin fibers are described. These ionic networks are compared to ionic liquids. Current ionic liquid compositions have a charge and molar ratio of 1:1 where an anionic species is matched with a cationic species; however, alteration of this molar ratio while maintaining the charge ratio of 1:1 by using multivalent cationic/anionic molecular pairs affords new ionically cross-linked networks with interesting properties.

Photo cross-linkable Biodendrimers as ophthalmic adhesives for central lacerations and penetrating keratoplasties.

PURPOSE: Biodendrimer-based hydrogel adhesives were derived from biocompatible building blocks and poly(ethylene glycol) of 3,400, 10,000 and 20,000 g/mole. The leaking pressures were determined for these adhesives when used to seal 4.1-mm central lacerations and penetrating keratoplasties (PKPs) in enucleated porcine eyes. METHODS: Three biodendrimers, ([G1]-PGLSA-MA)(2)-PEG(3,400), ([G1]-PGLSA-MA)(2)-PEG(10,000), and ([G1]-PGLSA-MA)(2)-PEG(20,000), at a range of weight percents were each photo cross-linked in the presence of a photo-initiator to form a hydrated network. These biodendrimer-based adhesives were applied directly to a 4.1-mm linear central laceration. In a PKP, the corneal button was initially secured with 8 or 16 sutures and then sealed with the adhesive. RESULTS: For the 4.1-mm central lacerations, the ([G1]-PGLSA-MA)(2)-PEG(3,400) at 20% and 40% wt/vol, the ([G1]-PGLSA-MA)(2)-PEG(10,000) at 10 and 20% wt/vol, and the ([G1]-PGLSA-MA)(2)-PEG(20,000) at 20% wt/vol held to leaking pressures above 200 mm Hg. In the autograft with 16 sutures, the 20% wt/vol of the ([G1]-PGLSA-MA)(2)-PEG(3,400), ([G1]-PGLSA-MA)(2)-PEG(10,000), and ([G1]-PGLSA-MA)(2)-PEG(20,000) held to a pressure at or above 100 mm Hg. In the autograft with eight sutures, the ([G1]-PGLSA-MA)(2)-PEG(10,000) and ([G1]-PGLSA-MA)(2)-PEG(20,000) formulations at 20% wt/vol held to leaking pressures of 85 +/- 22 and 80 +/- 30 mm Hg, respectively. CONCLUSIONS: The 10% wt/vol ([G1]-PGLSA-MA)(2)-PEG(10,000) formulation withheld leaking pressures above 200 mm Hg when used to secure a 4.1 mm central laceration. The 20% wt/vol ([G1]-PGLSA-MA)(2)-PEG(10,000) and ([G1]-PGLSA-MA)(2)-PEG(20,000) formulations, with 8 or 16 sutures, secured the PKP well above normal IOP. Biodendrimer-based adhesives are of potential use for repairing corneal wounds.

Immobilized hydrogels for screening of molecular interactions.

Spatially arrayed, high-density microarrays enable the rapid assessment of biological recognition events, and this information is of widespread interest for those working in basic research laboratories as well as in the clinic. Today, one can find DNA, protein, or small molecule arrays. Limitations with these systems include covalent modification of the target complement to the array substrate, array- and target-dependent setup conditions, multiple steps, and loss of hydration at the surface. To overcome these limitations, we have designed, prepared, and evaluated immobilized hydrogels as general screening chambers for small molecule-protein, protein-protein, and nucleic acid-nucleic acid interactions. This biomaterial-based approach is facile, rapid, requires only one setup protocol, and physically entraps the target complement within the polymer network and thus offers advantages over the conventional chips.

Dendrimer-encapsulated camptothecins: increased solubility, cellular uptake, and cellular retention affords enhanced anticancer activity in vitro.

A biocompatible polyester dendrimer composed of the natural metabolites, glycerol and succinic acid, is described for the encapsulation of the antitumor camptothecins, 10-hydroxycamptothecin and 7-butyl-10-aminocamptothecin. The cytotoxicity of the dendrimer-drug complex toward four different human cancer cell lines [human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non-small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268)] is also reported, and low nmol/L IC(50) values are measured. Cellular uptake and efflux measurements in MCF-7 cells show an increase of 16-fold for cellular uptake and an increase in drug retention within the cell when using the dendrimer vehicle.

Hydrogels formed by multiple peptide ligation reactions to fasten corneal transplants.

A stable cross-linked hydrogel was formed under mild aqueous conditions using pseudoproline peptide ligation chemistry. A cysteine-terminated lysine dendron containing four cysteines and a PEG macromolecule modified with terminal ester aldehydes were prepared. Upon mixing, the two macromers gave a stable hydrogel. This hydrogel along with sutures was used to successfully secure a corneal transplant in vitro.

Novel tissue adhesives to secure laser in situ keratomileusis flaps.

PURPOSE: To evaluate 2 novel biodendrimer tissue adhesives in sealing and securing laser in situ keratomileusis (LASIK) flaps. SETTING: Duke University Eye Center, Durham, North Carolina, USA. METHODS: Laser in situ keratomileusis flaps were created in 10 human eye-bank eyes using the Hansatome microkeratome system (Bausch & Lomb). These eyes were divided into 2 groups. Flaps in the first group (n=4) were secured with a laser-activated biodendrimer adhesive along the flap edge. In the second group (n=6), the flaps were secured with a self-gelling dendritic adhesive. Dry Merocel sponges (Medtronic Solan) were used to test the strength of flap adherence in both groups. Further testing was performed in the second group. The hinges of these flaps were cut with a scalpel blade and fluorescein dye was injected under the flap to observe potential dye leakage along the flap edge. RESULTS: Laser in situ keratomileusis flaps sealed with both adhesives were secure with no flap dislocation. There was no leakage of fluorescein dye observed in the second group. Both adhesives were easy to apply, clear when dry, and had a soft rubbery consistency. CONCLUSIONS: Two novel biodendrimer adhesives successfully sealed and secured LASIK flaps. These adhesives may prove to be an effective alternative for treating LASIK flap complications such as epithelial ingrowth or flap dislocation.

Dendritic macromers as in situ polymerizing biomaterials for securing cataract incisions.

Dendritic macromers are attractive macromolecules for hydrogel formation since high cross-linking densities at low polymer concentration can be obtained, varied physical properties can be observed based on the macromer structure, and low viscous aqueous solutions can be injected in an in vivo site of irregular shaped to form a well-integrated polymer network. A peptide dendron possessing terminal cysteine residues was synthesized and characterized. When this peptide dendron was mixed with poly(ethylene glycol dialdehyde) in aqueous solution at pH = 7.4, a hydrogel spontaneously formed as a consequence of thiazolidine linkages between the macromers. Such in situ polymerized hydrogels are of interest for tissue engineering and wound-repair applications. To evaluate the potential use of this hydrogel sealant in ophthalmic surgeries, a 3-mm clear corneal incision (i.e., the wounds created during a typical cataract surgery) was successfully sealed.

Change of cell wall chitin content in amphotericin B resistant Kluyveromyces strains.

The culture of two Kluyveromyces species, Kluyveromyces lactis (ATCC 96897) and Kluyveromyces bulgaricus (ATCC 96631), in the presence of subinhibitory doses of amphotericin B leads to the selection of mutants which are resistant to this polyene. The mutants show an alteration of their cell wall composition with the main change corresponding to an increase of chitin. The enzyme activities involved in the metabolism of this polymer, i.e. chitin synthases and chitinase, were measured. The results demonstrate that in both mutants the activity of chitinase was drastically decreased by 99% in comparison with the activity measured in the corresponding wild-type strain while no significant change of the chitin synthase I, II and III activities could be detected.