A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.

A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative chromatograms of biological samples containing these analytes are provided. In addition, the pharmacokinetic profile of these compounds in one human who daily consumed an over-the-counter curcuminoid product shows the peak and changes in circulating concentrations achieved by this mode of administration.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.

Hyaluronic Acid Microgels as Intracellular Endosomolysis Reagents.

Hyaluronic acid (HA) microgels were investigated as biocompatible and biodegradable reagents for facilitating endosomolysis in human cells. Employing inverse emulsion templates, HA microgels were prepared by cross-linking aqueous sodium hyaluronate droplets with divinyl sulfone (DVS). Introduction of ether sulfone cross-links was confirmed by infrared (IR) spectroscopy and elemental analysis. The degree of cross-linking of the microgels was estimated using high performance liquid chromatography (HPLC). The size distribution of the water-dispersible HA microgels was studied by laser diffraction analysis, and the gel morphology was investigated using scanning electron microscopy (SEM). Aqueous microgel suspensions were found to be well-tolerated in human cells at concentrations of up to 100 µg/mL. Endosome-rupturing properties of the HA microgels were evaluated in vitro using calcein internalization and Cre protein delivery assays. The results of this study serve as a proof-of-principle for the utility of cross-linked HA microgels as a new class of biocompatible and biodegradable endosomolytic reagents.

Glycan structure determinants for cation-independent mannose 6-phosphate receptor binding and cellular uptake of a recombinant protein.

The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a ß linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

Hyaluronan-tethered opioid depots: synthetic strategies and release kinetics in vitro and in vivo.

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

Synthesis and evaluation of hydrolyzable hyaluronan-tethered bupivacaine delivery systems.

Local anesthetics are useful for reducing acute pain, but their short duration precludes them from use in solely managing postoperative pain. To prolong the duration of local anesthesia, we conjugated bupivacaine to native hyaluronan (HA) and divinyl sulfone cross-linked Hylan A (Hylan B particles) using a hydrolyzable linker incorporating an imide. Bupivacaine was prepared for conjugation to HA by forming the acryl imide derivative. Separately, the carboxyl group of HA was reacted with nipsylethylamine (NEA) using carbodiimide-mediated coupling to provide HA-NEA that was subsequently reduced with tris(2-carboxyethylphosphine) hydrochloride to yield HA carrying a free sulfhydryl (HA-SH). The HA-bupivacaine conjugate was assembled by reacting HA-SH with acrylbupivacaine. Characterization of the conjugates showed 22% degree of modification by 1 mol of carboxyl. In vitro release studies comparing bupivacaine admixed in HA with bupivacaine conjugated to HA showed half-lives of 0.4 +/- 0.1 h, and 16.9 +/- 0.2 h, respectively, and the bupivacaine was released chemically unaltered as confirmed by LC-MS. In vivo studies to assess the duration of anesthetic activity were performed in a rat sciatic nerve blockade model. For these studies, bupivacaine was conjugated to Hylan B following a similar procedure, and the degree of modification obtained was 14%. Free bupivacaine (3 and 16 mg/kg) and free bupivacaine (3 mg/kg) admixed with Hylan B particles showed nerve block over 4, 9, and 6 h, respectively. Free bupivacaine (3 mg/kg) admixed with bupivacaine (13 mg/kg) conjugated to Hylan B particles showed a four to 5-fold longer impairment of motor function over the free bupivacaine formulations with a total block time of 19 h. Bupivacaine conjugated to Hylan B particles has the potential to prolong the duration of local anesthesia.