Tenofovir Douche as HIV Preexposure Prophylaxis for Receptive Anal Intercourse: Safety, Acceptability, Pharmacokinetics, and Pharmacodynamics (DREAM 01).

BACKGROUND: Despite highly effective HIV preexposure prophylaxis (PrEP) options, no options provide on-demand, nonsystemic, behaviorally congruent PrEP that many desire. A tenofovir-medicated rectal douche before receptive anal intercourse may provide this option. METHODS: Three tenofovir rectal douches-220 mg iso-osmolar product A, 660 mg iso-osmolar product B, and 660 mg hypo-osmolar product C-were studied in 21 HIV-negative men who have sex with men. We sampled blood and colorectal tissue to assess safety, acceptability, pharmacokinetics, and pharmacodynamics. RESULTS: The douches had high acceptability without toxicity. Median plasma tenofovir peak concentrations for all products were several-fold below trough concentrations associated with oral tenofovir disoproxil fumarate (TDF). Median colon tissue mucosal mononuclear cell (MMC) tenofovir-diphosphate concentrations exceeded target concentrations from 1 hour through 3 to 7 days after dosing. For 6-7 days after a single product C dose, MMC tenofovir-diphosphate exceeded concentrations expected with steady-state oral TDF 300 mg on-demand 2-1-1 dosing. Compared to predrug baseline, HIV replication after ex vivo colon tissue HIV challenge demonstrated a concentration-response relationship with 1.9 log10 maximal effect. CONCLUSIONS: All 3 tenofovir douches achieved tissue tenofovir-diphosphate concentrations and colorectal antiviral effect exceeding oral TDF and with lower systemic tenofovir. Tenofovir douches may provide a single-dose, on-demand, behaviorally congruent PrEP option, and warrant continued development. Clinical Trials Registration . NCT02750540.

Design of Thermoresponsive Polyamine Cross-Linked Perfluoropolyether Hydrogels for Imaging and Delivery Applications.

Perfluorocarbons are versatile compounds with applications in 19F magnetic resonance imaging (MRI) and chemical conjugation to drugs and pH sensors. We present a novel thermoresponsive perfluorocarbon emulsion hydrogel that can be detected by 19F MRI. The developed hydrogel contains perfluoro(polyethylene glycol dimethyl ether) (PFPE) emulsion droplets that are stabilized through ionic cross-linking with polyethylenimine (PEI). Specifically, PFPE ester undergoes hydrolysis upon contact with aqueous PEI solution, resulting in an ionic bond between the PFPE acid and charged PEI amino groups. Due to the ionic nature of the PFPE/PEI bond, potassium buffer is required to preserve the hydrogel's pH and rheological and emulsion droplet stability. The presence of the surface cross-linked PFPE droplets does not affect the hydrogel's rheological behavior, drug loading, or drug release, and the hydrogel is nontoxic. We propose that the presented hydrogel can be adapted to a broad range of biomedical imaging and delivery applications.

Development and characterization of resveratrol nanoemulsions carrying dual-imaging agents.

AIM: Delivery of the natural anti-inflammatory compound resveratrol with nanoemulsions can dramatically improve its tissue targeting, bioavailability and efficacy. Current assessment of resveratrol delivery efficacy is limited to indirect pharmacological measures. Molecular imaging solves this problem. Results/methodology: Nanoemulsions containing two complementary imaging agents, near-infrared dye and perfluoropolyether (PFPE), were developed and evaluated. Nanoemulsion effects on macrophage uptake, toxicity and NO production were also evaluated. The presence of PFPE did not affect nanoemulsion size, zeta potential, colloidal stability, drug loading or drug release. CONCLUSION: PFPE nanoemulsions can be used in future studies to evaluate nanoemulsion biodistribution without interfering with resveratrol delivery and pharmacological outcomes. Developed nanoemulsions show promise as a versatile treatment strategy for cancer and other inflammatory diseases. [Formula: see text].

Utilization of Near-Infrared Fluorescent Imaging for Pharmaceutically Relevant Applications.

Optical imaging can be utilized for several pharmaceutical applications involving near-infrared fluorescent (NIRF) dyes or NIRF moiety-containing products. Especially during the early phases of product development, NIRF dyes can be used as surrogates for drugs and optical imaging methods can be utilized to optimize the pharmaceutical product properties based on dye entrapment efficiency, in vitro dye release, cellular uptake, and in vivo biodistribution. Based on in vivo accumulation, product efficacy and toxicity can be evaluated in the early development stage. Compared to visible fluorescent dyes, NIRF offers advantages such as low background from formulation excipients as well as biological components.In this chapter, the utility of NIRF imaging methods for in vitro characterization (in vitro release and cellular uptake) and in vivo/ex vivo applicability of pharmaceutically relevant products is presented in detail. Specifically, the application of fluorescence imaging to characterize perfluorocarbon-based formulations for dye loading, in vitro release, cellular uptake, and in vivo imaging to assess target accumulation and biodistribution is discussed. These methods are widely applicable to other nanoparticle-based products involved in inflammation/cancer imaging and therapy. Overall, NIRF-based techniques are indispensible because they are relatively easy, fast, and cost effective to characterize and optimize pharmaceutical products at different stages of product development.

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a ß-fibrillizing peptide (ßFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a ßFP that forms ß-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose-response titration experiments showed that loading of IgG into the film was mediated by pAG(MG) bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAG(MG) after five days, compared to close to 90% in films without pAG(MG). Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAG(MG). When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

The First Scale-Up Production of Theranostic Nanoemulsions.

Theranostic nanomedicines are a promising new technological advancement toward personalized medicine. Although much progress has been made in pre-clinical studies, their clinical utilization is still under development. A key ingredient for successful theranostic clinical translation is pharmaceutical process design for production on a sufficient scale for clinical testing. In this study, we report, for the first time, a successful scale-up of a model theranostic nanoemulsion. Celecoxib-loaded near-infrared-labeled perfluorocarbon nanoemulsion was produced on three levels of scale (small at 54 mL, medium at 270 mL, and large at 1,000 mL) using microfluidization. The average size and polydispersity were not affected by the equipment used or production scale. The overall nanoemulsion stability was maintained for 90 days upon storage and was not impacted by nanoemulsion production scale or composition. Cell-based evaluations show comparable results for all nanoemulsions with no significant impact of nanoemulsion scale on cell toxicity and their pharmacological effects. This report serves as the first example of a successful scale-up of a theranostic nanoemulsion and a model for future studies on theranostic nanomedicine production and development.

Bioengineering mini functional thymic units with EAK16-II/EAKIIH6 self-assembling hydrogel.

Herein, we highlight the technical feasibility of generating a functional mini thymus with a novel hydrogel system, based on a peptide-based self-assembly platform that can induce the formation of 3-D thymic epithelial cell (TEC) clusters. Amphiphilic peptide EAK16-II co-assembled with its histidinylated analogue EAKIIH6 into beta-sheet fibrils. When adaptor complexes (recombinant protein A/G molecules loaded with both anti-His and anti-EpCAM IgGs) were added to the mix, TECs were tethered to the hydrogel and formed 3-D mini clusters. TECs bound to the hydrogel composites retained their molecular properties; and when transplanted into athymic nude mice, they supported the development of functional T-cells. These mini thymic units of TECs can be useful in clinical applications to reconstitute T-cell adaptive immunity.

Solvent-induced frequency shifts of 5-hydroxymethylfurfural deduced via infrared spectroscopy and ab initio calculations.

Solvent-induced frequency shifts (SIFS) of the carbonyl stretching vibration ν(C═O) of 5-hydroxymethylfurfural were measured in protic, polar aprotic, and nonpolar solvents. The Gutmann acceptor number (AN) was found to correlate with the measured frequency shifts. The SIFS in six solvents were investigated using ab initio electronic structure calculations, treating the solvent implicitly and with an explicit solvent ligand interacting with the carbonyl. The conductor-polarizable continuum model (CPCM) of solvation predicted that ν(C═O) shifted according with the dielectric constant as (ε - 1)/(2ε + 1), in agreement with the analytical predictions of the Kirkwood-Bauer-Magat (KBM) theory for a dipole in a dielectric continuum, but in disagreement with the experimental trend. The experimental SIFS were best predicted using gas-phase complexes of HMF and explicit solvent-ligand. Natural bond orbital (NBO) analysis and Bader's atoms in molecules theory were used to investigate the electronic structure of these complexes. Strong SIFS were found to arise from stronger H-bonding interactions, as observed in delocalization of carbonyl lone-pair electrons by H-bonding solvent σ*(X-H) orbitals, and an increase in charge density and a decrease in local potential energy at the H-bond (3, -1) critical point. Consequently, by predicting the experimental SIFS and examining the electronic structure, we find the first theoretical evidence for treating Gutmann's solvent AN as a measure of solvent Lewis acidity.

Antibody-functionalized peptidic membranes for neutralization of allogeneic skin antigen-presenting cells.

We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6 days. At the host-graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II major histocompatibility complex (MHC) molecule (I-A(d)) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with anti-I-A(d) antibodies than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on anti-I-A(d) antibody-loaded membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in allogeneic transplant models.

Design and formulation of nanoemulsions using 2-(poly(hexafluoropropylene oxide)) perfluoropropyl benzene in combination with linear perfluoro(polyethylene glycol dimethyl ether).

This is the first report where PFPAE aromatic conjugates and perfluoro(polyethylene glycol dimethyl ether) are combined and formulated as nanoemulsions with droplet size below 100 nm. A perfluoropolyalkylether (PFPAE) aromatic conjugate, 2-(poly(hexafluoropropylene oxide)) perfluoropropyl benzene, was used as fluorophilic-hydrophilic diblock (FLD) aimed at stabilizing perfluoro(polyethylene glycol dimethyl ether) nanoemulsions. Its effects on colloidal behaviors in triphasic (organic/fluorous/aqueous) nanoemulsions were studied. The addition of FLD construct to fluorous phase led to decrease in PFPAE nanoemulsion droplet size to as low as 85 nm. Prepared nanoemulsions showed high colloidal stability. Our results suggest that these materials represent viable novel approach to fluorous colloid systems design with potential for biomedical and synthetic applications.

Cannabinoid CB2 receptor as a new phototherapy target for the inhibition of tumor growth.

The success of targeted cancer therapy largely relies upon the selection of target and the development of efficient therapeutic agents that specifically bind to the target. In the current study, we chose a cannabinoid CB2 receptor (CB2R) as a new target and used a CB2R-targeted photosensitizer, IR700DX-mbc94, for phototherapy treatment. IR700DX-mbc94 was prepared by conjugating a photosensitizer, IR700DX, to mbc94, whose binding specificity to CB2R has been previously demonstrated. We found that phototherapy treatment using IR700DX-mbc94 greatly inhibited the growth of CB2R positive tumors but not CB2R negative tumors. In addition, phototherapy treatment with nontargeted IR700DX did not show significant therapeutic effect. Similarly, treatment with IR700DX-mbc94 without light irradiation or light irradiation without the photosensitizer showed no tumor-inhibitory effect. Taken together, IR700DX-mbc94 is a promising phototherapy agent with high target-specificity. Moreover, CB2R appears to have great potential as a phototherapeutic target for cancer treatment.

Determination of proton affinities and acidity constants of sugars.

Proton transfer reactions play a key role in the conversion of biomass derived sugars to chemicals. In this study, we employ high level ab initio theoretical methods, in tandem with solvation effects to calculate the proton affinities (PA) and acidity constants (pKa) of various d-glucose and d-fructose tautomers (protonation-deprotonation processes). In addition, we compare the theoretically derived pH values of sugar solutions against experimentally measured pH values in our lab. Our results demonstrate that the protonation of any of the O atoms of the sugars is thermodynamically preferred without any significant variation in the PA values. Intramolecular hydrogen transfers, dehydration reactions, and ring-opening processes were observed, resulting from the protonation of specific hydroxyl groups on the sugars. Regarding the deprotonation processes (pKa), we found that the sugars' anomeric hydroxyls exhibit the highest acidity. The theoretically calculated pH values of sugar solutions are in excellent agreement with experimental pH measurements at low sugar concentrations. At higher sugar concentrations the calculations predict less acidic solutions than the experiments. In this case, we expect the sugars to act as solvents increasing the proton solvation energy and the acidity of the solutions. We demonstrated through linear relationships that the pKa values are correlated with the relative stability of the conjugate bases. The latter is related to hydrogen bonding and polarization of the C-O(-) bond. A plausible explanation for the good performance of the direct method in calculating the pKa values of sugars can be the presence of intramolecular hydrogen bonds on the conjugate base. Both theory and experiments manifest that fructose is a stronger acid than glucose, which is of significant importance in self-catalyzed biomass-relevant dehydration reactions.

Synthesis and evaluation of molecularly imprinted polymers for enalapril and lisinopril, two synthetic peptide anti-hypertensive drugs.

Molecularly imprinted polymers (MIPs) for the recognition of enalapril and lisinopril were prepared using 4-vinylpyridine as the functional monomer. Following thermal polymerisation the resulting materials were crushed, ground and sieved. First generation MIPs were produced in protic polar porogenic solvents (mixture of methanol (MeOH) and acetonitrile (ACN)). These MIPs were used and validated as sorbents for solid phase extraction and binding assays. Second generation MIPs were produced with polar aprotic porogenic solvent (DMSO). These polymers were packed in HPLC columns in order to investigate their molecular recognition properties in a dynamic mode. The study of the mobile phase composition included two major parameters: organic modifier content and pH value. Retention factors illustrate selective binding of the template from the imprinted polymers, compared to structurally related compounds.