Dea Herrera-Ruiz

Spatial expression patterns of peptide transporters in the human and rat gastrointestinal tracts, Caco-2 In Vitro cell culture model, and multiple human tissues

This study sought to identify the spatial patterns of expression of peptide transporter 1 (PepT1), peptide transporter 3 (PTR3), peptide/histidine transporter 1 (PHT1), and the human peptide transporter 1 (HPT-1) mRNA in complementary DNA (cDNA) libraries of the human and rat gastrointestinal tracts (GIT), Caco-2 in vitro cell culture model, and in a human multiple tissue panel. Human PTR3 and PHT1 are putative peptide transporters recently discovered. Using sequence-specific primers designed to amplify regions of PepT1, PTR3, PHT1, and HPT-1, we were able to identify the expression of mRNA for each of these transporters in human cDNA panels (Clontech, Palo Alto, CA), the rat GIT, and in Caco-2 cDNA libraries by the polymerase chain reaction (PCR) and Southern Blot analysis. These studies suggest that in the human GIT, PepT1 appears to be localized predominantly in the duodenum, with decreasing expression in the jejunum and ileum. In contrast, PTR3 and HPT-1 were widely expressed in the human GIT, with predominant expression in the different regions of the colon. PHT1 appeared to be expressed in low levels throughout the human GI tract. Interestingly, the mRNAs for all 4 peptide transporters were expressed in Caco-2 cells throughout 30 days of culture. PepT1, PTR3, PHT1, and HPT-1 were also widely expressed in the rat GIT. Human tissue cDNA panel screening suggests that PTR3 and PHT1 are more uniformly expressed, whereas PepT1 and HPT-1 demonstrated site-specific expression. These results suggest that PepT1, PTR3, PHT1, and HPT-1 all may act to facilitate the diffusion of peptides and peptide-based pharmaceuticals in the GIT, PTR3, PHT1, and HPT-1 expressions in Caco-2 cell monolayers strongly suggest that their function needs to be further elucidated and their contribution to peptide transport not ignored. Taken together, these results demonstrate the potential for molecular biological characterization in localizing active transporter systems that can potentially be targeted for enhancing the absorption of peptide-based pharmaceuticals.

Formulation Approaches to Short Interfering RNA and MicroRNA: Challenges and Implications

RNA interference has emerged as a potentially powerful tool in the treatment of genetic and acquired diseases by delivering short interfering RNA (siRNA) or microRNA (miRNA) to target genes, resulting in their silencing. However, many physicochemical and biological barriers have to be overcome to obtain efficient in vivo delivery of siRNA and miRNA molecules to the organ/tissue of interest, thereby enabling their effective clinical therapy. This review discusses the challenges associated with the use of siRNA and miRNA and describes the nonviral delivery strategies used in overcoming these barriers. More specifically, emphasis has been placed on those technologies that have progressed to clinical trials for both local and systemic siRNA and miRNA delivery.

Design and In Vitro Evaluation of a New Nano-Microparticulate System for Enhanced Aqueous-Phase Solubility of Curcumin

Curcumin, a yellow polyphenol derived from the turmeric Curcuma longa, has been associated with a diverse therapeutic potential including anti-inflammatory, antioxidant, antiviral, and anticancer properties. However, the poor aqueous solubility and low bioavailability of curcumin have limited its potential when administrated orally. In this study, curcumin was encapsulated in a series of novel nano-microparticulate systems developed to improve its aqueous solubility and stability. The nano-microparticulate systems are based entirely on biocompatible, biodegradable, and edible polymers including chitosan, alginate, and carrageenan. The particles were synthesized via ionotropic gelation. Encapsulating the curcumin into the hydrogel nanoparticles yielded a homogenous curcumin dispersion in aqueous solution compared to the free form of curcumin. Also, the in vitro release profile showed up to 95% release of curcumin from the developed nano-microparticulate systems after 9 hours in PBS at pH 7.4 when freeze-dried particles were used.

On molecular complexes derived from amino acids and nicotinamides in combination with boronic acids

Cocrystallization experiments of three representative aromatic boronic acids, namely phenylboronic acid (PBA), 1,4-benzenediboronic acid (BDBA) and 4-iodophenylboronic acid (IPBA), with a series of essential amino acids, nicotinamide (NA) and isonicotinamide (INA) gave a total of nine molecular complexes of the compositions: PBA–PRO (1 : 1), PBA–PRO–H2O (1 : 1 : 1), α-BDBA–PRO (1 : 2), β-BDBA–PRO (1 : 2), α-IPBA–PRO (1 : 1), β-IPBA–PRO (1 : 1), BDBA–INA (1 : 2), IPBA–INA (1 : 1) and IPBA–NA (1 : 1), where PRO = L-proline. Of these, α-BDBA–PRO/β-BDBA–PRO and α-IPBA–PRO/β-IPBA–PRO were true polymorphs. Experiments varying the solvent in the screening process showed that the formation of the polymorphs is influenced strongly by the presence/absence of water. For the cocrystalline phases with L-proline, the structural characterization revealed that the molecular components were connected into high-dimensional hydrogen bonded networks. As expected, the charge-assisted –B(OH)2⋯carboxylate heterosynthon is dominant and was found to display varying degrees of distortion from planarity. The α-IPBA–PRO/β-IPBA–PRO polymorphs displayed a similar crystal packing, with differences only in one direction of the crystal lattice (AAAA versus ABAB stacking). The cocrystals with nicotinamide and isonicotinamide exhibited 1D or 2D hydrogen bonded layers, which were formed through (B)O–H⋯pyridine, B(OH)2⋯amide, amide⋯amide and/or single-bridged (B)O–H⋯O(H)B interactions. Comparison between the crystal structures of IPBA–INA and IPBA–NA revealed that the position of the heteroatom in the (iso)nicotinamide not only controlled the final crystalline form but also produced different hydrogen bonding interactions. In vitro toxicology studies showed that the cell viability is conserved when human kidney or hepatic cells are treated even with high concentrations of the boronic acids examined herein. However, some boronic acids exhibited teratogenicity in chicken embryos.

Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases. However, the siRNA therapy has been limited by its low stability and rapid degradation in presence of nucleases, low cellular uptake, and immune response activation. In order to overcome these drawbacks, we propose the synthesis and characterization of non-viral delivery systems using chitosan derivatives to obtain siRNA complexes (polyplexes). The non-viral delivery systems synthesized included PEG-g-OCs (oligochitosan) and PEG-g-Cs (chitosan medium molecular weight). Both systems allowed the formation of siRNA polyplexes, increased the stability of siRNA in the presence of nucleases, enhanced cellular internalization, and showed low toxicity in the A549 cell line. Finally, the complexes obtained with the PEG-g-OCs system showed silencing activity in a GFP model in the cell line A549 in comparison with naked siRNA.

A novel aerosol method for the production of hydrogel particles

A novel method of generating hydrogel particles for various applications including drug delivery purposes was developed. This method is based on the production of hydrogel particles from sprayed polymeric nano/microdroplets obtained by a nebulization process that is immediately followed by gelation in a crosslinking fluid. In this study, particle synthesis parameters such as type of nebulizer, type of crosslinker, air pressure, and polymer concentration were investigated for their impact on the mean particle size, swelling behavior, and morphology of the developed particles. Spherical alginate-based hydrogel particles with a mean particle size in the range from 842 to 886 nm were obtained. Using statistical analysis of the factorial design of experiment it was found that the main factors influencing the size and swelling values of the particles are the alginate concentration and the air pressure. Thus, it was demonstrated that the method described in the current study is promising for the generation of hydrogel particles and it constitutes a relatively simple and low-cost system.

Interrelation of the dissolution behavior and solid-state features of acetazolamide cocrystals

&NA; The thermal behavior, phase stability, indicative stability and intrinsic dissolution rates of a series of cocrystals and cocrystal hydrates derived from the pharmaceutically active ingredient acetazolamide (ACZ) and 2‐aminobenzamide (2ABAM), 2,3‐dihydroxybenzoic acid (23DHBA), 2‐hydroxybenzamide (2HBAM), 4‐hydroxybenzoic acid (4HBA), nicotinamide (NAM) and picolinamide (PAM) as cocrystal formers have been evaluated. Upon heating in an inert atmosphere most of the cocrystals tested demonstrated first the elimination of the crystal former, followed by ACZ degradation. Only in cocrystals with NAM was melting observed. Under controlled temperature and relative humidity conditions all cocrystals tested were stable. However, phase stability tests in a medium simulating physiological conditions (HCl 0.01 N, pH 2.0) indicated that cocrystals ACZ‐NAM‐H2O and ACZ‐PAM gradually transform into ACZ. All cocrystals examined gave enhanced intrinsic dissolution rates when compared to pure ACZ and the largest dissolution rate constants were measured for the cocrystals that transformed in the phase stability test (approximate two‐fold increase of the dissolution rate constants). The series of cocrystals examined herein exhibits an inverse correlation between the intrinsic dissolution rates and the melting/decomposition temperatures as well as the dimension of the hydrogen‐bonded ACZ aggregates found in the corresponding crystal structure, indicating that solid‐state stability is the major influence on dissolution performance. Graphical abstract Figure. No caption available.

Sulfonate salts of the therapeutic agent dapsone: 4-[(4-aminophenyl)sulfonyl]anilinium benzenesulfonate monohydrate and 4-[(4-aminophenyl)sulfonyl]anilinium methanesulfonate monohydrate.

Dapsone, formerly used to treat leprosy, now has wider therapeutic applications. As is the case for many therapeutic agents, low aqueous solubility and high toxicity are the main problems associated with its use. Derivatization of its amino groups has been widely explored but shows no significant therapeutic improvements. Cocrystals have been prepared to understand not only its structural properties, but also its solubility and dissolution rate. Few salts of dapsone have been described. The title salts, C12H13N2O2S(+)·C6H5O3S(-)·H2O and C12H13N2O2S(+)·CH3SO3(-)·H2O, crystallize as hydrates and both compounds exhibit the same space group (monoclinic, P21/n). The asymmetric unit of each salt consists of a 4-[(4-aminophenyl)sulfonyl]anilinium monocation, the corresponding sulfonate anion and a water molecule. The cation, anion and water molecule form hydrogen-bonded networks through N-H...O=S, N-H...Owater and Owater-H...O=S hydrogen bonds. For both salts, the water molecules interact with one sulfonate anion and two anilinium cations. The benzenesulfonate salt forms a two-dimensional network, while the hydrogen bonding within the methanesulfonate salt results in a three-dimensional network.